ELIZABETH GILLAM

Engineering Thermostable CYP2D Enzymes for Biocatalysis Using Combinatorial Libraries of Ancestors for Directed Evolution (CLADE)

Publisher: Wiley

Date: 09-01-2019

DOI: 10.1002/CCTC.201801644

Expression of Cytochrome P450 3A7 inEscherichia coli:Effects of 5′ Modification and Catalytic Characterization of Recombinant Enzyme Expressed in Bicistronic Format with NADPH-Cytochrome P450 Reductase

Publisher: Elsevier BV

Date: 10-1997

DOI: 10.1006/ABBI.1997.0286

Abstract: Cytochrome P450 3A7 is the major P450 form present in fetal liver tissue and may be responsible for the detoxification of many drugs that reach the fetal circulation. We report the development of bacterial expression systems for P450 3A7. Maximal yields (up to 50 nmol P450/liter culture) were obtained with a construct in which the 5'-terminus of the 3A7 cDNA was modified to include the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, Proc. Natl. Acad. Sci. USA 88, 5597-5601, 1991) and to incorporate several downstream amino acid substitutions derived from the P450 3A5 sequence. This sequence also appeared optimal for expression of P450 3A4 and 3A5. Recombinant P450 3A7 was partially purified using ion-exchange and hydroxylapatite chromatography and reconstituted with NADPH-cytochrome P450 reductase, cytochrome b5, and lipids. Activity comparable to that of P450 3A4 was demonstrated toward a number of procarcinogens. An alternative approach was used to further characterize recombinant 3A7 due to low yields of recombinant protein in the expression and poor recovery in the purification. P450 3A7 was subcloned into a bicistronic vector containing human NADPH-cytochrome P450 reductase and expressed in bacteria. Recombinant P450 3A7 coexpressed in bacterial membranes with NADPH-cytochrome P450 reductase showed similar levels of activity toward erythromycin (N-demethylation) and ethylmorphine (N-demethylation) to P450 3A4 and 3A5 expressed in the same system, whereas 3A7 was less active toward midazolam (1'- and 4-hydroxylation) and nifedipine (oxidation).

Exploiting photosynthesis-driven P450 activity to produce indican in tobacco chloroplasts

Publisher: Frontiers Media SA

Date: 09-01-2023

DOI: 10.3389/FPLS.2022.1049177

Abstract: Photosynthetic organelles offer attractive features for engineering small molecule bioproduction by their ability to convert solar energy into chemical energy required for metabolism. The possibility to couple biochemical production directly to photosynthetic assimilation as a source of energy and substrates has intrigued metabolic engineers. Specifically, the chemical ersity found in plants often relies on cytochrome P450-mediated hydroxylations that depend on reductant supply for catalysis and which often lead to metabolic bottlenecks for heterologous production of complex molecules. By directing P450 enzymes to plant chloroplasts one can elegantly deal with such redox prerequisites. In this study, we explore the capacity of the plant photosynthetic machinery to drive P450-dependent formation of the indigo precursor indoxyl-β-D-glucoside (indican) by targeting an engineered indican biosynthetic pathway to tobacco ( Nicotiana benthamiana ) chloroplasts. We show that both native and engineered variants belonging to the human CYP2 family are catalytically active in chloroplasts when driven by photosynthetic reducing power and optimize construct designs to improve productivity. However, while increasing supply of tryptophan leads to an increase in indole accumulation, it does not improve indican productivity, suggesting that P450 activity limits overall productivity. Co-expression of different redox partners also does not improve productivity, indicating that supply of reducing power is not a bottleneck. Finally, in vitro kinetic measurements showed that the different redox partners were efficiently reduced by photosystem I but plant ferredoxin provided the highest light-dependent P450 activity. This study demonstrates the inherent ability of photosynthesis to support P450-dependent metabolic pathways. Plants and photosynthetic microbes are therefore uniquely suited for engineering P450-dependent metabolic pathways regardless of enzyme origin. Our findings have implications for metabolic engineering in photosynthetic hosts for production of high-value chemicals or drug metabolites for pharmacological studies.

Ancestral Sequence Reconstruction of a Cytochrome P450 Family Involved in Chemical Defense Reveals the Functional Evolution of a Promiscuous, Xenobiotic-Metabolizing Enzyme in Vertebrates

Publisher: Oxford University Press (OUP)

Date: 26-05-2022

DOI: 10.1093/MOLBEV/MSAC116

Abstract: The cytochrome P450 family 1 enzymes (CYP1s) are a erse family of hemoprotein monooxygenases, which metabolize many xenobiotics including numerous environmental carcinogens. However, their historical function and evolution remain largely unstudied. Here we investigate CYP1 evolution via the reconstruction and characterization of the vertebrate CYP1 ancestors. Younger ancestors and extant forms generally demonstrated higher activity toward typical CYP1 xenobiotic and steroid substrates than older ancestors, suggesting significant ersification away from the original CYP1 function. Caffeine metabolism appears to be a recently evolved trait of the CYP1A subfamily, observed in the mammalian CYP1A lineage, and may parallel the recent evolution of caffeine synthesis in multiple separate plant species. Likewise, the aryl hydrocarbon receptor agonist, 6-formylindolo[3,2-b]carbazole (FICZ) was metabolized to a greater extent by certain younger ancestors and extant forms, suggesting that activity toward FICZ increased in specific CYP1 evolutionary branches, a process that may have occurred in parallel to the exploitation of land where UV-exposure was higher than in aquatic environments. As observed with previous reconstructions of P450 enzymes, thermostability correlated with evolutionary age the oldest ancestor was up to 35 °C more thermostable than the extant forms, with a 10T50 (temperature at which 50% of the hemoprotein remains intact after 10 min) of 71 °C. This robustness may have facilitated evolutionary ersification of the CYP1s by buffering the destabilizing effects of mutations that conferred novel functions, a phenomenon which may also be useful in exploiting the catalytic versatility of these ancestral enzymes for commercial application as biocatalysts.

Use of heterologously-expressed cytochrome P450 and glutathione transferase enzymes in toxicity assays

Publisher: Elsevier BV

Date: 12-2002

DOI: 10.1016/S0300-483X(02)00293-7

Abstract: Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1B1, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity.

Bioactivation of tamoxifen by recombinant human cytochrome P450 enzymes

Publisher: American Chemical Society (ACS)

Date: 17-04-2002

DOI: 10.1021/TX0100439

Abstract: Tamoxifen is a major drug used for adjuvant chemotherapy of breast cancer however, its use has been associated with a small but significant increase in risk of endometrial cancer. In rats, tamoxifen is a hepatocarcinogen, and DNA adducts have been observed in both rat and human tissues. Tamoxifen has been shown previously to be metabolized to reactive products that have the potential to form protein and DNA adducts. Previous studies have suggested a role for P450 3A4 in protein adduct formation in human liver microsomes, via a catechol intermediate however, no clear correlation was seen between P450 3A4 content of human liver microsomes and adduct formation. In the present study, we investigated the P450 forms responsible for covalent drug-protein adduct formation and the possibility that covalent adduct formation might occur via alternative pathways to catechol formation. Recombinant P450 3A4 catalyzed adduct formation, and this correlated with the level of uncoupling in the P450 incubation, consistent with a role of reactive oxygen species in potentiating adduct formation after enzymatic formation of the catechol metabolite. Whereas P450s 1A1, 2D6, and 3A5 generated catechol metabolite, no covalent adduct formation was observed with these forms. By contrast, P450 2B6, 2C19, and rat liver microsomes catalyzed drug-protein adduct formation but not catechol formation. Drug protein adducts formed specifically with P450 3A4 in incubations using membranes isolated from bacteria expressing P450 3A4 and reductase, as well as in reconstitutions of purified 3A4, suggesting that the electrophilic species reacted preferentially with the P450 enzymes concerned.

Direct electrochemistry of human and rat NADPH cytochrome P450 reductase

Publisher: Elsevier BV

Date: 12-2006

DOI: 10.1016/J.ELECOM.2006.08.020

Rabbit CYP4B1 engineered for high-level expression in Escherichia coli: ligand stabilization and processing of the N-terminus and heme prosthetic group

Publisher: Elsevier BV

Date: 08-2003

DOI: 10.1016/S0003-9861(03)00278-9

Abstract: Modifications at the N-terminus of the rabbit CYP4B1 gene resulted in expression levels in Escherichia coli of up to 660 nmol/L. Solubilization of the enzyme from bacterial membranes led to substantial conversion to cytochrome P420 unless alpha-naphthoflavone was added as a stabilizing ligand. Mass spectrometry analysis and Edman sequencing of purified enzyme preparations revealed differential N-terminal post-translational processing of the various constructs expressed. Notably, bacterial expression of CYP4B1 produced a holoenzyme with >98.5% of its heme prosthetic group covalently linked to the protein backbone. The near fully covalently linked hemoproteins exhibited similar rates and regioselectivities of lauric acid hydroxylation to that observed previously for the partially heme processed enzyme expressed in insect cells. These studies shed new light on the consequences of covalent heme processing in CYP4B1 and provide a facile system for future mechanistic and structural studies with the enzyme.

Cytochrome P450 Is Present in Both Ferrous and Ferric Forms in the Resting State within Intact Escherichia coli and Hepatocytes

Publisher: Elsevier BV

Date: 11-2011

DOI: 10.1074/JBC.M111.300871

P450 2C18 catalyzes the metabolic bioactivation of phenytoin

Publisher: American Chemical Society (ACS)

Date: 16-11-2005

DOI: 10.1021/TX050181O

Abstract: The safe clinical use of phenytoin (PHT) is compromised by a drug hypersensitivity reaction, hypothesized to be due to bioactivation of the drug to a protein-reactive metabolite. Previous studies have shown PHT is metabolized to the primary phenol metabolite, HPPH, then converted to a catechol which then autoxidizes to produce reactive quinone. PHT is known to be metabolized to HPPH by cytochromes P450 (P450s) 2C9 and 2C19 and then to the catechol by P450s 2C9, 2C19, 3A4, 3A5, and 3A7. However, the role of many poorly expressed or extrahepatic P450s in the metabolism and/or bioactivation of PHT is not known. The aim of this study was to assess the ability of other human P450s to catalyze PHT metabolism. P450 2C18 catalyzed the primary hydroxylation of PHT with a kcat (2.46 +/- 0.09 min-1) more than an order of magnitude higher than that of P450 2C9 (0.051 +/- 0.004 min-1) and P450 2C19 (0.054 +/- 0.002 min-1) and Km (45 +/- 5 microM) slightly greater than those of P450 2C9 (12 +/- 4 microM) and P450 2C19 (29 +/- 4 microM). P450 2C18 also efficiently catalyzed the secondary hydroxylation of PHT as well as covalent drug-protein adduct formation from both PHT and HPPH in vitro. While P450 2C18 is expressed poorly in the liver, significant expression has been reported in the skin. Thus, P450 2C18 may be important for the extrahepatic tissue-specific bioactivation of PHT in vivo.

A shuffled CYP1A library shows both structural integrity and functional diversity

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 06-09-2007

DOI: 10.1124/DMD.107.017939

Abstract: The cytochrome P450 enzymes (P450s) that mediate mammalian xenobiotic metabolism are highly versatile monooxygenases, which show wide and overlapping substrate ranges but generally poor catalytic rates. Re-engineering of these P450s may enable the development of useful biocatalysts for industrial applications. In the current study, restriction enzyme-mediated DNA family shuffling was used to create a library from human CYP1A1 and CYP1A2. Among sequenced clones (four randomly selected and eight functional clones), 5.9 +/- 2.3 crossovers and 1.5 +/- 1.5 spontaneous mutations (mean +/- S.D.) were detected per mutant. A high level of structural integrity as well as erse functionality were found, with 53% of clones expressed at significant levels (>50 nM P450 hemoprotein) and 23% of clones showing activity on one or more of the following compounds: luciferin 6'-chloroethyl ether (luciferin-CEE), luciferin 6'-methyl ether (luciferin-ME), 6'-deoxyluciferin (luciferin-H), the ethylene glycol ester of luciferin 6'-methyl ether, 7-ethoxyresorufin, and p-nitrophenol (PNP). Different activity profiles were seen with higher specific activity on in idual compounds (e.g., clone 22 9 times the CYP1A1 specific activity toward luciferin-CEE), novel activities (e.g., clone 35 activity toward luciferin-H and PNP), and broadening of substrate range observed in particular clones (e.g., clone 9 activity toward both selective substrates luciferin-ME and luciferin-CEE as well as toward luciferin-H and PNP). In summary, forms were found with distinct and novel activity profiles, despite the relatively small number of mutants examined. In addition, the whole-cell metabolic assays described here provide simple, high-throughput methods useful for screening larger libraries.

Strigolactones and Shoot Branching: What Is the Real Hormone and How Does It Work?

Publisher: Oxford University Press (OUP)

Date: 08-2023

DOI: 10.1093/PCP/PCAD088

Abstract: There have been substantial advances in our understanding of many aspects of strigolactone regulation of branching since the discovery of strigolactones as phytohormones. These include further insights into the network of phytohormones and other signals that regulate branching, as well as deep insights into strigolactone biosynthesis, metabolism, transport, perception and downstream signaling. In this review, we provide an update on recent advances in our understanding of how the strigolactone pathway co-ordinately and dynamically regulates bud outgrowth and pose some important outstanding questions that are yet to be resolved.

An Inexpensive, Efficient Alternative to NADPH to Support Catalysis by Thermostable Cytochrome P450 Enzymes

Publisher: Wiley

Date: 19-02-2020

DOI: 10.1002/CCTC.201902235

Opportunities for Accelerating Drug Discovery and Development by Using Engineered Drug-Metabolizing Enzymes

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 02-12-2023

DOI: 10.1124/DMD.121.000743

‘S-SXR-RMs’? Selective SXR response modulators – the future of designer drug interactions?

Publisher: Elsevier BV

Date: 08-2002

DOI: 10.1016/S0165-6147(02)02092-8

Chemical Defense and Exploitation. Biotransformation of Xenobiotics by Cytochrome P450 Enzymes

Publisher: Wiley

Date: 23-03-2007

DOI: 10.1002/9780470028155.CH15

Assessment of arginine 97 and lysine 72 as determinants of substrate specificity in cytochrome P450 2C9 (CYP2C9).

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 23-03-2004

DOI: 10.1124/DMD.32.4.431

Rational evolution of the cofactor‐binding site of cytochrome P450 reductase yields variants with increased activity towards specific cytochrome P450 enzymes

Publisher: Wiley

Date: 26-07-2019

DOI: 10.1111/FEBS.14982

Abstract: NADPH-cytochrome P450 reductase (CPR) is the natural redox partner of microsomal cytochrome P450 enzymes. CPR shows a stringent preference for NADPH over the less expensive cofactor, NADH, economically limiting its use as a biocatalyst. The complexity of cofactor-linked CPR protein dynamics and the incomplete understanding of the interaction of CPR with both cofactors and electron acceptors present challenges for the successful rational engineering of a CPR with enhanced activity with NADH. Here, we report a rational evolution approach to enhance the activity of CPR with NADH, in which mutations were introduced into the NADPH-binding flavin adenine dinucleotide (FAD) domain. Multiple CPR mutants that used NADH more effectively than the wild-type CPR in the reduction of the surrogate electron acceptor, cytochrome c were found. However, most were inactive in supporting P450 activity, arguing against the use of cytochrome c as a surrogate electron acceptor. Unexpectedly, several mutants showed significantly improved activity towards CYP2C9 (mutant 1-014) and/or CYP2A6 (mutants 1-014, 1-015, 1-053 and 1-077) using NADPH, even though the mutations were introduced at locations remote from the putative CPR-P450 interaction face. Therefore, mutations at sites in the FAD domain of CPR may be promising future engineering targets to enhance P450-mediated substrate turnover. ENZYMES: NADPH-cytochrome P450 reductase - EC 1.6.2.4 cytochrome P450 - EC 1.14.14.1.

Extending the capabilities of nature’s most versatile catalysts: Directed evolution of mammalian xenobiotic-metabolizing P450s

Publisher: Elsevier BV

Date: 08-2007

DOI: 10.1016/J.ABB.2007.04.033

Abstract: Cytochrome P450 enzymes are amongst the most versatile enzymatic catalysts known. The ability to introduce a single atom of oxygen into an organic substrate has led to the ersification and exploitation of these enzymes throughout nature. Nowhere is this versatility more apparent than in the mammalian liver, where P450 monooxygenases catalyze the metabolic clearance of innumerate drugs and other environmental chemicals. In addition to the aromatic and aliphatic hydroxylations, N- and O-dealkylations, and heteroatom oxidations that are common in drug metabolism, many more unusual reactions catalyzed by P450s have been discovered, including reductions, group transfers and other biotransformations not typically associated with monooxygenases. A research area that shows great potential for development over the next few decades is the directed evolution of P450s as biocatalysts. Mammalian xenobiotic-metabolizing P450s are especially well suited to such protein engineering due to their ability to interact with relatively wide ranges of substrates with marked differences in structure and physicochemical properties. Typical characteristics, such as the low turnover rates and poor coupling seen during the metabolism of xenobiotics, as well as the enzyme specificity towards particular substrates and reactions, can be improved by directed evolution. This mini-review will cover the fundamental enabling technologies required to successfully engineer P450s, examine the work done to date on the directed evolution of mammalian forms, and provide a perspective on what will be required for the successful implementation of engineered enzymes.

Deep mutational scanning of CYP2C19 reveals a substrate specificity-abundance tradeoff

Publisher: Cold Spring Harbor Laboratory

Date: 08-10-2023

DOI: 10.1101/2023.10.06.561250

F1000Prime Recommendation

Publisher: No publisher found

Date: 2013

DOI: 10.3410/F.718026768.793481287

Characterization of the human cytochrome P450 forms involved in metabolism of tamoxifen to its α-hydroxy and α,4-dihydroxy derivatives

Publisher: American Chemical Society (ACS)

Date: 30-09-2005

DOI: 10.1021/TX050140S

Abstract: Tamoxifen is a known hepatocarcinogen in rats and is associated with an increased incidence of endometrial cancer in patients. One mechanism for these actions is via bioactivation, where reactive metabolites are generated that are capable of binding to DNA or protein. Several metabolites of tamoxifen have been identified that appear to predispose to adduct formation. These include alpha-hydroxytamoxifen, alpha,4-dihydroxytamoxifen, and alpha-hydroxy-N-desmethyltamoxifen. Previous studies have shown that cytochrome P450 (P450) enzymes play an important role in the biotransformation of tamoxifen. The aim of our work was to determine which P450 enzymes were capable of producing alpha-hydroxylated metabolites from tamoxifen. When tamoxifen (18 or 250 microM) was used as the substrate, P450 3A4, and to a lesser extent, P450 2D6, P450 2B6, P450 3A5, P450 2C9, and P450 2C19 all produced a metabolite with the same HPLC retention time as alpha-hydroxytamoxifen at either substrate concentration tested. This peak was well-separated from 4-hydroxy-N-desmethyltamoxifen, which eluted substantially later under the chromatographic conditions used. No alpha,4-dihydroxytamoxifen was detected in incubations with any of the forms with tamoxifen as substrate. However, when 4-hydroxytamoxifen (100 microM) was used as the substrate, P450 2B6, P450 3A4, P450 3A5, P450 1B1, P450 1A1, and P450 2D6 all produced detectable concentrations of alpha,4-dihydroxytamoxifen. These studies demonstrate that multiple human P450s, including forms found in the endometrium, may generate reactive metabolites in women undergoing tamoxifen therapy, which could subsequently play a role in the development of endometrial cancer.

Bioactivation of Phenytoin by Human Cytochrome P450:  Characterization of the Mechanism and Targets of Covalent Adduct Formation

Publisher: American Chemical Society (ACS)

Date: 09-1997

DOI: 10.1021/TX9700836

Exploiting the Versatility of Human Cytochrome P450 Enzymes: The Promise of Blue Roses From Biotechnology

Publisher: Wiley

Date: 12-2001

DOI: 10.1080/152165401317291110

Abstract: The cytochrome P450 (P450) enzymes involved in drug metabolism are among the most versatile biological catalysts known. A small number of discrete forms of human P450 are capable of catalyzing the monooxygenation of a practically unlimited variety of xenobiotic substrates, with each enzyme showing a more or less wide and overlapping substrate range. This versatility makes P450s ideally suited as starting materials for engineering designer catalysts for industrial applications. In the course of heterologous expression of P450s in bacteria, we observed the unexpected formation of blue pigments. Although this was initially assumed to be an artifact, subsequent work led to the discovery of a new function of P450s in intermediary metabolism and toxicology, new screens for protein engineering, and potential applications in the dye and horticulture industries.

Immunotoxic Side-Effects of Drug Therapy

Publisher: Springer Science and Business Media LLC

Date: 1990

DOI: 10.2165/00002018-199005030-00002

Abstract: Adverse reactions to drugs in which an immune mechanism is responsible for toxicity have been described as idiosyncratic. Understanding these toxic effects is important to enable the identification of patients at risk. The specific toxic side effects considered are heparin-induced thrombocytopenia, penicillin-induced haemolytic anaemia, hepatitis as a result of halothane and tienilic acid therapy, quinine- and quinidine-dependent thrombocytopenia, methyldopa-induced haemolytic anaemia and immune-complex disease following administration of hydralazine, procainamide and penicillamine. The molecular mechanisms of immunotoxicity are presented where such information is available although more than one effect may contribute to the observed pattern of toxicity. The initial events leading to antibody production in certain in iduals in response to drug therapy are not understood and, in many of the ex les described, antibody production occurs in some patients who do not subsequently experience clinical problems. Clinically serious adverse effects involving immune reactions are infrequent, and a range of genetic and environmental circumstances need to be present simultaneously in an in idual before toxicity develops. The ability to metabolise a particular drug has been shown to be one major predisposing factor in toxicity the immunocompetence of the patient is likely to be another. Both of these considerations are subject to genetic and environmental controls, including infection and disease.

Metabolism of Benzo[a]pyrene totrans-7,8-Dihydroxy-7,8-dihydrobenzo[a]pyrene by Recombinant Human Cytochrome P450 1B1 and Purified Liver Epoxide Hydrolase†

Publisher: American Chemical Society (ACS)

Date: 17-06-1999

DOI: 10.1021/TX990028S

Differential inhibition of human liver phenacetin o-deethylation by histamine and four histamine h2-receptor antagonists

Publisher: Informa UK Limited

Date: 1988

DOI: 10.3109/00498258809041674

Abstract: 1. The effects of histamine and four histamine H-2 receptor antagonists on phenacetin O-deethylation by microsomal preparations of four human livers was quantified by a radiometric-thin layer chromatographic method. 2. Histamine and three of these drugs, namely cimetidine, ranitidine and famotidine, were weak inhibitors of this cytochrome P-450-catalysed O-deethylation, but mifentidine was a potent competitive inhibitor with a Ki in the range 40-70 microM. 3. Cimetidine, histamine and mifentidine are all 4(5)-substituted imidazole derivatives, and the contrast between the very weak inhibitory effects of cimetidine and histamine, and the more potent effect of mifentidine, suggests that the imidazole moiety may play little role in the inhibition of phenacetin O-deethylase by mifentidine. 4. The demonstration that cimetidine, ranitidine and histamine were all poor inhibitors of phenacetin oxidation further suggests the possible lack of identity between the human liver cytochrome P-450 isoenzymes responsible for catalyzing the oxidation of metoprolol and phenacetin. This follows from recognizing that metoprolol oxidation is known, from both in vivo and in vitro studies, to be strongly inhibited by both of these H-2 receptor antagonists and from in vitro studies also to be inhibited by histamine.

Requirements for cytochrome b5 in the oxidation of 7-ethoxycoumarin, chlorzoxazone, aniline, and N-nitrosodimethylamine by recombinant cytochrome P450 2E1 and by human liver microsomes

Publisher: Elsevier BV

Date: 07-1996

DOI: 10.1016/0006-2952(96)00208-0

Abstract: NADH-dependent 7-ethoxycoumarin O-deethylation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 reductase, and bacterial recombinant P450 2E1 in 100 mM potassium phosphate buffer (pH 7.4) containing a synthetic phospholipid mixture and cholate. Replacement of NADH-b5 reductase with NADPH-P450 reductase yielded a 4-fold increase in 7-ethoxycoumarin O-deethylation activity, and further stimulation (approximately 1.5-fold) could be obtained when NADPH was used as an electron donor. Removal of b5 from the NADH- and NADPH-supported systems caused a 90% loss of 7-ethoxycoumarin O-deethylation activities in the presence of NADPH-P450 reductase, but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. Km values were increased and Vmax values were decreased for 7-ethoxycoumarin O-deethylation when b5 was omitted from the NADPH-supported P450 2E1-reconstituted systems. Requirements for b5 in P450 2E1 systems were also observed in chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation. In human liver microsomes, NADH-dependent 7-ethoxycoumarin O-deethylation, chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation activities were found to be about 55, 41, 33, and 50%, respectively, of those catalyzed by NADPH-supported systems. Anti-rat NADPH-P450 reductase immunoglobulin G inhibited 7-ethoxycoumarin O-deethylation activity catalyzed by human liver microsomes more strongly in NADPH- than NADH-supported reactions, while anti-human b5 immunoglobulin G inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. These results suggest that b5 is an essential component in P450 2E1-catalyzed oxidations of several substrates used, that about 10% of the activities occur via P450 2E1 reduction by NADPH-P450 reductase in the absence of b5, and that the NADH-supported system contributes, in part, to some reactions catalyzed by P450 2E1 in human liver microsomes.

F1000Prime Recommendation

Publisher: No publisher found

Date: 2013

DOI: 10.3410/F.718038874.793485652

Expression of Modified Human Cytochrome P450 3A4 in Escherichia coli and Purification and Reconstitution of the Enzyme

Publisher: Elsevier BV

Date: 08-1993

DOI: 10.1006/ABBI.1993.1401

Abstract: A full-length human cytochrome P450 (P450) 3A4 cDNA clone and four derivatives in which the N-terminus was modified were inserted into a pCW vector and used to transform Escherichia coli DH5 alpha cells. Little expression was seen with the native sequence the highest level of expression (range of 40-110 membrane-bound nmol P450 liter-1) was achieved with a construct (NF14) in which residues 3-12 were deleted. In all of the constructs P450 was found primarily in the membranes. The modified P450 3A4 (construct NF14) showed typical P450 hemoprotein spectra. The protein was purified to electrophoretic homogeneity in a five-step procedure [nominally 23 nmol P450 (mg protein)-1]. For most purposes it was found to be more practical to purify the modified P450 3A4 to approximately 70% homogeneity [nominally 15 nmol P450 (mg protein)-1] in a simple two-step process. The modified P450 3A4 (NF14) or P450 3A4 purified from human liver could be mixed with rabbit liver NADPH-P450 reductase to achieve catalytic activities nearly as high as those found in human liver microsomes (on a nmol P450 basis), but the optimal reconstitution conditions included not only a mixture of phosphatidylserine, L-alpha-dilauroyl- and L-alpha-dioleoyl-sn-glycero-3-phosphocholines, cholate, and cytochrome b5 suggested by others but also glutathione during the preincubation. Several other thiols were found not to substitute in this role. Good catalytic activity was seen for nifedipine oxidation, testosterone 6 beta-hydroxylation, and the 8,9-epoxidation and 3 alpha-hydroxylation of aflatoxin B1, reactions previously ascribed to the enzyme. These procedures provide a relatively convenient and reliable means of producing, purifying, and reconstituting a catalytically active and useful derivative of P450 3A4, a human P450 enzyme that has many roles in the oxidation of drugs and other xenobiotic chemicals.

STAR: predicting recombination sites from amino acid sequence

Publisher: Springer Science and Business Media LLC

Date: 08-10-2006

DOI: 10.1186/1471-2105-7-437

Cytochrome P450 enzyme-mediated degradation of Echinacea alkylamides in human liver microsomes

Publisher: Elsevier BV

Date: 06-2005

DOI: 10.1016/J.CBI.2005.04.003

Abstract: Echinacea preparations are widely used herbal remedies for the prevention and treatment of colds. In this study we have investigated the metabolism by human liver microsomes of the alkylamide components from an Echinacea preparation as well as that of pure synthetic alkylamides. No significant degradation of alkylamides was evident in cytosolic fractions. Time- and NADPH-dependent degradation of alkylamides was observed in microsomal fractions suggesting they are metabolised by cytochrome P450 (P450) enzymes in human liver. There was a difference in the susceptibility of 2-ene and 2,4-diene pure synthetic alkylamides to microsomal degradation with (2E)-N-isobutylundeca-2-ene-8,10-diynamide (1) metabolised to only a tenth the extent of (2E,4E,8Z,10Z)-N-isobutyldodeca-2,4,8,10-tetraenamide (3) under identical incubation conditions. Markedly less degradation of 3 was evident in the mixture of alkylamides present in an ethanolic Echinacea extract, suggesting that metabolism by liver P450s was dependent both on their chemistry and the combination present in the incubation. Co-incubation of 1 with 3 at equimolar concentrations resulted in a significant decrease in the metabolism of 3 by liver microsomes. This inhibition by 1, which has a terminal alkyne moiety, was found to be time- and concentration-dependent, and due to a mechanism-based inactivation of the P450s. Alkylamide metabolites were detected and found to be the predicted epoxidation, hydroxylation and dealkylation products. These findings suggest that Echinacea may effect the P450-mediated metabolism of other concurrently ingested pharmaceuticals.

Bacterial expression of two human aryl sulfotransferases

Publisher: Elsevier BV

Date: 02-1998

DOI: 10.1016/S0009-2797(97)00128-2

Abstract: The effect of replacing a single codon in the N-terminal of human aryl sulfotransferase (HAST) 1 and 3 with one that is more commonly found in E. coli genes was assessed. The pKK233-2 E. coli expression vector was employed and the polymerase chain reaction (PCR) was used to introduce the 5' nucleotide substitution, at the same time maintaining the fidelity of the amino acid sequence. The data indicates that this change had a minimal effect on protein production, subcellular localization or, in the case of HAST3, catalytic activity. In general, the pKK233-2 E. coli vector has been less than optimal for expressing human sulfotransferase cDNAs.

Cytochrome P450 1B1: A target for inhibition in anticarcinogenesis strategies

Publisher: Elsevier BV

Date: 02-2003

DOI: 10.1016/S0027-5107(02)00333-0

Abstract: Cytochrome P450 (P450) 1B1 is expressed in a number of human tissues in which cancers occur (e.g. prostate, ovary, uterus, mammary gland). P450 1B1 activates many environmental mutagens and also catalyzes the 4-hydroxylation of estrogens, considered to be an important step in hormonal carcinogenesis. We have examined the activities of several of the major allelic variants of human P450 1B1 in these reactions. Another interest has been the development of chemical inhibitors of P450 1B1. 2-Ethynylpyrene and alpha-naphthoflavone preferentially inhibit human P450 1B1 compared to P450 1A1, which may be present in the same tissue sites. The natural product resveratrol is also an inhibitor of P450 1B1. Further studies with rhapontigenin and synthetic stilbenes led to the discovery of 2,4,3',5'-tetramethoxystilbene, a selective inhibitor of P450 1B1 relative to other P450s. Inhibition is competitive, with a K(i) value of 3nM, and the inhibitor is resistant to metabolism. In addition to blocking 17beta-estradiol 4-hydroxylation, this stilbene also inhibited the activation of heterocyclic amines to mutagens. 2,4,3',5'-Tetramethoxystilbene also suppressed expression of P450 1B1 and growth of human mammary tumor cells. 3,3',4',5,5'-Pentamethoxystilbene was a selective inhibitor of P450 1A1, showing mixed inhibition, and also suppressed P450 1A1 expression in HepG2 cells. Substituted stilbenes may be useful in preventing cancer caused by estrogens and xenobiotics.

The PXR ligand-binding domain: how to be picky and promiscuous at the same time

Publisher: Elsevier BV

Date: 09-2001

DOI: 10.1016/S0165-6147(00)01847-2

Physical partitioning as the major source of metoprolol uptake by hepatic microsomes

Publisher: Elsevier BV

Date: 12-1987

DOI: 10.1016/0006-2952(87)90576-4

Facile production of minor metabolites for drug development using a CYP3A shuffled library

Publisher: Elsevier BV

Date: 11-2011

DOI: 10.1016/J.YMBEN.2011.09.001

Abstract: Metabolic profiling of new drugs is limited by the difficulty in obtaining sufficient quantities of minor metabolites for definitive structural identification. Biocatalytic methods offer the potential to produce metabolites that are difficult to synthesize by traditional medicinal chemistry. We hypothesized that the regioselectivity of the drug metabolizing cytochrome P450s could be altered by directed evolution to produce minor metabolites of drugs in development. A biocatalyst library was constructed by DNA shuffling of four CYP3A forms. The library contained 11 ± 4 (mean ± SD) recombinations and 1 ± 1 spontaneous mutations per mutant. On expression in Escherichia coli, 96% of mutants showed detectable activity to at least one probe substrate. Using testosterone as a model drug-like substrate, mutants were found that preferentially formed metabolites produced in only trace amounts by parental forms. A single 1.6L batch culture of one such mutant enabled the facile isolation of 0.3mg of the minor metabolite 1β-hydroxytestosterone and its ab initio structural determination by 1D- and 2D-NMR spectroscopy.

Cytochrome P450-mediated metabolism of haloperidol and reduced haloperidol to pyridinium metabolites

Publisher: American Chemical Society (ACS)

Date: 13-06-2006

DOI: 10.1021/TX0600090

Abstract: Haloperidol (HP) has been reported to undergo cytochrome P450 (P450)-mediated metabolism to potentially neurotoxic pyridinium metabolites however, the chemical pathways and specific enzymes involved in these reactions remain to be identified. The aims of the current study were to (i) fully identify the cytochrome P450 enzymes capable of metabolizing HP to the pyridinium metabolite, 4-(4-chlorophenyl)-1-(4-fluorophenyl)-4-oxobutylpyridinium (HPP(+)), and reduced HP (RHP) to 4-(4-chlorophenyl)-1-(4-fluorophenyl)-4-hydroxybutylpyridinium (RHPP(+)) and (ii) determine whether 4-(4-chlorophenyl)-1-(4-fluorophenyl)-4-oxobutyl-1,2,3,6-tetrahydropyridine (HPTP) and 4-(4-chlorophenyl)-1-(4-fluorophenyl)-4-hydroxybutyl-1,2,3,6-tetrahydropyridine (RHPTP) were metabolic intermediates in these pathways. In vitro studies were conducted using human liver microsomal preparations and recombinant human cytochrome P450 enzymes (P450s 1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19 2D6, 2E1, 3A4, 3A5, and 3A7) expressed in bicistronic format with human NADPH cytochrome P450 reductase in Escherichia coli membranes. Pyridinium formation from HP and RHP was highly correlated across liver preparations, suggesting the same enzyme or enzymes were responsible for both reactions. Cytochrome P450s 3A4, 3A5, and 3A7 were the only recombinant enzymes which demonstrated significant catalytic activity under optimized conditions, although trace levels of activity could be catalyzed by NADPH-P450 reductase alone. NADPH-P450 reductase-mediated activity was inhibited by reduced glutathione but not catalase or superoxide dismutase, suggesting O(2)-dependent oxidation. No evidence was obtained to support the contention that HPTP and RHPTP are intermediates in these pathways. K(m) values for HPP(+) (34 +/- 5 microM) and RHPP(+) (64 +/- 4 microM) formation by recombinant P450 3A4 agreed well with those obtained with human liver microsomes, consistent with P450 3A4 being the major catalyst of pyridinium metabolite formation in human liver.

Volume 317, Number 2 (1995), in the article Expression of Cytochrome P450-3A5 in Escherichia-Coli: Effects of 5′ Modification, Purification, Spectral Characterization, Reconstitution Conditions, and

Publisher: Elsevier BV

Date: 04-1995

DOI: 10.1006/ABBI.1995.1259

Restriction enzyme-mediated DNA family shuffling

Publisher: Springer New York

Date: 2014

DOI: 10.1007/978-1-4939-1053-3_12

Abstract: DNA shuffling is an established recombinatorial method that was originally developed to increase the speed of directed evolution experiments beyond what could be accomplished using error-prone PCR alone. To achieve this, mutated copies of a protein-coding sequence are fragmented with DNase I and the fragments are then reassembled in a PCR without primers. The fragments anneal where there is sufficient sequence identity, resulting in full-length variants of the original gene that have inherited mutations from multiple templates. Subsequent studies demonstrated that directed evolution could be further accelerated by shuffling similar native protein-coding sequences from the same gene family, rather than mutated variants of a single gene. Generally at least 65-75 % global identity between parental sequences is required in DNA family shuffling, with recombination mostly occurring at sites with at least five consecutive nucleotides of local identity. Since DNA shuffling was originally developed, many variations on the method have been published. In particular, the use of restriction enzymes in the fragmentation step allows for greater customization of fragment lengths than DNase I digestion and avoids the risk that parental sequences may be over-digested into unusable very small fragments. Restriction enzyme-mediated fragmentation also reduces the occurrence of undigested parental sequences that would otherwise reduce the number of unique variants in the resulting library. In the current chapter, we provide a brief overview of the alternative methods currently available for DNA shuffling as well as a protocol presented here that improves on several previous implementations of restriction enzyme-mediated DNA family shuffling, in particular with regard to purification of DNA fragments for reassembly.

Evaluation of recombinant cytochromes P450 activity in metabolic pathways

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 2003

DOI: 10.1124/DMD.31.1.145

The GMC superfamily of oxidoreductases revisited: analysis and evolution of fungal GMC oxidoreductases

Publisher: Springer Science and Business Media LLC

Date: 10-05-2019

DOI: 10.1186/S13068-019-1457-0

Engineering highly functional thermostable proteins using ancestral sequence reconstruction

Publisher: Springer Science and Business Media LLC

Date: 22-10-2018

DOI: 10.1038/S41929-018-0159-5

Using the Evolutionary History of Proteins to Engineer Insertion-Deletion Mutants from Robust, Ancestral Templates Using Graphical Representation of Ancestral Sequence Predictions (GRASP)

Publisher: Springer US

Date: 24-11-2022

DOI: 10.1007/978-1-0716-1826-4_6

Abstract: Analyzing the natural evolution of proteins by ancestral sequence reconstruction (ASR) can provide valuable information about the changes in sequence and structure that drive the development of novel protein functions. However, ASR has also been used as a protein engineering tool, as it often generates thermostable proteins which can serve as robust and evolvable templates for enzyme engineering. Importantly, ASR has the potential to provide an insight into the history of insertions and deletions that have occurred in the evolution of a protein family. Indels are strongly associated with functional change during enzyme evolution and represent a largely unexplored source of genetic ersity for designing proteins with novel or improved properties. Current ASR methods differ in the way they handle indels inclusion or exclusion of indels is often managed subjectively, based on assumptions the user makes about the likelihood of each recombination event, yet most currently available ASR tools provide limited, if any, opportunities for evaluating indel placement in a reconstructed sequence. Graphical Representation of Ancestral Sequence Predictions (GRASP) is an ASR tool that maps indel evolution throughout a reconstruction and enables the evaluation of indel variants. This chapter provides a general protocol for performing a reconstruction using GRASP and using the results to create indel variants. The method addresses protein template selection, sequence curation, alignment refinement, tree building, ancestor reconstruction, evaluation of indel variants and approaches to library development.

EXPRESSION OF HUMAN CYTOCHROME-P450 ENZYMES IN YEAST AND BACTERIA AND RELEVANCE TO STUDIES ON CATALYTIC SPECIFICITY

Publisher: Elsevier BV

Date: 10-1993

DOI: 10.1016/0300-483X(93)90057-Y

Abstract: Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 (P450) and other enzymes involved in the biotransformation of drugs and other xenobiotics. The list of studies made possible with the technology includes discernment of catalytic specificity, elucidation of structure-activity relationships, and various biophysical measurements. There are advantages and disadvantages to each of the vector systems and choices must be made on the basis of needs. Yeast expression systems were used to establish that different P450 2C enzymes are involved in the hydroxylations of tolbutamide and (S)-mephenytion. P450 3A4 was also expressed in yeast and its very broad catalytic specificity was confirmed. Recently, it has been possible to express P450 3A4 as well as other human and animal P450s in bacteria after slight modification of their 5'-coding sequences.

Modified nicotine metabolism in transgenic tobacco plants expressing the human cytochrome P450 2A6 cDNA

Publisher: Wiley

Date: 07-04-2005

DOI: 10.1016/J.FEBSLET.2005.02.082

Abstract: In this study, the human cytochrome P450 (CYP) 2A6 was used in order to modify the alkaloid production of tobacco plants. The cDNA for human CYP2A6 was placed under the control of the constitutive 35S promoter and transferred into Nicotiana tabacum via Agrobacterium-mediated transformation. Transgenic plants showed formation of the recombinant CYP2A6 enzyme but no obvious phenotypic changes. Unlike wild-type tobacco, the transgenic plants accumulated cotinine, a metabolite which is usually formed from nicotine in humans. This result substantiates that metabolic engineering of the plant secondary metabolism via mammalian P450 enzymes is possible in vivo.

Catalytic properties of polymorphic human cytochrome P4501B1 variants

Publisher: Oxford University Press (OUP)

Date: 08-1999

DOI: 10.1093/CARCIN/20.8.1607

Abstract: Four polymorphic human cytochrome P450 (CYP) 1B1 allelic variants, namely Arg48,Ala119,Leu432,Asn453, Arg48,Ser119,Leu432,Asn453, Arg48, Ala119,Val432,Asn-453 and Arg48,Ser119,Val432,Asn453, were expressed in Escherichia coli together with human NADPH-P450 reductase and the recombinant proteins (in bacterial membranes) were used to assess whether CYP1B1 polymorphisms affect catalytic activities towards a variety of P450 substrates, including erse procarcinogens and steroid hormones. Activities for activation of 19 procarcinogens to DNA-damaging products by these four CYP1B1 variants in a Salmonella typhimurium NM2009 umu response system were found to be essentially similar, except that a Arg48, Ser119,Leu432,Asn453 variant was slightly more active (1.2- to 1.5-fold) than the other three CYP1B1 enzymes in catalyzing activation of (+)- and (-)-benzo[a]pyrene-7, 8-diols, 7,12-dimethylbenz[a]anthracene-3,4-diol, benzo[g]chrysene-11,12-diol, benzo[b]fluoranthene-9,10-diol, 2-amino-3,5-dimethylimidazo[4,5-f]quinoline, 2-amino-3-methylimidazo[4,5-f]quinoline and 2-aminofluorene. Kinetic analysis of 17beta-estradiol hydroxylation showed that V(max) values for 4-hydroxylation ranged between 0.9 and 1.5 nmol/min/nmol P450 for 4-hydroxylation and 0.3 and 0.6 nmol/min/nmol P450 for 2-hydroxylation in these CYP1B1 variants, with K(m) values ranging from 1 to 9 microM. Interestingly, the ratio of product formation of 4-hydroxyestradiol to 2-hydroxyestradiol was higher for the Val432 variants of CYP1B1 variants than the Leu432 variants of the enzyme. The same trend was noted in the ratio of estrone 4-hydroxylation to estrone 2-hydroxylation catalyzed by CYP1B1 variants. Mutation in the CYP1B1 genes also affected the K(m) and V(max) values in the 6beta-hydroxylation of testosterone and 6beta- and 16alpha-hydroxylation of progesterone. These results indicate that the polymorphisms in the human CYP1B1 gene cause some alterations in catalytic function towards procarcinogens and steroid hormones and thus may make some contribution to susceptibilities of in iduals towards mammary and lung cancers in humans.

Membrane integration of recombinant human P450 forms

Publisher: Informa UK Limited

Date: 17-06-2009

DOI: 10.1080/00498250902934884

Abstract: Amino terminal sequence modification of cytochrome P450 enzymes is often necessary to achieve expression in bacteria. The aim of this study was to examine the effect of such modifications on membrane integration and P450 activity. Forms that retained substantial N-terminal hydrophobic sequences remained unaffected by treatments to remove peripheral membrane proteins and were released only by detergent. Truncated P450s 2A13, 2C9 (delta 3-20), 2C19 (delta 3-20), 2D6 (DB11) and 2E1 remained principally membrane-bound, but some P450 was found in the soluble fraction and could be partially extracted by alkaline and high salt treatments. The subcellular localization of P450s 2C9 and 2C19 assessed by fluorescence microscopy mirrored the distribution between subcellular fractions. The MALLLAVFL modified forms of P450 2C9 YFP, P450 2C18 YFP and P450 2C19 YFP were found primarily at the periphery of the cells, whereas the truncated forms of P450 2C9 (delta 3-20) YFP and 2C19 (delta 3-20) YFP were observed at the periphery as well as inside the cells. N-terminal variants of P450s 2C9 and 2C19 showed altered kinetics towards form-selective substrates. Rates of diclofenac 4 -hydroxylation by P450 2C9 and luciferin H-EGE metabolism by P450 2C19 were higher for the MALLLAVFL-modified forms compared with the (delta 3-20) truncated forms despite supplementation of truncated form incubations with additional reductase. Thus, N-terminal sequence modifications changed the degree of membrane integration, potentially affecting subcellular localization, interactions with redox partners, and hence enzymatic activity.

The evolution of cytochrome P450 enzymes as biocatalysts in drug discovery and development.

Publisher: Bentham Science Publishers Ltd.

Date: 10-2013

DOI: 10.2174/15680266113136660158

Abstract: Engineered biocatalysts offer the opportunity to introduce modifications into complex lead molecules and drug candidates in a chemo-, regio- and stereoselective manner that is difficult to accomplish using traditional synthetic organic chemistry. As candidate biocatalysts, the cytochrome P450 enzymes that metabolize drugs and other xenobiotics are amongst the most versatile agents known. Not only can they mediate an exceptional range of biotransformation reactions, but they act on an unparalleled ersity of substrates. However, this versatility comes at the cost of relatively poor catalytic efficiency and low rates of coupling of cofactor consumption to product formation. Directed evolution is being used to redefine the substrate specificity of P450 enzymes while at the same time improving their efficiency, thermostability and other properties. This review will outline the key successes with bacterial P450s used as biocatalysts, examine the studies done to date with mammalian forms, and assess the prospects for exploiting xenobiotic-metabolizing P450s for applications in medicinal chemistry.

Prospects for applying synthetic biology to toxicology: Future opportunities and current limitations for the repurposing of cytochrome P450 systems

Publisher: American Chemical Society (ACS)

Date: 13-12-2016

DOI: 10.1021/ACS.CHEMRESTOX.6B00396

Abstract: The 30 years since the inception of Chemical Research in Toxicology, game-changing advances in chemical and molecular biology, the fundamental disciplines underpinning molecular toxicology, have been made. While these have led to important advances in the study of mechanisms by which chemicals damage cells and systems, there has been less focus on applying these advances to prediction, detection, and mitigation of toxicity. Over the last ∼15 years, synthetic biology, the repurposing of biological "parts" in systems engineered for useful ends, has been explored in other areas of the biomedical and life sciences, for such applications as detecting metabolites, drug discovery and delivery, investigating disease mechanisms, improving medical treatment, and producing useful chemicals. These ex les provide models for the application of synthetic biology to toxicology, which, for the most part, has not yet benefited from such approaches. In this perspective, we review the synthetic biology approaches that have been applied to date and speculate on possible short to medium term and "blue sky" aspirations for synthetic biology, particularly in clinical and environmental toxicology. Finally, we point out key hurdles that must be overcome for the full potential of synthetic biology to be realized.

Association of CYP1B1 genetic polymorphism with incidence to breast and lung cancer

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 02-2000

DOI: 10.1097/00008571-200002000-00004

Abstract: Cytochrome P450 1B1 (CYP1B1) participates in the metabolic activation of a number of procarcinogens including benzo[a]pyrene and the hydroxylation of 17beta-estradiol at the C-4 position. In this study, we investigated the association between CYP1B1 genetic polymorphism and breast or lung cancer incidence. The Ala-Ser polymorphism at codon 119 in presumed substrate recognition site 1 was significantly associated with the incidence of breast or squamous cell carcinoma of the lung. On the other hand, Leu-Val polymorphism at codon 432 did not show any association to the cancers. An allele containing both Ala and Leu simultaneously, comprised 75% of alleles among 315 Japanese healthy controls, was significantly inversely associated with breast cancer incidence. When expressed in a recombinant system, this CYP1B1 cDNA showed the lowest 17beta-estradiol 4-hydroxylase activity among four different variant forms of CYP1B1. Thus, inter-in idual differences in activation of procarcinogens or metabolism of oestrogen originating from genetic polymorphisms of the human CYP1B1 gene may contribute to the susceptibility of human cancers.

Formation of Indigo by Recombinant Mammalian Cytochrome P450

Publisher: Elsevier BV

Date: 11-1999

DOI: 10.1006/BBRC.1999.1702

Transgenic xenosensors: humanizing mice.

Publisher: Elsevier BV

Date: 09-2000

DOI: 10.1016/S0165-6147(00)01532-7

Computational tools for directed evolution: A comparison of prospective and retrospective strategies

Publisher: Springer New York

Date: 2014

DOI: 10.1007/978-1-4939-1053-3_21

Abstract: Directed evolution methods have proved to be highly effective in the design of novel proteins and in the generation of large libraries of erse sequences. However, searching through the vast number of mutants produced during such experiments in order to find the best represents a daunting and difficult task. In recent years, a number of computational tools have been developed to provide guidance during this exploratory process. It can, however, be unclear as to which tool or tools best complement the chosen library design strategy. In this review, we describe and critically evaluate some of the more notable tools in this area, discussing the rationale behind each, the requirements for their implementation, and potential issues faced when using them. Some ex les of their application in an experimental setting are also provided. The tools have been classified based on contrasting strategies as to how they function: prospective tools SCHEMA and OPTCOMB use extant sequence and structural data to predict optimal locations for crossover sites, whereas retrospective tools ProSAR and ASRA use property data from the mutant library to predict beneficial mutations and features. From our evaluation, we suggest that each tool can play a role in the design process however this is largely dictated by the data available and the desired experimental strategy for the project.

Phenacetin o-deethylation by human liver microsomes: Kinetics and propranolol inhibition

Publisher: Informa UK Limited

Date: 1988

DOI: 10.3109/00498258809055140

Abstract: 1. Phenacetin O-deethylation catalysed by human liver microcomes has been examined over a substrate concentration range of 2.5 to 700 microM using preparations of eight human liver s les. Michaelis-Menten kinetics described phenacetin oxidation satisfactorily in five of these s les apparent Km values were in the range of 17.7 to 38.4 microM. 2. In the three other livers a single rectangular hyperbolic relationship did not describe the substrate saturation data adequately analyses in these three cases requiring two classes of catalytic site. The apparent Km value for the higher affinity class of site in these three s les was within the range quoted above, but limitations imposed by assay sensitivity and phenacetin solubility obviated accurate characterization of the lower affinity class. 3. Estimates of Vmax for the high affinity class of site in the eight livers varied eleven-fold and there was no correlation between either Km or Vmax and microsomal cytochrome P-450 specific content, NADPH cytochrome c (P-450) reductase specific activity or ethylmorphine demethylase activity. 4. Propranolol was a potent competitive inhibitor of phenacetin deethylation with apparent Ki values of 2 to 7 microM describing its effect on the higher affinity class of activity. Propranolol was also an inhibitor of the lower affinity phenacetin deethylase identified in three of the livers, however the mechanism of inhibition could not be characterized. 5. These data suggest the possibility that propranolol oxidation may be mediated in part, by one or more human liver cytochrome P-450 species catalyzing phenacetin oxidation.

Quantitative whole-cell cytochrome P450 measurement suitable for high-throughput application

Publisher: Elsevier BV

Date: 02-2008

DOI: 10.1177/1087057107312780

Abstract: The recombinant expression of cytochrome P450 enzymes involved in drug metabolism is of interest to the pharmaceutical and biotechnological industries due to the versatile catalytic properties of these enzymes. Accurate quantification of cytochrome P450 enzymes expressed in bacterial culture generally depends on disruption and fractionation of cells to prepare membranes for spectral analysis. Although whole-cell methods for spectral determination have been reported, problems with poor reproducibility and low signal-to-noise ratio confound the use of such techniques where P450 hemoprotein expression levels are relatively low, such as in cultures of certain mammalian forms. In particular, interference from bacterial hemoproteins often obscures the P450 peak. In the current study, the combination of culture concentration, incubation under microaerobic conditions, and a modified method of baseline correction enabled reproducible quantification of cytochrome P450s in whole cells. This whole-cell method is well suited to high-throughput application, as large sets or libraries of enzymes can be expressed in parallel and relative expression levels measured without downstream cell processing.

κ-Carrageenan Gel Modified Mesoporous Gold Chronocoulometric Sensor for Ultrasensitive Detection of MicroRNA

Publisher: The Chemical Society of Japan

Date: 15-01-2022

DOI: 10.1246/BCSJ.20210286

Effect of tamoxifen on the enzymatic activity of human cytochrome CYP2B6

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 06-2002

DOI: 10.1124/JPET.301.3.945

Abstract: Tamoxifen is primarily used in the treatment of breast cancer. It has been approved as a chemopreventive agent for in iduals at high risk for this disease. Tamoxifen is metabolized to a number of different products by cytochrome P450 enzymes. The effect of tamoxifen on the enzymatic activity of bacterially expressed human cytochrome CYP2B6 in a reconstituted system has been investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified CYP2B6 was inactivated by tamoxifen in a time- and concentration-dependent manner. Enzymatic activity was lost only in s les that were incubated with both tamoxifen and NADPH. The inactivation was characterized by a K(I) of 0.9 microM, a k(inact) of 0.02 min(-1), and a t(1/2) of 34 min. The loss in the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity did not result in a similar percentage loss in the reduced carbon monoxide spectrum, suggesting that the heme moiety was not the major site of modification. The activity of CYP2B6 was not recovered after removal of free tamoxifen using spin column gel filtration. The loss in activity seemed to be due to a modification of the CYP2B6 and not reductase because adding fresh reductase back to the inactivated s les did not restore enzymatic activity. A reconstituted system containing purified CYP2B6, NADPH-reductase, and NADPH-generating system was found to catalyze tamoxifen metabolism to 4-OH-tamoxifen, 4'-OH-tamoxifen, and N-desmethyl-tamoxifen as analyzed by high-performance liquid chromatography analysis. Preliminary studies showed that tamoxifen had no effect on the activities of CYP1B1 and CYP3A4, whereas CYP2D6 and CYP2C9 exhibited a 25% loss in enzymatic activity.

Expression of modified human cytochrome P450 1A1 in Escherichia coli: Effects of 5' substitution, stabilization, purification, spectral characterization, and catalytic properties

Publisher: Elsevier BV

Date: 08-1994

DOI: 10.1006/ABBI.1994.1330

Abstract: Human cytochrome P450 (P450) 1A1 is primarily an extrahepatic enzyme and is important because of its roles in the activation of polycyclic hydrocarbons and other xenobiotic chemicals. Purification of active enzyme from human tissues has not been successful. We report the expression and purification of the recombinant enzyme from Escherichia coli. A full-length cDNA of human cytochrome P450 1A1 and several modified constructs were engineered into a pCW vector and used to transform E. coli cells. Little expression was observed with the native sequence and several modified constructs, but successful expression (20-25 nmol membrane-bound P450 1A1 per liter of culture) was achieved with a construct in which the Ala codon GCT was placed in the second position and the 5'-terminal codons were maximized for AT content and minimized for the potential of secondary structure formation of the mRNA transcript. alpha-Naphthoflavone was found to protect against denaturation by detergents during solubilization and was added to buffers used for purification. The recombinant P450 1A1 was purified to electrophoretic homogeneity after two ion-exchange chromatography steps in approximately 50% yield. N-Terminal amino acid sequence analysis verified the expected first 21 residues, with the exception of the terminal Met. The isolated human ferric P450 1A1 was predominantly in the high spin state, in contrast to the orthologous rat and rabbit enzymes. Recombinant P450 1A1 catalyzed 7-ethoxyresorufin O-deethylation and benzo[a]pyrene 3-hydroxylation with Km values of 0.58 and 15 microM and Vmax values of 8.3 and 2.5 nmol min-1 (nmol P450 1A1)-1, respectively. The successful expression and purification of human P450 1A1 should increase the availability of this enzyme and the generation of antibodies for further biochemical and other biological studies.

Reconstitution of Recombinant Cytochrome P450 2C10(2C9) and Comparison with Cytochrome P450 3A4 and Other Forms: Effects of Cytochrome P450–P450 and Cytochrome P450–b5Interactions

Publisher: Elsevier BV

Date: 06-1997

DOI: 10.1006/ABBI.1997.0125

Abstract: Tolbutamide methyl hydroxylation and S-warfarin 7-hydroxylation activities were reconstituted in systems containing recombinant human cytochrome P450 (P450 or CYP) 2C10(2C9) and the optimal conditions for the systems were compared with those of bufuralol 1'-hydroxylation by CYP1A1, theophylline 8-hydroxylation by CYP1A2, bufuralol 1'-hydroxylation by CYP2D6, chlorzoxazone 6-hydroxylation by CYP2E1, and testosterone 6 beta-hydroxylation by CYP3A4. CYP2C10 required cytochrome b5 (b5) for optimal rates of tolbutamide and S-warfarin oxidations and b5 could be replaced by apo-b5 apo-b5 and b5 effects on the reconstituted systems have already been reported in systems containing CYP3A4 for the oxidation of testosterone and nifedipine and for the rapid reduction of CYP3A4 by NADPH-P450 reductase (H. Yamazaki et al., 1996, J. Biol. Chem. 271, 27438-27444). Stopped-flow studies, however, suggested that apo-b5 as well as b5 did not cause stimulation of the reduction of CYP2C10 by NADPH-P450 reductase, while the reduction rates were dependent on the substrates in reconstituted systems. Chlorzoxazone 6-hydroxylation by CYP2E1 was stimulated by b5, but not by apo-b5, in reconstituted systems. Neither apo- nor holo-b5 increased bufuralol 1'-hydroxylation activity by CYP1A1 or 2D6 or theophylline 8-hydroxylation by CYP1A2. Interestingly, we found that testosterone 6 beta-hydroxylation by CYP3A4 was stimulated by CYP1A2 (and also by a modified form in which the first 36 residues of the native human protein were removed) and CYP1A1 as well as by b5, and such stimulations were not seen when other P450 proteins (e.g., CYP2C10, 2D6, or 2E1) were added to the reconstituted systems. In contrast, substrate oxidations by CYP2C10 and CYP2E1 were not stimulated by other P450 proteins. The present results suggest that there are differences in optimal conditions for reconstitution of substrate oxidations by various forms of human P450 enzymes, and in some P450-catalyzed reactions protein-protein interactions between P450 and b5 and other P450 proteins are very important in some oxidations catalyzed by CYP2C10, 2E1, and 3A4.

Oxidation of indole by cytochrome P450 enzymes

Publisher: American Chemical Society (ACS)

Date: 13-10-2000

DOI: 10.1021/BI001229U

Abstract: Indole is a product of tryptophan catabolism by gut bacteria and is absorbed into the body in substantial amounts. The compound is known to be oxidized to indoxyl and excreted in urine as indoxyl (3-hydroxyindole) sulfate. Further oxidation and dimerization of indoxyl leads to the formation of indigoid pigments. We report the definitive identification of the pigments indigo and indirubin as products of human cytochrome P450 (P450)-catalyzed metabolism of indole by visible, (1)H NMR, and mass spectrometry. P450 2A6 was most active in the formation of these two pigments, followed by P450s 2C19 and 2E1. Additional products of indole metabolism were characterized by HPLC/UV and mass spectrometry. Indoxyl (3-hydroxyindole) was observed as a transient product of P450 2A6-mediated metabolism isatin, 6-hydroxyindole, and dioxindole accumulated at low levels. Oxindole was the predominant product formed by P450s 2A6, 2E1, and 2C19 and was not transformed further. A stable end product was assigned the structure 6H-oxazolo[3,2-a:4, 5-b']diindole by UV, (1)H NMR, and mass spectrometry, and we conclude that P450s can catalyze the oxidative coupling of indoles to form this dimeric conjugate. On the basis of these results, we propose that the P450/NADPH-P450 reductase system can catalyze oxidation of indole to a variety of products.

Directed evolution of cytochrome P450 enzymes for biocatalysis: exploiting the catalytic versatility of enzymes with relaxed substrate specificity.

Publisher: Portland Press Ltd.

Date: 20-03-2015

DOI: 10.1042/BJ20141493

Abstract: Cytochrome P450 enzymes are renowned for their ability to insert oxygen into an enormous variety of compounds with a high degree of chemo- and regio-selectivity under mild conditions. This property has been exploited in Nature for an enormous variety of physiological functions, and representatives of this ancient enzyme family have been identified in all kingdoms of life. The catalytic versatility of P450s makes them well suited for repurposing for the synthesis of fine chemicals such as drugs. Although these enzymes have not evolved in Nature to perform the reactions required for modern chemical industries, many P450s show relaxed substrate specificity and exhibit some degree of activity towards non-natural substrates of relevance to applications such as drug development. Directed evolution and other protein engineering methods can be used to improve upon this low level of activity and convert these promiscuous generalist enzymes into specialists capable of mediating reactions of interest with exquisite regio- and stereo-selectivity. Although there are some notable successes in exploiting P450s from natural sources in metabolic engineering, and P450s have been proven repeatedly to be excellent material for engineering, there are few ex les to date of practical application of engineered P450s. The purpose of the present review is to illustrate the progress that has been made in altering properties of P450s such as substrate range, cofactor preference and stability, and outline some of the remaining challenges that must be overcome for industrial application of these powerful biocatalysts.

Exploring the past and the future of protein evolution with ancestral sequence reconstruction: the ' retro' approach to protein engineering

Publisher: Portland Press Ltd.

Date: 22-12-2017

DOI: 10.1042/BCJ20160507

Abstract: A central goal in molecular evolution is to understand the ways in which genes and proteins evolve in response to changing environments. In the absence of intact DNA from fossils, ancestral sequence reconstruction (ASR) can be used to infer the evolutionary precursors of extant proteins. To date, ancestral proteins belonging to eubacteria, archaea, yeast and vertebrates have been inferred that have been hypothesized to date from between several million to over 3 billion years ago. ASR has yielded insights into the early history of life on Earth and the evolution of proteins and macromolecular complexes. Recently, however, ASR has developed from a tool for testing hypotheses about protein evolution to a useful means for designing novel proteins. The strength of this approach lies in the ability to infer ancestral sequences encoding proteins that have desirable properties compared with contemporary forms, particularly thermostability and broad substrate range, making them good starting points for laboratory evolution. Developments in technologies for DNA sequencing and synthesis and computational phylogenetic analysis have led to an escalation in the number of ancient proteins resurrected in the last decade and greatly facilitated the use of ASR in the burgeoning field of synthetic biology. However, the primary challenge of ASR remains in accurately inferring ancestral states, despite the uncertainty arising from evolutionary models, incomplete sequences and limited phylogenetic trees. This review will focus, firstly, on the use of ASR to uncover links between sequence and phenotype and, secondly, on the practical application of ASR in protein engineering.

In silico characterization of protein chimeras: Relating sequence and function within the same fold

Publisher: Wiley

Date: 04-05-2009

DOI: 10.1002/PROT.22422

Abstract: The exploration of novel proteins via recombination of fragments derived from structurally homologous proteins has enormous potential for medicine and biotechnology. This modular exchange of sequence material puts novel activities, substrate specificities, and stability within reach of a semi-random search. This article takes stock of the growing resource of experimentally characterized chimeric proteins within a homologous protein family to build sequence-function models that can effectively guide the construction of new libraries. A novel framework for predicting structural viability of chimeric proteins, only assuming knowledge of their sequence and their parental structure, is presented. Removing a major barrier in previous work, the model processes any sequence that derives from parents with similar folds. The method naturally mixes test and training data from site-directed recombination, DNA shuffling, or random mutagenesis experiments. We train a model from a site-directed recombination library with state-of-the-art prediction accuracy on hold-out test data from the same experimental source and convincing performance on chimeras with a different origin. Specifically, the model is used to assess the structural viability of P450 chimeras deriving from proteins with only 18% sequence similarity to those used for model tuning.

Identification, Synthesis, and Biological Evaluation of the Major Human Metabolite of NLRP3 Inflammasonne Inhibitor MCC950

Publisher: American Chemical Society (ACS)

Date: 30-09-2016

DOI: 10.1021/ACSMEDCHEMLETT.6B00198

Engineering cytochrome p450 enzymes.

Publisher: American Chemical Society (ACS)

Date: 08-12-2008

DOI: 10.1021/TX7002849

Abstract: The last 20 years have seen the widespread and routine application of methods in molecular biology such as molecular cloning, recombinant protein expression, and the polymerase chain reaction. This has had implications not only for the study of toxicological mechanisms but also for the exploitation of enzymes involved in xenobiotic clearance. The engineering of P450s has been performed with several purposes. The first and most fundamental has been to enable successful recombinant expression in host systems such as bacteria. This in turn has led to efforts to solubilize the proteins as a prerequisite to crystallization and structure determination. Lagging behind has been the engineering of enzyme activity, h ered in part by our still-meager comprehension of fundamental structure-function relationships in P450s. However, the emerging technique of directed evolution holds promise in delivering both engineered enzymes for use in biocatalysis and incidental improvements in our understanding of sequence-structure and sequence-function relationships, provided that data mining can extract the fundamental correlations underpinning the data. From the very first studies on recombinant P450s, efforts were directed toward constructing fusions between P450s and redox partners in the hope of generating more efficient enzymes. While this aim has been allowed to lie fallow for some time, this area merits further investigation as does the development of surface-displayed P450 systems for biocatalytic and biosensor applications. The final application of engineered P450s will require other aspects of their biology to be addressed, such as tolerance to heat, solvents, and high substrate and product concentrations. The most important application of these enzymes in toxicology in the near future is likely to be the biocatalytic generation of drug metabolites for the pharmaceutical industry. Further tailoring will be necessary for specific toxicological applications, such as in bioremediation.

Structure of an ancestral mammalian family 1B1 cytochrome P450 with increased thermostability

Publisher: Elsevier BV

Date: 04-2020

DOI: 10.1074/JBC.RA119.010727

A shuffled CYP2C library with a high degree of structural integrity and functional versatility

Publisher: Elsevier BV

Date: 11-2007

DOI: 10.1016/J.ABB.2007.08.023

Abstract: Cytochrome P450 (CYP) enzymes involved in mammalian xenobiotic metabolism are attractive targets for the engineering of biocatalysts since they have broad and overlapping substrate and reaction substrate specificities. In this report, a library of chimeric mutants was prepared from CYP2C8, CYP2C9, CYP2C18 and CYP2C19 by DNA family shuffling. Twelve randomly selected clones were fully sequenced and showed 9+/-2 crossovers and 1.5+/-0.5 spontaneous mutations per approximately 1.5kbp open reading frame. CYP hemoprotein expression was observed in 50% (microaerobic culture) to 54% (aerobic culture) of clones. The functional ersity of the library was assessed using three luminogenic substrates, diclofenac and indole as probe substrates. A random s le of 26 clones revealed two clones with activity towards luciferin ME, one towards luciferin H and five towards diclofenac 4'-hydroxylation. One mutant showed activity towards all three substrates. Of 96 clones screened on solid media, one showed elevated indigo production compared to the parental forms. Turnover rates for luciferin ME and H metabolism by CYP2C9 and mutants were at least one order of magnitude higher in experiments with membranes compared to whole cells, consistent with impaired product egress from cells. Apparent K(m) values were increased in whole cell incubations with luciferin H suggesting impaired access of the substrate to the active site of the enzymes in whole cells. Finally screening with a panel of CYP2C ligands using CYP2C9 or active mutants revealed different patterns of inhibition and heteroactivation of metabolism of luciferin analogs.

Formation of the indigo precursor indican in genetically engineered tobacco plants and cell cultures

Publisher: Wiley

Date: 22-11-2007

DOI: 10.1111/J.1467-7652.2006.00231.X

Abstract: The production of the blue dye indigo in plants has been assumed to be a possible route to the introduction of novel coloration into flowers or fibres. As the human cytochrome P450 mono-oxygenase 2A6 (CYP2A6) can form indigo in bacterial cultures, we investigated whether the expression of the corresponding cDNA in transgenic plants could lead to indigo formation. In a first attempt, we generated tobacco cell suspension cultures expressing the cDNA encoding human CYP2A6. Supplementation of the medium with indole led to the generation of indican (3-hydroxyindole-beta-d-glucoside), a metabolite usually exclusively present in indigoferous dye plants. Hence, the recombinant CYP2A6 converted indole to the reactive metabolite 3-hydroxyindole (indoxyl), whereas rapid glucosylation is obviously conducted by ubiquitous plant glucosyl transferases (GTs). Interestingly, of nine additionally tested plant cell suspension cultures from various plant families, five were also capable of the formation of indican after indole supplementation, although this metabolism was more pronounced in transgenic tobacco cell suspension cultures expressing CYP2A6 cDNA. To evaluate whether indican or even indigo could be produced in whole plants, we generated transgenic tobacco plants harbouring active CYP2A6 together with an indole synthase (BX1) from maize. The genetically engineered tobacco plants accumulated indican, but did not develop a blue coloration. Although the de novo formation of indican in transgenic tobacco plants h ered indigo formation, it supports the contention that biosynthetic pathways can be efficiently mimicked by metabolic engineering.

Functional characterisation of an engineered multidomain human P450 2E1 by molecular Lego

Publisher: Springer Science and Business Media LLC

Date: 09-11-2005

DOI: 10.1007/S00775-005-0033-1

Abstract: The human cytochrome P450s constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. We present here results of a fusion between a human P450 enzyme and a bacterial reductase that for the first time is shown does not require the addition of lipids or detergents to achieve wild-type-like activities. The fusion enzyme, P450 2E1-BMR, contains the N-terminally modified residues 22-493 of the human P450 2E1 fused at the C-terminus to residues 473-1049 of the P450 BM3 reductase (BMR). The P450 2E1-BMR enzyme is active, self-sufficient and presents the typical marker activities of the native human P450 2E1: the hydroxylation of p-nitrophenol (KM=1.84+/-0.09 mM and kcat of 2.98+/-0.04 nmol of p-nitrocatechol formed per minute per nanomole of P450) and chlorzoxazone (KM=0.65+/-0.08 mM and kcat of 0.95+/-0.10 nmol of 6-hydroxychlorzoxazone formed per minute per nanomole of P450). A 3D model of human P450 2E1 was generated to rationalise the functional data and to allow an analysis of the surface potentials. The distribution of charges on the model of P450 2E1 compared with that of the FMN domain of BMR provides the ground for the understanding of the interaction between the fused domains. The results point the way to successfully engineer a variety of catalytically self-sufficient human P450 enzymes for drug metabolism studies in solution.

Engineering indel and substitution variants of diverse and ancient enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP)

Publisher: Public Library of Science (PLoS)

Date: 24-10-2022

DOI: 10.1371/JOURNAL.PCBI.1010633

Abstract: Ancestral sequence reconstruction is a technique that is gaining widespread use in molecular evolution studies and protein engineering. Accurate reconstruction requires the ability to handle appropriately large numbers of sequences, as well as insertion and deletion (indel) events, but available approaches exhibit limitations. To address these limitations, we developed Graphical Representation of Ancestral Sequence Predictions (GRASP), which efficiently implements maximum likelihood methods to enable the inference of ancestors of families with more than 10,000 members. GRASP implements partial order graphs (POGs) to represent and infer insertion and deletion events across ancestors, enabling the identification of building blocks for protein engineering. To validate the capacity to engineer novel proteins from realistic data, we predicted ancestor sequences across three distinct enzyme families: glucose-methanol-choline (GMC) oxidoreductases, cytochromes P450, and dihydroxy/sugar acid dehydratases (DHAD). All tested ancestors demonstrated enzymatic activity. Our study demonstrates the ability of GRASP (1) to support large data sets over 10,000 sequences and (2) to employ insertions and deletions to identify building blocks for engineering biologically active ancestors, by exploring variation over evolutionary time.

The concept lens diagram: A new mechanism for presenting biochemistry content in terms of “big ideas”

Publisher: Wiley

Date: 07-2011

DOI: 10.1002/BMB.20517

Abstract: A strong, recent movement in tertiary education is the development of conceptual, or "big idea" teaching. The emphasis in course design is now on promoting key understandings, core competencies, and an understanding of connections between different fields. In biochemistry teaching, this radical shift from the content-based tradition is being driven by the "omics" information explosion we can no longer teach all the information we have available. Biochemistry is a core, enabling discipline for much of modern scientific research, and biochemistry teaching is in urgent need of a method for delivery of conceptual frameworks. In this project, we aimed to define the key concepts in biochemistry. We find that the key concepts we defined map well onto the core science concepts recommended by the Vision and Change project. We developed a new method to present biochemistry through the lenses of these concepts. This new method challenged the way we thought about biochemistry as teachers. It also stimulated the majority of the students to think more deeply about biochemistry and to make links between biochemistry and material in other courses. This method is applicable to the full spectrum of content usually taught in biochemistry.

Engineering indel and substitution variants of diverse and ancient enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP)

Publisher: Cold Spring Harbor Laboratory

Date: 31-12-2019

DOI: 10.1101/2019.12.30.891457

Abstract: Ancestral sequence reconstruction is a technique that is gaining widespread use in molecular evolution studies and protein engineering. Accurate reconstruction requires the ability to handle appropriately large numbers of sequences, as well as insertion and deletion (“indel”) events, but available approaches exhibit limitations. To address these limitations, we developed Graphical Representation of Ancestral Sequence Predictions (GRASP), which efficiently implements maximum likelihood methods to enable the inference of ancestors of families with more than 10,000 members. GRASP implements partial order graphs (POGs) to represent and infer insertion and deletion events across ancestors, enabling the identification of building blocks for protein engineering. To validate the capacity to engineer novel proteins from realistic data, we predicted ancestor sequences across three distinct enzyme families: glucose-methanol-choline (GMC) oxidoreductases, cytochromes P450, and dihydroxy/sugar acid dehydratases (DHAD). All tested ancestors demonstrated enzymatic activity. Our study demonstrates the ability of GRASP (1) to support large data sets over 10,000 sequences and (2) to employ insertions and deletions to identify building blocks for engineering biologically active ancestors, by exploring variation over evolutionary time. Massive sequencing projects expose the extent of natural, genetic ersity. Here, we describe a method with capacity to perform ancestor sequence reconstruction from data sets in excess of 10,000 sequences, poised to recover ancestral ersity, including the evolutionary events that determine present-time biological function and structure. We introduce a novel strategy for suggesting “indel variants” that are distinct from, but can be explored alongside, substitution variants for creating ancestral libraries. We demonstrate how indels can be used as building blocks to form “hybrid ancestors” based on this strategy, we synthesise ancestor variants, with varying enzymatic activities, for wide-ranging applications in the biotechnology sector.

EXPRESSION OF MODIFIED HUMAN CYTOCHROME-P450 2E1 IN ESCHERICHIA-COLI, PURIFICATION, AND SPECTRAL AND CATALYTIC PROPERTIES

Publisher: Elsevier BV

Date: 07-1994

DOI: 10.1006/ABBI.1994.1280

Abstract: Human cytochrome P450 (P450) 2E1 is of interest because of its role in the oxidation of numerous drugs and carcinogens. The purification of the protein from human liver is difficult, and we report the development of a system for relatively high-level expression in Escherichia coli. A cDNA was prepared from liver cDNA by polymerase chain reaction methods and several variants with modified 5'-termini were constructed. Analysis of seven of these indicated that the highest levels of expression were found when the first 21 codons of the native sequence were deleted and the Trp immediately following the resulting N-terminal Met was changed to Ala (GCT). Levels of 40-nmol membrane-bound P450 2E1 (liter culture)-1 were routinely recovered. The recombinant P450 2E1 was purified to electrophoretic homogeneity from the bacterial membranes in two ion-exchange steps in > 80% yield. Ferric P450 2E1 was isolated in a mixed spin state. The enzyme was active in chlorzoxazone 6-hydroxylation the addition of human liver cytochrome b5 lowered the Km for the substrate and increased Vmax. N-Terminal amino acid sequence analysis yielded the expected first 21 residues. The expression system should facilitate the availability of human P450 2E1 and antibodies for studies of the enzyme.

Recombinant Human Cytochrome P450 1B1 Expression inEscherichia coli

Publisher: Elsevier BV

Date: 09-1998

DOI: 10.1006/ABBI.1998.0808

Abstract: Human cytochrome P450 (P450) 1B1 was expressed in Escherichia coli at a level of 200 nmol/liter culture using a pCW vector by removal of codons 2-4 and modification of the nucleotide sequence of the resulting N-terminal seven codons a similar level of expression was found with a bicistronic construct that also expressed human NADPH-P450 reductase. P450 1B1 was purified (from the monocistronic system) to electrophoretic homogeneity and a specific content of 9.2 nmol P450/mg protein using DEAE, CM, and hydroxylapatite chromatography. The absolute spectra showed a considerable fraction of high-spin iron and little cytochrome P420. The catalytic activity of the purified enzyme was considerably enhanced in the presence of cholate. Both reconstituted P450 1B1 and the bacterial membranes prepared from the bicistronic vector system had similar7-ethoxyresorufin O-deethylation activities as expected, 17beta-estradiol was hydroxylated primarily at the 4-position. The ability of human P450 1B1 to activate several heterocyclic amines and polycyclic hydrocarbon dihydrodiols was confirmed with reconstituted P450 1B1 and the P450 1B1 membranes in which NADPH-P450 reductase was coexpressed.

Drug metabolism by escherichia coli expressing human cytochromes P450

Publisher: Springer Science and Business Media LLC

Date: 08-1997

DOI: 10.1038/NBT0897-784

Abstract: The broad substrate specificity of the cytochrome P450 (P450) enzyme superfamily of heme-thiolate proteins lends itself to erse environmental and pharmaceutical applications. Until recently, the primary drawback in using living bacteria to catalyze mammalian P450-mediated reactions has been the paucity of electron transport from NADPH to P450 via endogenous flavoproteins. We report the functional expression in Escherichia coli of bicistronic constructs consisting of a human microsomal P450 enzyme encoded by the first cistron and the auxiliary protein NADPH-P450 reductase by the second. Expression levels of P450s ranged from 35 nmol per liter culture to 350 nmol per liter culture, with expression of NADPH-P450 reductase typically ranging from 50% to 100% of that of P450. Transformed bacteria metabolized a number of typical P450 substrates at levels comparable to isolated bacterial membranes fortified with an NADPH-generating system. These rates compare favorably with those obtained using human liver microsomes as well as those of reconstituted in vitro systems composed of purified proteins, lipids, and cofactors.

Engineering functional thermostable proteins using ancestral sequence reconstruction

Publisher: Elsevier BV

Date: 10-2022

DOI: 10.1016/J.JBC.2022.102435

Measurement of P450 difference spectra using intact cells

Publisher: Humana Press

Date: 2013

DOI: 10.1007/978-1-62703-321-3_17

Abstract: Whole-cell assays provide a rapid means of determining expression and substrate binding for cytochrome P450 enzymes expressed heterologously in Escherichia coli and, potentially, other host cells. Such assays are particularly useful for screening large libraries of mutant P450s, where rapid, high-throughput assays are needed for first-tier screens that can, firstly, quantify any P450 form independent of P450 subfamily and, secondly, suggest possible ligands before more labor-intensive direct measurement of substrate turnover. Whole-cell spectral techniques are derived from methods that have been used for a long time to study P450s in microsomal or other subcellular fractions (Omura T and Sato R, J Biol Chem 239:2370-2378, 1964 Schenkman JB et al., Biochemistry 11:4243-4251, 1972), but recent studies have detailed important modifications which allow quantitative results to be obtained in whole cells (Otey CR, Methods in Molecular Biology, vol. 230, Humana, Totowa, NJ, pp. 137-139, 2003 Johnston WA et al., J Biomol Screen 13:135-141, 2008). A general method is presented here for the measurement of difference spectra on recombinant P450 cultures that can be applied to both carbon monoxide and any number of alternative ligands that alter the characteristic spectral signature of P450s.

DNA shuffling of cytochrome P450 enzymes

Publisher: Humana Press

Date: 2013

DOI: 10.1007/978-1-62703-321-3_16

Abstract: DNA family shuffling is an efficient method for creating libraries of novel enzymes, in which a high proportion of mutants exhibit correct folding and possess catalytic properties distinct from the starting material. The evolutionary arrangement of cytochromes P450 into subfamilies of enzymes with highly similar nucleotide sequences but distinct catalytic properties renders them excellent starting material for DNA family shuffling experiments. This chapter provides a general method for creating libraries of shuffled P450s from two or more related sequences and incorporates several recent improvements to previously published methods.

Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: formation of the 4-hydroxy, 4'-hydroxy and N-desmethyl metabolites and isomerization of trans-4-hydroxytamoxifen.

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 08-2002

DOI: 10.1124/DMD.30.8.869

Abstract: The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 microM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 microM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 microM tamoxifen 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 microM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.

Expression of Cytochrome-P450-3A5 in Escherichia Coli: Effects of 5′ Modification, Purification, Spectral Characterization, Reconstitution Conditions, and Catalytic Activities

Publisher: Elsevier BV

Date: 03-1995

DOI: 10.1006/ABBI.1995.1177

Abstract: Cytochrome P450 (P450) 3A5 is a human enzyme with 85% amino acid sequence identity to the more predominantly expressed P450 3A4 and has been reported to have overlapping catalytic specificity. The 5'-terminus of a P450 3A5 cDNA was modified for optimal expression in Escherichia coli using the vector pCW, by aligning the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, (1991) Proc. Natl. Acad. Sci. USA 88, 5597-5601) to the 3A5 cDNA. Two constructs were made, differing by their identity with the modified 3A4 N-terminal sequence (E. M. J. Gillam, T. Baba, B-R. Kim, S. Ohmori, and F. P. Guengerich, (1993) Arch. Biochem. Biophys. 305, 123-131). The first modified sequence (3A5#1) was identical to recombinant P450 3A4 up to codon 15, the 3A5 sequence being introduced thereafter. In the other (3A5#2), the successful 3A4 N-terminal nucleotide sequence was attached to codon 30. The yield was greater than fourfold higher in the first construct [up to 260 nmol (liter culture)-1]. The recombinant P450 3A5 (construct 1) was purified to electrophoretic homogeneity using a variation of a three-step procedure developed previously for P450 3A4, with an overall yield of approximately 40%. Purified P450 3A5 was active in nifedipine oxidation, testosterone 6 beta-hydroxylation, aflatoxin 3 alpha-hydroxylation and 8,9-epoxidation, ethylmorphine N-demethylation, erythromycin N-demethylation, and d-benzphetamine N-demethylation. The reconstitution of nifedipine oxidation, testosterone 6 beta-hydroxylation, and the aflatoxin oxidation activities showed dependence upon the presence of cytochrome b5, alent cations, phospholipid mixtures, glutathione, and cholate similar to that previously found for purified P450 3A4. However, rates of the N-demethylations of ethylmorphine, erythromycin, and d-benzphetamine were as high or higher for P450 3A5 than P450 3A4 and were not particularly dependent upon modifications of reconstitution systems [corrected].

Soluble and membrane-bound Drosophila melanogaster CYP6G1 expressed in Escherichia coli: Purification, activity, and binding properties toward multiple pesticides

Publisher: Elsevier BV

Date: 05-2013

DOI: 10.1016/J.IBMB.2013.02.003

Abstract: Cytochrome P450 CYP6G1 has been implicated in the resistance of Drosophila melanogaster to numerous pesticides. While in vivo and in vitro studies have provided insight to the erse functions of this enzyme, direct studies on the isolated CYP6G1 enzyme have not been possible due to the need for a source of recombinant enzyme. In the current study, the Cyp6g1 gene was isolated from D. melanogaster and re-engineered for heterologous expression in Escherichia coli. Approximately 460 nmol L⁻¹ of P450 holoenzyme were obtained in 500 mL cultures. The recombinant enzyme was located predominantly within the bacterial cytosol. A two-step purification protocol using Ni-chelate affinity chromatography followed by removal of detergent on a hydroxyapatite column produced essentially homogenous enzyme from both soluble and membrane fractions. Recombinant CYP6G1 exhibited p-nitroanisole O-dealkylation activity but was not active against eleven other typical P450 marker substrates. Substrate-induced binding spectra and IC₅₀ values for inhibition of p-nitroanisole O-dealkylation were obtained for a wide selection of pesticides, namely DDT, imidacloprid, chlorfenvinphos, malathion, endosulfan, dieldrin, dicyclanil, lufenuron and carbaryl, supporting previous in vivo and in vitro studies on Drosophila that have suggested that the enzyme is involved in multi-pesticide resistance in insects.

Role of Glutamic Acid 216 in Cytochrome P450 2D6 Substrate Binding and Catalysis†

Publisher: American Chemical Society (ACS)

Date: 17-01-2003

DOI: 10.1021/BI027085W

Abstract: Human cytochrome P450 (P450) 2D6 is an important enzyme involved in the metabolism of drugs, many of which are amines or contain other basic nitrogen atoms. Asp301 has generally been considered to be involved in electrostatic docking with the basic substrates, on the basis of previous modeling studies and site-directed mutagenesis. Substitution of Glu216 with a residue other than Asp strongly attenuated the binding of quinidine, bufuralol, and several other P450 2D6 ligands. Catalytic activity with the substrates bufuralol and 4-methoxyphenethylamine was strongly inhibited by neutral or basic mutations at Glu216 (>95%), to the same extent as the substitution of Asn at Asp301. Unlike the Asp301 mutants, the Gln216 mutant (E216Q) retained 40% enzyme efficiency with the substrate spirosulfonamide, devoid of basic nitrogen, suggesting that the substitutions at Glu216 affect binding of amine substrates more than other catalytic steps. Attempts to induce catalytic specificity toward new substrates by substitutions at Asp301 and Glu216 were unsuccessful. Collectively, the results provide evidence for electrostatic interaction of amine substrates with Glu216, and we propose that both of these acidic residues plus at least another residue(s) is (are) involved in binding the repertoire of P450 2D6 ligands.

Use of engineered cytochromes P450 for accelerating drug discovery and development

Publisher: Elsevier

Date: 2022

DOI: 10.1016/BS.APHA.2022.06.001

Abstract: Numerous steps in drug development, including the generation of authentic metabolites and late-stage functionalization of candidates, necessitate the modification of often complex molecules, such as natural products. While it can be challenging to make the required regio- and stereoselective alterations to a molecule using purely chemical catalysis, enzymes can introduce changes to complex molecules with a high degree of stereo- and regioselectivity. Cytochrome P450 enzymes are biocatalysts of unequalled versatility, capable of regio- and stereoselective functionalization of unactivated CH bonds by monooxygenation. Collectively they catalyze over 60 different biotransformations on structurally and functionally erse organic molecules, including natural products, drugs, steroids, organic acids and other lipophilic molecules. This catalytic versatility and substrate range makes them likely candidates for application as potential biocatalysts for industrial chemistry. However, several aspects of the P450 catalytic cycle and other characteristics have limited their implementation to date in industry, including: their lability at elevated temperature, in the presence of solvents, and over lengthy incubation times the typically low efficiency with which they metabolize non-natural substrates and their lack of specificity for a single metabolic pathway. Protein engineering by rational design or directed evolution provides a way to engineer P450s for industrial use. Here we review the progress made to date toward engineering the properties of P450s, especially eukaryotic forms, for industrial application, and including the recent expansion of their catalytic repertoire to include non-natural reactions.

Exploring the potential of xenobiotic-metabolising enzymes as biocatalysts: Evolving designer catalysts from polyfunctional cytochrome P450 enzymes

Publisher: Wiley

Date: 03-2005

DOI: 10.1111/J.1440-1681.2005.04165.X

Abstract: 1. Biological catalysts have the advantage of being able to catalyse chemical reactions with an often exquisite degree of regio- and stereospecificity in contrast with traditional methods of organic synthesis. 2. The cytochrome P450 enzymes involved in human drug metabolism are ideal starting materials for the development of designer biocatalysts by virtue of their catalytic versatility and extreme substrate ersity. Applications can be envisaged in fine chemical synthesis, such as in the pharmaceutical industry and bioremediation. 3. A variety of techniques of enzyme engineering are currently being applied to P450 enzymes to explore their catalytic potential. Although most studies to date have been performed with bacterial P450s, reports are now emerging of work with mammalian forms of the enzymes. 4. The present minireview will explore the rationale and general techniques for redesigning P450s, review the results obtained to date with xenobiotic-metabolising forms and discuss strategies to overcome some of the logistic problems limiting the full exploitation of these enzymes as industrial-scale biocatalysts.

Recombinant expression and characterization of Lucilia cuprina CYP6G3: Activity and binding properties toward multiple pesticides

Publisher: Elsevier BV

Date: 11-2017

DOI: 10.1016/J.IBMB.2017.09.004

Abstract: The Australian sheep blowfly, Lucilia cuprina, is a primary cause of sheep flystrike and a major agricultural pest. Cytochrome P450 enzymes have been implicated in the resistance of L. cuprina to several classes of insecticides. In particular, CYP6G3 is a L. cuprina homologue of Drosophila melanogaster CYP6G1, a P450 known to confer multi-pesticide resistance. To investigate the basis of resistance, a bicistronic Escherichia coli expression system was developed to co-express active L. cuprina CYP6G3 and house fly (Musca domestica) P450 reductase. Recombinant CYP6G3 showed activity towards the high-throughput screening substrates, 7-ethoxycoumarin and p-nitroanisole, but not towards p-nitrophenol, coumarin, 7-benzyloxyresorufin, or seven different luciferin derivatives (P450-Glo™ substrates). The addition of house fly cytochrome b

Roles of NADPH-P450 Reductase in the O-Deethylation of 7-Ethoxycoumarin by Recombinant Human Cytochrome P450 1B1 Variants in Escherichia coli

Publisher: Elsevier BV

Date: 10-2000

DOI: 10.1006/PREP.2000.1302

Direct electrochemistry of enzymes from the cytochrome P4502C family

Publisher: Elsevier BV

Date: 04-2005

DOI: 10.1016/J.ELECOM.2005.02.021

Expression of cytochrome P450 2D6 in escherichia coli, purification, and spectral and catalytic characterization

Publisher: Elsevier BV

Date: 06-1995

DOI: 10.1006/ABBI.1995.1329

Abstract: Cytochrome P450 (P450) 2D6 is the classic human liver debrisoquine 4-hydroxylase, the first human P450 for which genetic polymorphism was clearly demonstrated. We prepared 11 different constructs of P450 2D6, with modification at the N-terminus, for expression in Escherichia coli with the vector pCW. These varied considerably in levels of expression of apo- and holoprotein, with the best yield being obtained in a system in which much of the N-terminal hydrophobic segment was removed. Production of holoprotein was highly dependent upon the addition of delta-aminolevulinic acid and FeCl3 to cultures, even though heme production should not be limiting in this system. The expressed protein was not tightly bound to the "heavier" membrane fraction but did not appear to behave as a soluble protein either. A purification strategy was developed involving fractional centrifugation, Triton X-114 phase separation, and flavodoxin affinity chromatography, which led to recovery of apparently electrophoretically homogeneous protein in good yield. Purified P450 2D6 had the expected N-terminal amino acid sequence and catalytic activities toward debrisoquine (4-hydroxylation) and bufuralol (1'-hydroxylation). The availability of a ready source of the recombinant protein should facilitate physical as well as functional studies and antibody production for other uses.

Molecular modelling of human CYP1B1 substrate interactions and investigation of allelic variant effects on metabolism

Publisher: Elsevier BV

Date: 06-2003

DOI: 10.1016/S0009-2797(03)00021-8

Abstract: Molecular modelling of human CYP1B1 based on homology with the mammalian P450, CYP2C5, of known three-dimensional structure is reported. The enzyme model has been used to investigate the likely mode of binding for selected CYP1B1 substrates, particularly with regard to the possible effects of allelic variants of CYP1B1 on metabolism. In general, it appears that the CYP1B1 model is consistent with known substrate selectivity for the enzyme, and the sites of metabolism can be rationalized in terms of specific contacts with key amino acid residues within the CYP1B1 heme locus. Furthermore, a mode of binding interaction for the inhibitor, alpha-naphthoflavone, is presented which accords with currently available information. The current paper shows that a combination of molecular modelling and experimental determinations on the substrate metabolism for CYP1B1 allelic variants can aid in the understanding of structure-function relationships within P450 enzymes.

Determinants of thermostability in the cytochrome P450 fold

Publisher: Elsevier BV

Date: 2018

DOI: 10.1016/J.BBAPAP.2017.08.003

Abstract: Cytochromes P450 are found throughout the biosphere in a wide range of environments, serving a multitude of physiological functions. The ubiquity of the P450 fold suggests that it has been co-opted by evolution many times, and likely presents a useful compromise between structural stability and conformational flexibility. The ersity of substrates metabolized and reactions catalyzed by P450s makes them attractive starting materials for use as biocatalysts of commercially useful reactions. However, process conditions impose different requirements on enzymes to those in which they have evolved naturally. Most natural environments are relatively mild, and therefore most P450s have not been selected in Nature for the ability to withstand temperatures above ~40°C, yet industrial processes frequently require extended incubations at much higher temperatures. Thus, there has been considerable interest and effort invested in finding or engineering thermostable P450 systems. Numerous P450s have now been identified in thermophilic organisms and analysis of their structures provides information as to mechanisms by which the P450 fold can be stabilized. In addition, protein engineering, particularly by directed or artificial evolution, has revealed mutations that serve to stabilize particular mesophilic enzymes of interest. Here we review the current understanding of thermostability as it applies to the P450 fold, gleaned from the analysis of P450s characterized from thermophilic organisms and the parallel engineering of mesophilic forms for greater thermostability. We then present a perspective on how this information might be used to design stable P450 enzymes for industrial application. This article is part of a Special Issue entitled: Cytochrome P450 bio ersity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Oxygen Surrogate Systems for Supporting Human Drug-Metabolizing Cytochrome P450 Enzymes

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 04-2020

DOI: 10.1124/DMD.120.090555

Resurrection and characterization of ancestral CYP11A1 enzymes

Publisher: Wiley

Date: 27-06-2021

DOI: 10.1111/FEBS.16054

Abstract: Mitochondrial cytochromes P450 presumably originated from a common microsomal P450 ancestor. However, it is still unknown how ancient mitochondrial P450s were able to retain their oxygenase function following relocation to the mitochondrial matrix and later emerged as enzymes specialized for steroid hormone biosynthesis in vertebrates. Here, we used the approach of ancestral sequence reconstruction (ASR) to resurrect ancient CYP11A1 enzymes and characterize their unique biochemical properties. Two ancestral CYP11A1 variants, CYP11A_Mammal_N101 and CYP11A_N1, as well as an extant bovine form were recombinantly expressed and purified to homogeneity. All enzymes showed characteristic P450 spectral properties and were able to convert cholesterol as well as other sterol substrates to pregnenolone, yet with different specificities. The vertebrate CYP11A_N1 ancestor preferred the cholesterol precursor, desmosterol, as substrate suggesting a convergent evolution of early cholesterol metabolism and CYP11A1 enzymes. Both ancestors were able to withstand increased levels of hydrogen peroxide but only the ancestor CYP11A_N1 showed increased thermostability (˜ 25 °C increase in T 50 ) compared with the extant CYP11A1. The extraordinary robustness of ancient mitochondrial P450s, as demonstrated for CYP11A_N1, may have allowed them to stay active when presented with poorly compatible electron transfer proteins and resulting harmful ROS in the new environment of the mitochondrial matrix. To the best of our knowledge, this work represents the first study that describes the resurrection of ancient mitochondrial P450 enzymes. The results will help to understand and gain fundamental functional insights into the evolutionary origins of steroid hormone biosynthesis in animals.

Directed Evolution Reveals Requisite Sequence Elements in the Functional Expression of P450 2F1 in Escherichia coli

Publisher: American Chemical Society (ACS)

Date: 29-08-2012

DOI: 10.1021/TX300281G

Metabolic activation of heterocyclic amines and other procarcinogens in Salmonella typhimurium umu tester strains expressing human cytochrome P4501A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4 and human NADPH-P450 reductase and bacterial O-acetyltransferase

Publisher: Elsevier BV

Date: 05-2001

DOI: 10.1016/S1383-5718(01)00154-1

Abstract: We investigated roles of different forms of cytochrome P450 (P450 or CYP) in the metabolic activation of heterocyclic amines (HCAs) and other procarcinogens to genotoxic metabolite(s) in the newly developed umu tester strains Salmonella typhimurium (S. typhimurium) OY1002/1A1, OY1002/1A2, OY1002/1B1, OY1002/2C9, OY1002/2D6, OY1002/2E1 and OY1002/3A4, which express respective human P450 enzymes and NADPH-cytochrome P450 reductase (reductase) and bacterial O-acetyltransferase (O-AT). These strains were established by introducing two plasmids into S. typhimurium TA1535, one carrying both P450 and the reductase cDNA in a bicistronic construct under control of an IPTG-inducible double tac promoter and the other, pOA102, carrying O-AT and umuC"lacZ fusion genes. Expression levels of CYP were found to range between 35 to 550 nmol/l cell culture in the strains tested. O-AT activities in different strains ranged from 52 to 125 nmol isoniazid acetylated/min/mg protein. All HCAs tested, and 2-aminoanthracene and 2-aminofluorene exhibited high genotoxicity in the OY1002/1A2 strain, and genotoxicity of 2-amino-3-methylimidazo [4,5-f]quinoline was detected in both the OY1002/1A1 and OY1002/1A2 strains. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole and 3-amino-1-methyl-5H-pyrido[4,3-b]-indole were activated in the OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4 strains. Aflatoxin B(1) exhibited genotoxicity in the OY1002/1A2, OY1002/1A1, and OY1002/3A4 strains. beta-Naphthylamine and benzo[a]pyrene did not exhibit genotoxicity in any of the strains. These results suggest that CYP1A2 is the major cytochrome P450 enzyme involved in bioactivation of HCAs.

Extending the diversity of cytochrome P450 enzymes by DNA family shuffling

Publisher: Elsevier BV

Date: 06-2007

DOI: 10.1016/J.GENE.2007.01.031

Abstract: The cytochrome P450 enzymes involved in xenobiotic metabolism are an excellent starting point for the directed evolution of novel biocatalysts due to their wide substrate specificity. A shuffled library of three highly homologous mammalian genes (for P450 2C9, P450 2C11 and P450 2C19) was constructed by applying a modified DNA family shuffling procedure. The modifications made to the traditional DNA shuffling protocols involved non-random digestion via the use of different combinations of restriction enzymes (REs) followed by isolation of fragments under 300 bp by size-selective filtration. Shuffled cytochrome P450 mutants were co-expressed in Escherichia coli with their redox partner, NADPH-cytochrome P450 reductase (NPR). We report here how non-random fragmentation may help in chimeragenesis within the areas of low sequence similarity such as substrate recognition sites (SRSs) that are generally underrepresented in recombination using the random fragmentation process. Size-selective filtration was used to limit recovery of incompletely digested fragments and consequently minimize the chances for contamination of the shuffled library with parental forms. No parental forms could be detected in the shuffled library using restriction fragment length polymorphism (RFLP) analysis, suggesting the library was free of parental contamination. Sequencing of randomly selected mutants demonstrated a high level of chimeragenesis with on average of 8.0+/-2.2 crossovers and a low level of mutagenesis with 5.2+/-2.8 spontaneous mutations per approximately 1.5 kbp of the full-length P450 sequence. The proportion of properly folded protein as indicated by the observation of characteristic Fe(II).CO vs. Fe(II) difference spectra was 15% (4/27) of analysed mutants. Screening of the shuffled library for indole oxidation revealed four clones with similar or higher levels of indigo pigment production to those of the parental P450s and two clones with elevated P450 expression. In this paper we present a method for the effective family shuffling of cytochrome P450 enzymes, applicable to the creation of mutant libraries with expanded metabolic ersity and with a significant proportion of functional clones.

In vitro metabolism of the anti-inflammatory clerodane diterpenoid polyandric acid A and its hydrolysis product by human liver microsomes and recombinant cytochrome P450 and UDP-glucuronosyltransferase enzymes

Publisher: Informa UK Limited

Date: 14-07-2016

DOI: 10.1080/00498254.2016.1203041

Abstract: 1. The metabolism of the anti-inflammatory diterpenoid polyandric acid A (PAA), a constituent of the Australian Aboriginal medicinal plant Dodonaea polyandra, and its de-esterified alcohol metabolite, hydrolysed polyandric acid A (PAAH) was studied in vitro using human liver microsomes (HLM) and recombinant UDP-glucuronosyltransferase (UGT) and cytochrome P450 (CYP) enzymes. 2. Hydrolysis of PAA to yield PAAH occurred upon incubation with HLM. Further incubations of PAAH with HLM in the presence of UGT and CYP cofactors resulted in significant depletion, with UGT-mediated depletion as the major pathway. 3. Reaction phenotyping utilising selective enzyme inhibitors and recombinant human UGT and CYP enzymes revealed UGT2B7 and UGT1A1, and CYP2C9 and CYP3A4 as the major enzymes involved in the metabolism of PAAH. 4. Analysis of incubations of PAAH with UDP-glucuronic acid-supplemented HLM and recombinant enzymes by UPLC/MS/MS identified three glucuronide metabolites. The metabolites were further characterised by β-glucuronidase and mild alkaline hydrolysis. The acyl glucuronide of PAAH was shown to be the major metabolite. 5. This study demonstrates the in vitro metabolism of PAA and PAAH and represents the first systematic study of the metabolism of an active constituent of an Australian Aboriginal medicinal plant.

Is the undergraduate research experience (URE) always best?: The power of choice in a bifurcated practical stream for a large introductory biochemistry class

Publisher: Wiley

Date: 2012

DOI: 10.1002/BMB.20576

WHAT MAKES P450s WORK? SEARCHES FOR ANSWERS WITH KNOWN AND NEW P450s1*

Publisher: Informa UK Limited

Date: 2000

DOI: 10.1081/DMR-100102334

Abstract: Random mutagenesis has been developed as an approach for the study of human cytochrome P450 (P450) enzymes and their structure and function. Sensitive screening methods are critical for the success of this approach. We have developed one system that takes advantage of the ability of human P450 1A2 to activate heterocyclic amines to mutagenic products [A. Parikh, P. D. Josephy, and F. P. Guengerich, Biochemistry, 38, 5283-5289 (1999)]. Mutants with both attenuated and enhanced activity have been recovered and subjected to further kinetic analysis. For phenacetin O-deethylation, the E225I mutant had kcat 6x > wild type D320A had kcat 1/10x wild type). With all three P450s, the rate of first electron reduction was similar, and all had similar binding constants for phenacetin (approximately 15 microM). All three forms yielded intermolecular, noncompetitive kinetic deuterium isotope effects of 1.5-2 [DV and D(V/K)] for O-deethylation of [OCD2CH3]-phenacetin. All three forms of P450 1A2 also formed a minor product, the acetol (C-hydroxylation of the acetyl group). This reaction had a deuterium isotope effect of approximately 14 with all three forms of the enzyme, and C-H bond breaking is the rate-determining step. Another approach to P450 2A6 involves the recent observation that this P450 can accumulate indigo [E. M. J. Gillam, A. M. A. Aguinaldo, L. M. Notley, D. Kim, R. G. Mundkowski, A. A. Volkov, F. H. Arnold, P. Soucek, J. T. DeVoss, and F. P. Guengerich, Biochem. Biophys. Res. Commun 265, 469-472 (1999)]. Current results indicate that this process involves the conversion of endogenous indole to indoxyl by the P450. The reaction may be used in assays of random mutants and has some potential applications in industry.

Activation of procarcinogens by human cytochrome P450 enzymes expressed in escherichia coli. simplified bacterial systems for genotoxicity assays

Publisher: Oxford University Press (OUP)

Date: 1994

DOI: 10.1093/CARCIN/15.11.2523

Abstract: Bacterial assays were used to examine the activation of 14 known procarcinogens by cytochrome P450 (P450) enzymes. Human P450s 1A1, 1A2 and 3A4 were expressed in Escherichia coli with slight modification of their N-terminal sequences. Genotoxicity was measured by the induction of the SOS response in Salmonella typhimurium NM2009 (TA1535 SK1002 NM12), which contains a umuC regulatory sequence attached to the lacZ reporter gene. Conditions for analysis were examined using E. coli membranes and purified enzymes. Membrane fractions, fortified with NADPH-P450 reductase, were found to be useful preparations for measuring activation of the procarcinogens. Conditions of linearity were established for these assays and the systems were applied to several particular problems related to bioactivation of procarcinogens by P450s. The patterns of activation of the 14 in idual chemicals were consistent with the literature developed using human liver microsomes, purified liver P450s and other approaches. The P450s expressed in bacterial membranes could be inhibited by antibodies. 7,8-Benzoflavone inhibited P450s 1A1 and 1A2 and stimulated P450 3A4 in the membranes. The contributions of P450s 1A1 and 1A2 were distinguished with some of the arylamines and 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Recombinant P450 3A4 was found to be more active than P450 1A2 in the activation of aflatoxin B1 at all substrate concentrations examined.

Specificity of 17β-oestradiol and benzo[a]pyrene oxidation by polymorphic human cytochrome P4501B1 variants substituted at residues 48, 119 and 432

Publisher: Informa UK Limited

Date: 2001

DOI: 10.1080/00498250110043490

Abstract: 1. Eight human cytochrome P4501B1 (CYP1B1) allelic variants, namely Arg48 Ala119 Leu432, Arg48 Ala119 Val432 Gly48 Ala119 Leu432, Gly48 Ala119 Val432, Arg48 Ser119 Leu432, Arg48 Ser119 Val432, Gly48 Ser119 Leu432 and Gly48 Ser119 Va1432 (all with Asn453), were expressed in Escherichia coli together with human NADPH-P450 reductase and their catalytic specificities towards oxidation of 17beta-oestradiol and benzo[a]pyrene were determined. 2. All of the CYP1B1 variants expressed in bacterial membranes showed Fe2+.CO versus Fe2+ difference spectra with wavelength maxima at 446 nm and they reacted with antibodies raised against recombinant human CYP1B1 in immunoblots. The ratio of expression of the reductase to CYP1B1 in these eight preparations ranged from 0.2 to 0.5. 3. CYP1B1 Arg48 variants tended to have higher activities for 17beta-oestradiol 4-hydroxylation than Gly48 variants, although there were no significant variations in 17beta-oestradiol 2-hydroxylation activity in these eight CYP1B1 variants. Interestingly, ratios of formation of 17beta-oestradiol 4-hydroxylation to 2-hydroxylation by these CYP1B1 variants were higher in all of the Val432 forms than the corresponding Leu432 forms. 4. In contrast, Leu432 forms of CYP1B1 showed higher rates of oxidation of benzo[a]pyrene (to the 7,8-dihydoxy-7,8-dihydrodiol in the presence of epoxide hydrolase) than did the Val432 forms. 5. These results suggest that polymorphic human CYP1B1 variants may cause some altered catalytic specificity with 17beta-oestradiol and benzo[a]pyrene and may influence susceptibilities of in iduals towards endogenous and exogenous carcinogens.

Re-engineering of CYP2C9 to probe acid-base substrate selectivity.

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 07-07-2008

DOI: 10.1124/DMD.108.022186

A novel CYP2A6 allele, CYP2A6*23, impairs enzyme function in vitro and in vivo and decreases smoking in a population of Black-African descent

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 2008

DOI: 10.1097/FPC.0B013E3282F3606E

Directed Evolution of Enzymes

Publisher: Elsevier

Date: 2010

DOI: 10.1016/B978-008045382-8.00675-4

SeqScrub: a web tool for automatic cleaning and annotation of FASTA file headers for bioinformatic applications

Publisher: Future Science Ltd

Date: 08-2019

DOI: 10.2144/BTN-2018-0188

Abstract: Data consistency is necessary for effective bioinformatic analysis. SeqScrub is a web tool that parses and maintains consistent information about protein and DNA sequences in FASTA file format, checks if records are current, and adds taxonomic information by matching identifiers against entries in authoritative biological sequence databases. SeqScrub provides a powerful, yet simple workflow for managing, enriching and exchanging data, which is crucial to establish a record of provenance for sequences found from broad and varied searches for ex le, using BLAST on continually updated genome sequence sets. Headers standardized using SeqScrub can be parsed by a majority of bioinformatic tools, stay uniformly named between collaborators and contain informative labels to aid management of reproducible, scientific data. SeqScrub is available at bioinf.scmb.uq.edu.au/seqscrub

ReX: A suite of computational tools for the design, visualization, and analysis of Chimeric protein libraries

Publisher: Future Science Ltd

Date: 02-2016

DOI: 10.2144/000114381

Abstract: Directed evolution has greatly facilitated protein engineering and provided new insights into protein structure—function relationships. DNA shuffling using restriction enzymes is a particularly simple and cost-effective means of recombinatorial evolution that is well within the capability of most molecular biologists, but tools for the design and analysis of such experiments are limited. Here we introduce a suite of freely available online tools to make the construction and analysis of chimeric libraries readily accessible to the novice. REcut ( qpmf.rx.umaryland.edu/REcut.html ) facilitates the choice of DNA fragmentation strategy, while Xover ( qpmf.rx.umaryland.edu/Xover.html ) analyzes chimeric mutants to reveal recombination patterns and extract quantitative data.

Ancestral sequence reconstruction of the CYP711 family reveals functional divergence in strigolactone biosynthetic enzymes associated with gene duplication events in monocot grasses.

Publisher: Wiley

Date: 25-06-2022

DOI: 10.1111/NPH.18285

Abstract: The strigolactone (SL) class of phytohormones shows broad chemical ersity, the functional importance of which remains to be fully elucidated, along with the enzymes responsible for the ersification of the SL structure. Here we explore the functional evolution of the highly conserved CYP711A P450 family, members of which catalyze several key monooxygenation reactions in the strigolactone pathway. Ancestral sequence reconstruction was utilized to infer ancestral CYP711A sequences based on a comprehensive set of extant CYP711 sequences. Eleven ancestral enzymes, corresponding to key points in the CYP711A phylogenetic tree, were resurrected and their activity was characterized towards the native substrate carlactone and the pure enantiomers of the synthetic strigolactone analogue, GR24. The ancestral and extant CYP711As tested accepted GR24 as a substrate and catalyzed several ersifying oxidation reactions on the structure. Evidence was obtained for functional ergence in the CYP711A family. The monocot group 3 ancestor, arising from gene duplication events within monocot grasses, showed both increased catalytic activity towards GR24 and high stereoselectivity towards the GR24 isomer resembling strigol-type SLs. These results are consistent with a role for CYP711As in strigolactone ersification in early land plants, which may have extended to the ersification of strigol-type SLs.

Using force-displacement relations to obtain actuation parameters from artificial muscles

Publisher: Elsevier BV

Date: 05-2019

DOI: 10.1016/J.SNA.2019.03.012

University Of Queensland

Location: Australia

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University Of Oxford

Location: United Kingdom of Great Britain and Northern Ireland

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Vanderbilt University School Of Medicine

Location: United States of America

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Discovery Projects - Grant ID: DP200102837

Start Date: 07-2020

End Date: 12-2024

Amount: $451,284.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP170100717

Start Date: 01-2019

End Date: 12-2022

Amount: $460,800.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0210635

Start Date: 2002

End Date: 12-2004

Amount: $155,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0772404

Start Date: 2007

End Date: 12-2010

Amount: $263,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0560595

Start Date: 07-2005

End Date: 07-2008

Amount: $461,454.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP160100865

Start Date: 2016

End Date: 06-2019

Amount: $330,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP120101772

Start Date: 03-2012

End Date: 12-2015

Amount: $320,000.00

Funder: Australian Research Council