ORCID Profile
0000-0002-7457-9727
Current Organisation
University of Adelaide
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Enzymes | Characterisation of Biological Macromolecules | Transition Metal Chemistry | Macromolecular and Materials Chemistry | Synthesis of Materials | Inorganic Chemistry | Organic Chemical Synthesis | Bioinorganic Chemistry | Nanochemistry and Supramolecular Chemistry | Characterisation of biological macromolecules | Medicinal and Biomolecular Chemistry | Structure and dynamics of materials | Medicinal and Biomolecular Chemistry not elsewhere classified | Macromolecular and materials chemistry | Electrochemical energy storage and conversion
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Publisher: Wiley
Date: 10-12-2011
Abstract: CYP101C1 from Novosphingobium aromaticivorans DSM12444 is a homologue of CYP101D1 and CYP101D2 enzymes from the same bacterium and CYP101A1 from Pseudomonas putida. CYP101C1 does not bind c hor but is capable of binding and hydroxylating ionone derivatives including α- and β-ionone and β-damascone. The activity of CYP101C1 was highest with β-damascone (k(cat)=86 s(-1)) but α-ionone oxidation was the most regioselective (98 % at C3). The crystal structures of hexane-2,5-diol- and β-ionone-bound CYP101C1 have been solved both have open conformations and the hexanediol-bound form has a clear access channel from the heme to the bulk solvent. The entrance of this channel is blocked when β-ionone binds to the enzyme. The heme moiety of CYP101C1 is in a significantly different environment compared to the other structurally characterised CYP101 enzymes. The likely ferredoxin binding site on the proximal face of CYP101C1 has a different topology but a similar overall positive charge compared to CYP101D1 and CYP101D2, all of which accept electrons from the ArR/Arx class I electron transfer system.
Publisher: Elsevier BV
Date: 07-2023
Publisher: Elsevier BV
Date: 04-2018
DOI: 10.1016/J.ENZMICTEC.2018.01.002
Abstract: The stereoselective oxidation of hydrocarbons is an area of research where enzyme biocatalysis can make a substantial impact. The cyclic ketone isophorone was stereoselectively hydroxylated (≥95%) by wild-type CYP102A1 to form (R)-4-hydroxyisophorone, an important chiral synthon and flavour and fragrance compound. CYP102A1 variants were also selective for 4-hydroxyisophorone formation and the product formation rate increased over the wild-type enzyme by up to 285-fold, with the best mutants being R47L/Y51F/I401P and A74G/F87V/L188Q. The latter variant, which contained mutations in the distal substrate binding pocket, was marginally less selective. Combining perfluorodecanoic acid decoy molecules with the rate accelerating variant R47L/Y51F/I401P engendered further improvement with the purified enzymes. However when the decoy molecules were used with A74G/F87V/L188Q the amount of product generated by the enzyme was reduced. Addition of decoy molecules to whole-cell turnovers did not improve the productivity of these CYP102A1 systems. WT CYP101A1 formed significant levels of 7-hydroxyisophorone as a minor product alongside 4-hydroxyisophorone. However the F87W/Y96F/L244A/V247L CYP101A1 mutant was ≥98% selective for (R)-4-hydroxyisophorone. A comparison of the two enzyme systems using whole-cell oxidation reactions showed that the best CYP101A1 variant was able to generate more product. We also characterised that the further oxidation metabolite 4-ketoisophorone was produced and then subsequently reduced to levodione by an endogenous Escherichia coli ene reductase.
Publisher: International Union of Crystallography (IUCr)
Date: 30-04-2014
DOI: 10.1107/S139900471400474X
Abstract: Rhodopseudomonas palustris HaA2 contains a gene, RPB3630 , encoding a ferredoxin, HaPuxC, with an atypical C XX H XX C( X ) n CP iron–sulfur cluster-binding motif. The ferredoxin gene is associated with a cytochrome P450 (CYP) monooxygenase-encoding gene, CYP194A3, an arrangement which is conserved in several strains of bacteria. Similar ferredoxin genes are found in other bacteria, such as Mycobacterium tuberculosis , where they are also associated with CYP genes. The crystal structure of HaPuxC has been solved at 2.3 Å resolution. The overall fold of this [3Fe–4S] cluster-containing ferredoxin is similar to other [3Fe–4S] and [4Fe–4S] species, with the loop around the iron–sulfur cluster more closely resembling those of [3Fe–4S] ferredoxins. The side chain of His17 from the cluster-binding motif in HaPuxC points away from the vacant site of the cluster and interacts with Glu61 and one of the sulfide ions of the cluster. This is the first cytochrome P450 electron-transfer partner of this type to be structurally characterized and will provide a better understanding of the electron-transfer processes between these ferredoxins and their CYP enzymes.
Publisher: American Chemical Society (ACS)
Date: 17-05-2018
Publisher: Springer Science and Business Media LLC
Date: 04-04-2020
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C4RA14010A
Abstract: CYP101B1 from Novosphingobium aromaticivorans oxidises ionone derivatives and phenylcyclohexane with high activity and regioselectivity.
Publisher: American Chemical Society (ACS)
Date: 14-02-2020
Publisher: Springer Science and Business Media LLC
Date: 22-07-2013
DOI: 10.1007/S00253-012-4278-7
Abstract: CYP101D2 is a cytochrome P450 monooxygenase from Novosphingobium aromaticivorans which is closely related to CYP101A1 (P450cam) from Pseudomonas putida. Both enzymes selectively hydroxylate c hor to 5-exo-hydroxyc hor, and the residues that line the active sites of both enzymes are similar including the pre-eminent Tyr96 residue. However, Met98 and Leu253 in CYP101D2 replace Phe98 and Val247 in CYP101A1, and c hor binding only results in a maximal change in the spin state to 40 % high-spin. Substitutions at Tyr96, Met98 and Leu253 in CYP101D2 reduced both the spin state shift on c hor binding and the c hor oxidation activity. The Tyr96Ala mutant increased the affinity of CYP101D2 for hydrocarbon substrates including adamantane, cyclooctane, hexane and 2-methylpentane. The monooxygenase activity of the Tyr96Ala variant towards alkane substrates was also enhanced compared with the wild-type enzyme. The crystal structure of the substrate-free form of this variant shows the enzyme in an open conformation (PDB: 4DXY), similar to that observed with the wild-type enzyme (PDB: 3NV5), with the side chain of Ala96 pointing away from the heme. Despite this, the binding and activity data suggest that this residue plays an important role in substrate binding, evidencing that the enzyme probably undergoes catalysis in a more closed conformation, similar to those observed in the crystal structures of CYP101A1 (PDB: 2CPP) and CYP101D1 (PDB: 3LXI).
Publisher: International Union of Crystallography (IUCr)
Date: 14-02-2012
Publisher: American Chemical Society (ACS)
Date: 21-12-2003
DOI: 10.1021/JA028460A
Abstract: Oxygenated derivatives of the monoterpene (+)-alpha-pinene are found in plant essential oils and used as fragrances and flavorings. (+)-alpha-Pinene is structurally related to (+)-c hor, the natural substrate of the heme monooxygenase cytochrome P450(cam) from Pseudomonas putida. The aim of the present work was to apply the current understanding of P450 substrate binding and catalysis to engineer P450(cam) for the selective oxidation of (+)-alpha-pinene. Consideration of the structures of (+)-c hor and (+)-alpha-pinene lead to active-site mutants containing combinations of the Y96F, F87A, F87L, F87W, and V247L mutations. All mutants showed greatly enhanced binding and rate of oxidation of (+)-alpha-pinene. Some mutants had tighter (+)-alpha-pinene binding than c hor binding by the wild-type. The most active was the Y96F/V247L mutant, with a (+)-alpha-pinene oxidation rate of 270 nmol (nmol of P450(cam))(-)(1) min(-)(1), which was 70% of the rate of c hor oxidation by wild-type P450(cam). C hor is oxidized by wild-type P450(cam) exclusively to 5-exo-hydroxyc hor. If the gem dimethyl groups of (+)-alpha-pinene occupied similar positions to those found for c hor in the wild-type structure, (+)-cis-verbenol would be the dominant product. All P450(cam) enzymes studied gave (+)-cis-verbenol as the major product but with much reduced selectivity compared to c hor oxidation by the wild-type. (+)-Verbenone, (+)-myrtenol, and the (+)-alpha-pinene epoxides were among the minor products. The crystal structure of the Y96F/F87W/V247L mutant, the most selective of the P450(cam) mutants initially examined, was determined to provide further insight into P450(cam) substrate binding and catalysis. (+)-alpha-Pinene was bound in two orientations which were related by rotation of the molecule. One orientation was similar to that of c hor in the wild-type enzyme while the other was significantly different. Analysis of the enzyme/substrate contacts suggested rationalizations of the product distribution. In particular competition rather than cooperativity between the F87W and V247L mutations and substrate movement during catalysis were proposed to be major factors. The crystal structure lead to the introduction of the L244A mutation to increase the selectivity of pinene oxidation by further biasing the binding orientation toward that of c hor in the wild-type structure. The F87W/Y96F/L244A mutant gave 86% (+)-cis-verbenol and 5% (+)-verbenone. The Y96F/L244A/V247L mutant gave 55% (+)-cis-verbenol but interestingly also 32% (+)-verbenone, suggesting that it may be possible to engineer a P450(cam) mutant that could oxidize (+)-alpha-pinene directly to (+)-verbenone. Verbenol, verbenone, and myrtenol are naturally occurring plant fragrance and flavorings. The preparation of these compounds by selective enzymatic oxidation of (+)-alpha-pinene, which is readily available in large quantities, could have applications in synthesis. The results also show that the protein engineering of P450(cam) for high selectivity of substrate oxidation is more difficult than achieving high substrate turnover rates because of the subtle and dynamic nature of enzyme-substrate interactions.
Publisher: Wiley
Date: 21-06-2007
Publisher: Wiley
Date: 12-2008
Publisher: International Union of Crystallography (IUCr)
Date: 25-03-2009
Publisher: American Chemical Society (ACS)
Date: 15-12-2021
DOI: 10.1021/ACS.INORGCHEM.1C02786
Abstract: The cytochrome P450 (CYP) superfamily of heme monooxygenases is involved in a range of important chemical biotransformations across nature. Azole-containing molecules have been developed as drugs that bind to the heme center of these enzymes, inhibiting their function. The optical spectrum of CYP enzymes after the addition of these inhibitors is used to assess how the molecules bind. Here we use the bacterial CYP199A4 enzyme, from
Publisher: Springer Science and Business Media LLC
Date: 25-09-2010
DOI: 10.1007/S00253-009-2234-Y
Abstract: Cytochrome P450 (CYP) enzymes of the CYP101 and CYP111 families from Novosphingobium aromaticivorans are heme monooxygenases that catalyze the hydroxylation of a range of terpenoid compounds. CYP101D1 and CYP101D2 oxidized c hor to 5-exo-hydroxyc hor. CYP101B1 and CYP101C1 oxidized beta-ionone to predominantly 3-R-hydroxy-beta-ionone and 4-hydroxy-beta-ionone, respectively. CYP111A2 oxidized linalool to 8-hydroxylinalool. Physiologically, these CYP enzymes could receive electrons from Arx, a [2Fe-2S] ferredoxin equivalent to putidaredoxin from the CYP101A1 system from Pseudomonas putida. A putative ferredoxin reductase (ArR) in the N. aromaticivorans genome, with high amino acid sequence homology to putidaredoxin reductase, has been over-produced in Escherichia coli and found to support substrate oxidation by these CYP enzymes via Arx with both high activity and coupling of product formation to NADH consumption. The ArR/Arx electron-transport chain has been co-expressed with the CYP enzymes in an E. coli host to provide in vivo whole-cell substrate oxidation systems that could produce up to 6.0 g L(-1) of 5-exo-hydroxyc hor at rates of up to 64 microM (gram of cell dry weight)(-1) min(-1). These efficient biocatalytic systems have potential uses in preparative scale whole-cell biotransformations.
Publisher: Royal Society of Chemistry (RSC)
Date: 2008
DOI: 10.1039/B718124H
Abstract: The evolution of CYP102A1 variants with enhanced activity and altered specificity characteristics.
Publisher: Elsevier BV
Date: 06-1999
DOI: 10.1016/S0028-3932(98)00067-0
Abstract: On the covert orienting of visual attention task (COVAT), responses to targets appearing at the location indicated by a non-predictive spatial cue are faster than responses to targets appearing at uncued locations when stimulus onset asynchrony (SOA) is less than approximately 200 ms. For longer SOAs, this pattern reverses and RTs to targets appearing at uncued locations become faster than RTs to targets appearing at the cued location. This facilitation followed by inhibition has been termed the biphasic effect of non-predictive peripheral spatial cues. Currently, there is debate about whether these two processes are independent. This issue was addressed in a series of experiments where the temporal overlap between the peripheral cue and target was manipulated at both short and long SOAs. Results showed that facilitation was present only when the SOA was short and there was temporal overlap between cue and target. Conversely, inhibition occurred only when the SOA was long and there was no temporal overlap between cue and target. The biphasic effect, with an early facilitation followed by a later inhibition, occurred only when the cue duration was fixed such that there was temporal overlap between the cue and target at short but not long SOAs. In a final experiment, the duration of targets the temporal overlap between cue and target and the SOA were manipulated factorially. The results showed that facilitation occurred only when the SOA was short, there was temporal overlap between cue and target and the target remained visible until the subject responded. These results suggest that the facilitation and inhibition found on COVATs which use non-informative peripheral cues are independent processes and their presence and magnitude is related to the temporal properties of cues and targets.
Publisher: Wiley
Date: 21-10-2010
DOI: 10.1002/PROT.22627
Publisher: MDPI AG
Date: 28-01-2023
DOI: 10.3390/IJMS24032519
Abstract: Platelets play a vital role in regulating hemostasis and thrombosis. Rho GTPases are well known as molecular switches that control various cellular functions via a balanced GTP-binding/GTP-hydrolysis cycle and signaling cascade through downstream effectors. In platelets, Rho GTPases function as critical regulators by mediating signal transduction that drives platelet activation and aggregation. Mostly by gene targeting and pharmacological inhibition approaches, Rho GTPase family members RhoA, Rac1, and Cdc42 have been shown to be indispensable in regulating the actin cytoskeleton dynamics in platelets, affecting platelet shape change, spreading, secretion, and aggregation, leading to thrombus formation. Additionally, studies of Rho GTPase function using platelets as a non-transformed model due to their anucleated nature have revealed valuable information on cell signaling principles. This review provides an updated summary of recent advances in Rho GTPase signaling in platelet regulation. We also highlight pharmacological approaches that effectively inhibited platelet activation to explore their possible development into future antiplatelet therapies.
Publisher: Springer Science and Business Media LLC
Date: 11-11-2009
DOI: 10.1007/S00775-009-0604-7
Abstract: CYP199A2 from Rhodopseudomonas palustris CGA009 is a heme monooxygenase that catalyzes the oxidation of para-substituted benzoic acids. CYP199A2 activity is reconstituted by a class I electron transfer chain consisting of the associated [2Fe-2S] ferredoxin palustrisredoxin (Pux) and a flavoprotein palustrisredoxin reductase (PuR). Another [2Fe-2S] ferredoxin, palustrisredoxin B (PuxB RPA3956) has been identified in the genome. PuxB shares sequence identity and motifs with vertebrate-type ferredoxins involved in Fe-S cluster assembly but also 50% identity with Pux and it mediates electron transfer from PuR to CYP199A2, albeit with lower steady-state turnover activity: 99 nmol (nmol P450)(-1)min(-1) for 4-methoxybenzoic acid oxidation compared with 1,438 nmol (nmol P450)(-1 )min(-1) for Pux. This difference mainly arises from weak CYP199A2-PuxB binding (K (m) 34.3 vs. 0.45 microM for Pux) rather than slow electron transfer (k (cat) 19.1 vs. 37.9 s(-1) for Pux). Comparison of the 2.0-A-resolution crystal structure of the PuxB A105R mutant with other vertebrate-type, P450-associated ferredoxins revealed similar protein folds but also significant differences in some loop regions. Therefore, PuxB offers a platform for studying ferredoxin-P450 recognition in class I P450 systems. Substitution of PuxB residues at key locations with those in Pux shows that Ala42, Cys43, and Ala44 in the [2Fe-2S] cluster binding loop and Met66 are important in electron transfer from PuxB to CYP199A2, whereas Phe73 and the C-terminal Ala105 were involved in both protein binding and electron transfer.
Publisher: Wiley
Date: 22-07-2009
DOI: 10.1002/PROT.22510
Abstract: Cytochrome P450-199A2 from Rhodopseudomonas palustris oxidizes para-substituted benzoic acids and may play a role in lignin and aromatic acid degradation pathways in the bacterium. CYP199A2 has an associated [2Fe-2S] ferredoxin, palustrisredoxin (Pux) but not a ferredoxin reductase. A genome search identified the palustrisredoxin reductase (PuR) gene. PuR was produced in Escherichia coli and shown to be a flavin-dependent protein that supports efficient electron transfer from NADH to Pux, thus reconstituting CYP199A2 monooxygenase activity (k(cat) = 37.9 s(-1) with 4-methoxybenzoic acid). The reduction of Pux by PuR shows K(m) = 4.2 microM and k(cat) = 262 s(-1) in 50 mM Tris, pH 7.4. K(m) is increased to 154 microM in the presence of 200 mM KCl, indicating the importance of ionic interactions in PuR/Pux binding. The crystal structure of PuR has been determined at 2.2 A resolution and found to be closely related to that of other oxygenase-coupled NADH-dependent ferredoxin reductases. Residues on the surface that had been proposed to be involved in ferredoxin reductase-ferredoxin binding are conserved in PuR. However, Lys328 in PuR lies over the FAD isoalloxazine ring and, together with His11 and Gln41, render the electrostatic potential of the surface more positive and may account for the greater involvement of electrostatic interactions in ferredoxin binding by PuR. Consistent with these observations the K328G mutation weakened Pux binding and virtually eliminated the dependence of PuR/Pux binding on salt concentration, thus confirming that the FAD si side surface in the vicinity of Lys328 is the ferredoxin binding site.
Publisher: Elsevier BV
Date: 10-2002
Publisher: American Chemical Society (ACS)
Date: 19-08-2019
DOI: 10.1021/JACS.9B06589
Abstract: Protection of biological assemblies is critical to applications in biotechnology, increasing the durability of enzymes in biocatalysis or potentially stabilizing biotherapeutics during transport and use. Here we show that a porous hydrogen-bonded organic framework (HOF) constructed from water-soluble tetra-amidinium (
Publisher: American Chemical Society (ACS)
Date: 14-01-2019
Publisher: Royal Society of Chemistry (RSC)
Date: 2011
DOI: 10.1039/C1DT10098J
Abstract: The substrate-free crystal structure of a five-mutation directed evolution variant of CYP102A1 (P450(BM3)) with generic activity-enhancing properties ("KT2") has been determined to 1.9-Å resolution. There is a close resemblance to substrate-bound structures of the wild-type enzyme (WT). The disruption of two salt bridges that link the G- and I-helices in WT causes conformational changes that break several hydrogen bonds and reduce the angle of the kink in the I-helix where dioxygen activation is thought to take place. The side-chain of a key active site residue, Phe87, is rotated in one molecule of the asymmetric unit, and the side-chains of Phe158 and Phe261 cascade into the orientations found in fatty-acid-bound forms of the enzyme. The iron is out of the porphyrin plane, towards the proximal cysteine. Unusually, the axial water ligand to the haem iron is not hydrogen-bonded to Ala264. The first electron transfer from the reductase domain to the haem domain of substrate-free KT2 is almost as fast as in palmitate-bound WT even though the reduction potential of the haem domain is only slightly more oxidising than that of substrate-free WT. However, NADPH is turned over slowly in the absence of substrate, so the catalytic cycle is gated by a step subsequent to the first electron transfer-a contrast to WT. Propylbenzene binding slightly raises the first electron transfer rate in WT but not in KT2. It is proposed that the generic rate accelerating properties of KT2 arise from the substrate-free form being in a catalytically ready conformation, such that substrate-induced changes to the structure play a less significant role in promoting the first electron transfer than in WT.
Publisher: Oxford University Press (OUP)
Date: 10-2001
DOI: 10.1093/PROTEIN/14.10.797
Abstract: The protein engineering of CYP enzymes for structure-activity studies and the oxidation of unnatural substrates for biotechnological applications will be greatly facilitated by the availability of functional, whole-cell systems for substrate oxidation. We report the construction of a tricistronic plasmid that expresses the CYP101 monooxygenase from Pseudomonas putida, and its physiological electron transfer co-factor proteins putidaredoxin reductase and putidaredoxin in Escherichia coli, giving a functional in vivo catalytic system. Wild-type CYP101 expressed in this system efficiently transforms c hor to 5-exo-hydroxyc hor without further oxidation to 5-oxo-c hor until >95% of c hor has been consumed. CYP101 mutants with increased activity for the oxidation of diphenylmethane (the Y96F-I395G mutant), styrene and ethylbenzene (the Y96F-V247L mutant) have been engineered. In particular, the Y96F-V247L mutant shows coupling efficiency of approximately 60% for styrene and ethylbenzene oxidation, with substrate oxidation rates of approximately 100/min. Escherichia coli cells transformed with tricistronic plasmids expressing these mutants readily gave 100-mg quantities of 4-hydroxydiphenylmethane and 1-phenylethanol in 24-72 h. This new in vivo system can be used for preparative scale reactions for product characterization, and will greatly facilitate directed evolution of the CYP101 enzyme for enhanced activity and selectivity of substrate oxidation.
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C8SC01286E
Abstract: Unusual ferredoxins with different iron–sulfur cluster binding motifs support natural product biosynthesis in a wide range of bacteria.
Publisher: Wiley
Date: 28-12-2018
Abstract: The first total synthesis of bruceol has been achieved using a biomimetic cascade cyclization initiated by a stereoselective Jacobsen-Katsuki epoxidation (and kinetic resolution) of racemic protobruceol-I. A bacterial cytochrome P450 monooxygenase was also found to catalyze the conversion of protobruceol-I into bruceol. The first full analysis of the NMR data of natural bruceol suggested that "isobruceol" was a previously unrecognized natural product also isolated from Philotheca brucei. This was confirmed by the re-isolation, synthesis, and X-ray analysis of isobruceol. In total, eight stereoisomers and structural isomers of bruceol have been synthesized in a highly ergent approach.
Publisher: Wiley
Date: 23-03-2007
Publisher: Wiley
Date: 15-09-2003
DOI: 10.1046/J.1432-1033.2003.03799.X
Abstract: Cytochrome p450BM3 is a self-sufficient fatty acid monooxygenase consisting of a diflavin (FAD/FMN) reductase domain and a heme domain fused together in a single polypeptide chain. The multidomain structure makes it an ideal model system for studying the mechanism of electron transfer and for understanding p450 systems in general. Here we report the redox properties of the cytochrome p450BM3 wild-type holoenzyme, and its isolated FAD reductase and p450 heme domains, when immobilized in a didodecyldimethylammonium bromide film cast on an edge-plane graphite electrode. The holoenzyme showed cyclic voltammetric peaks originating from both the flavin reductase domain and the FeIII/FeII redox couple contained in the heme domain, with formal potentials of -0.388 and -0.250 V with respect to a saturated calomel electrode, respectively. When measured in buffer solutions containing the holoenzyme or FAD-reductase domain, the reductase response could be maintained for several hours as a result of protein reorganization and refreshing at the didodecyldimethylammonium modified surface. When measured in buffer solution alone, the cyclic voltammetric peaks from the reductase domain rapidly diminished in favour of the heme response. Electron transfer from the electrode to the heme was measured directly and at a similarly fast rate (ks' = 221 s-1) to natural biological rates. The redox potential of the FeIII/FeII couple increased when carbon monoxide was bound to the reduced heme, but when in the presence of substrate(s) no shift in potential was observed. The reduced heme rapidly catalysed the reduction of oxygen to hydrogen peroxide.
Publisher: Walter de Gruyter GmbH
Date: 26-05-2017
Abstract: Cytochrome P450s belong to a superfamily of enzymes that catalyse a wide variety of oxidative transformations. Hydroxylation is one the most thoroughly investigated of all identified P450-catalysed reactions whilst dehydrogenation has been relatively much less explored to date. P450-catalysed dehydrogenation is often found to occur with hydroxylation and thus, it was initially suspected to be a stepwise process consisting of hydroxylation and subsequent dehydration to yield the final olefin product. This theory has been proven to be invalid and the olefin was shown to be the direct product of a P450-catalysed reaction. This interesting reaction plays a vital role in the metabolism of xenobiotics and the biosynthesis of endogenous compounds, including a number of steroids. A number of well-known ex les of P450 mediated dehydrogenation, including those in the metabolism of valproic acid, capsaicin and 3-methylindole and those in the biosynthesis of plant and fungal sterols are discussed in this review.
Publisher: Elsevier BV
Date: 06-2018
DOI: 10.1016/J.JINORGBIO.2018.03.001
Abstract: The wild-type cytochrome P450 (CYP) monooxygenase enzyme CYP102A1 (P450Bm3) has low activity for cycloalkane oxidation. The oxidation of these substrates by variants of this enzyme in combination with perfluorinated decoy molecules (PFCs) was investigated to improve productivity. The use of rate accelerating variants, which have mutations located outside of the substrate binding pocket as well as an active site variant of CYP102A1 (A74G/F87V/L188Q) all enhanced cycloalkane oxidation (C5 to C10). The addition of the decoy molecules to the wild-type and the rate accelerating mutants of CYP102A1 boosted the substrate oxidation rates even further. However, the levels of cycloalkanol product decreased with the larger alkanes when the decoy molecules were used with the variant A74G/F87V/L188Q, which contained mutations within the substrate binding pocket. For the majority of the enzymes and PFC decoy molecule combinations the highest levels of oxidation were obtained with cyclooctane. When larger second generation decoy molecules, based on modified amino acids were utilised there was a significant improvement in the oxidation of the smaller cycloalkanes by the wild-type enzyme and one other variant. This resulted in significant improvements in biocatalytic oxidation of cyclopentane and cyclohexane. However, the use of these optimised decoy molecules did not significantly improve cycloalkane oxidation over the fluorinated fatty acid derivatives when combined with the best rate accelerating variant, R47L/Y51F/I401P. Overall our approach enabled the cycloalkanes to be oxidised 300- to 8000-fold more efficiently than the wild-type enzyme at product formation rates in excess of 500 and up to 1700 nmol·nmol-CYP
Publisher: Elsevier BV
Date: 06-2016
Publisher: Royal Society of Chemistry (RSC)
Date: 2014
DOI: 10.1039/C3OB42417K
Abstract: Protected cyclohexanol and cyclohex-2-enol substrates were efficiently and selectively oxidised by different P450cam mutants providing a general methodology for generating substituted diols using biocatalysts.
Publisher: International Union of Crystallography (IUCr)
Date: 30-03-2007
Publisher: Wiley
Date: 26-04-2011
Abstract: The oxidation of o-xylene by P450(BM3) from Bacillus megaterium yields, in addition to the products formed by microsomal P450s, two metabolites containing an NIH-shifted methyl group, one of which lacks the aromatic character of the substrate. The failure of the epoxide precursor of these two products to rearrange to the more stable 2,7-dimethyloxepin suggests that ring opening is P450-mediated. With m-xylene, the principal metabolite is 2,4-dimethylphenol. The partition between aromatic and benzylic hydroxylation is primarily governed by the steric prescriptions of the active site rather than by C-H bond reactivity. It is also substrate-dependent, o- and m-xylene appearing to bind to the enzyme in different orientations. The product distributions given by variants containing the F87A mutation, which creates additional space in the active site, resemble those reported for microsomal systems.
Publisher: Elsevier BV
Date: 03-2006
DOI: 10.1016/J.BBRC.2006.01.133
Abstract: Four (CYP195A2, CYP199A2, CYP203A1, and CYP153A5) of the seven P450 enzymes, and palustrisredoxin A, a ferredoxin associated with CYP199A2, from the metabolically erse bacterium Rhodopseudomonas palustris have been expressed and purified. A range of substituted benzenes, phenols, benzaldehydes, and benzoic acids was shown to bind to the four P450 enzymes. Monooxygenase activity of CYP199A2 was reconstituted with palustrisredoxin A and putidaredoxin reductase of the P450cam system from Pseudomonas putida. We found that 4-ethylbenzoate and 4-methoxybenzoate were oxidized to single products, and 4-methoxybenzoate was demethylated to form 4-hydroxybenzoate. Crystals of substrate-free CYP199A2 which diffracted to approximately 2.0A have been obtained.
Publisher: Bentham Science Publishers Ltd.
Date: 05-2008
DOI: 10.2174/092986608784246470
Abstract: Cytochrome P450 monooxygenases are a superfamily of heme-thiolate proteins involved in the metabolism of a wide variety of endogenous and xenobiotic compounds. The P450 enzyme CYP195A2 from Rhodopseudomonas palustris CGA009, a metabolically versatile bacterium, was overproduced in E. coli and purified. Two distinct crystal forms were obtained under separately optimized conditions by the hanging-drop vapor-diffusion method. Native data sets extending to resolutions of 2.3 A and 2.8 A have been collected and processed in space groups P222 and C2221 respectively.
Publisher: Wiley
Date: 2013
DOI: 10.1002/BAB.1084
Abstract: CYP238A1, one of the two P450 enzymes in the genome of Pseudomonas putida KT2440, has been produced heterologously in Escherichia coli, purified, and found to bind acyclic and cyclic terpene alcohols such as farnesol, nerolidol, linalool, and terpineol. The other P450 enzyme in this organism (gene locus: PP1950) was also produced in E. coli but no substrate has been identified from a limited screen. A phthalate family oxygenase reductase (PFOR) encoded by the PP1957 gene, just downstream of the PP1955 gene for CYP238A1, accepts electrons from the reduced form of both nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate and is able to support monooxygenase activity of CYP238A1, both in vitro and in E. coli, in which both enzymes are produced. CYP238A1 oxidizes cis- and trans-nerolidol to the 9-hydroxy product, with no evidence of attack at the olefinic double bonds. The NADH turnover rate of 170 nmol(nmol-P450)⁻¹ Min⁻¹ for CYP238A1 with cis-nerolidol as substrate at a PP1957:CYP238A1 concentration ratio of 8:1 suggests that this PFOR could function as the physiological redox partner for CYP238A1. The physiological role of CYP238A1 may be related to the PP1955 gene being part of an island/cluster of inducible genes associated with energy metabolism and response to xenobiotics.
Publisher: Elsevier BV
Date: 05-2019
Publisher: Elsevier BV
Date: 10-1997
Publisher: Elsevier BV
Date: 12-2007
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C5CC09247G
Abstract: The addition of perfluorinated fatty acids to the rate accelerating KT2 mutant of P450Bm3 resulted in the highly active oxidation of cyclohexane and benzenes whilst maintaining the product selectivity.
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C4MB00665H
Abstract: The structure of CYP109B1 from Bacillus subtilis , which catalyses the oxidation of ionones, has been determined. This will allow the future design of more efficient biocatalytic monooxygenase systems.
Publisher: Elsevier BV
Date: 12-2003
Publisher: American Chemical Society (ACS)
Date: 31-01-2018
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D3CY01223A
Publisher: Wiley
Date: 02-08-2023
Abstract: The cytochrome P450 (CYP) superfamily of monooxygenase enzymes play important roles in the metabolism of molecules which contain heterocyclic, aromatic functional groups. Here we study how oxygen‐ and sulfur‐containing heterocyclic groups interact with and are oxidized using the bacterial enzyme CYP199A4. This enzyme oxidized both 4‐(thiophen‐2‐yl)benzoic acid and 4‐(thiophen‐3‐yl)benzoic acid almost exclusively via sulfoxidation. The thiophene oxides produced were activated towards Diels‐Alder dimerization after sulfoxidation, forming dimeric metabolites. Despite X‐ray crystal structures demonstrating that the aromatic carbon atoms of the thiophene ring were located closer to the heme than the sulfur, sulfoxidation was still favoured with 4‐(thiophen‐3‐yl)benzoic acid. These results highlight a preference of this cytochrome P450 enzyme for sulfoxidation over aromatic hydroxylation. Calculations predict a strong preference for homodimerization of the enantiomers of the thiophene oxides and the formation of a single major product, in broad agreement with the experimental data. 4‐(Furan‐2‐yl)benzoic acid was oxidized to 4‐(4′‐hydroxybutanoyl)benzoic acid using a whole‐cell system. This reaction proceeded via a γ‐keto‐α,β‐unsaturated aldehyde species which could be trapped in vitro using semicarbazide to generate a pyridazine species. The combination of the enzyme structures, the biochemical data and theoretical calculations provides detailed insight into the formation of the metabolites formed from these heterocyclic compounds.
Publisher: American Chemical Society (ACS)
Date: 19-11-2019
DOI: 10.1021/JACS.9B08064
Abstract: The highly strained cubylmethyl radical undergoes one of the fastest radical rearrangements known (reported
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C8CE00746B
Abstract: Nanoscale structuralisation is demonstrated to influence the stability and catalytic properties of zeolitic imidazolate framework-8.
Publisher: Royal Society of Chemistry (RSC)
Date: 07-02-2002
DOI: 10.1039/B110957J
Abstract: The haem monooxygenase cytochrome P450cam has been engineered to oxidise the gaseous alkanes butane and propane to butan-2-ol and propan-2-ol, respectively, by the use of bulky amino acid substitutions to reduce the volume of the substrate pocket and thus improve the enzyme-substrate fit: the F87W/Y96F/T101L/V247L mutant oxidizes butane with a turnover rate of 750 min-1 and 95% yield based on NADH consumed while the wild-type enzyme has an activity of 0.4 min-1 with 4% yield.
Publisher: American Chemical Society (ACS)
Date: 23-08-2016
Publisher: American Chemical Society (ACS)
Date: 12-01-2022
Publisher: Wiley
Date: 05-08-2016
Publisher: Wiley
Date: 09-11-2022
Abstract: Cytochrome P450 (CYP) heme‐thiolate monooxygenases catalyze the hydroxylation of the C−H bonds of organic molecules. This reaction is initiated by a ferryl‐oxo heme radical cation (Cpd I). These enzymes can also catalyze sulfoxidation reactions and the ferric‐hydroperoxy complex (Cpd 0) and the Fe(III)‐H 2 O 2 complex have been proposed as alternative oxidants for this transformation. To investigate this, the oxidation of 4‐alkylthiobenzoic acids and 4‐methoxybenzoic acid by the CYP199A4 enzyme from Rhodopseudomonas palustris HaA2 was compared using both monooxygenase and peroxygenase pathways. By examining mutants at the mechanistically important, conserved acid alcohol‐pair (D251N, T252A and T252E) the relative amounts of the reactive intermediates that would form in these reactions were disturbed. Substrate binding and X‐ray crystal structures helped to understand changes in the activity and enabled an attempt to evaluate whether multiple oxidants can participate in these reactions. In peroxygenase reactions the T252E mutant had higher activity towards sulfoxidation than O ‐demethylation but in the monooxygenase reactions with the WT enzyme the activity of both reactions was similar. The peroxygenase activity of the T252A mutant was greater for sulfoxidation reactions than the WT enzyme, which is the reverse of the activity changes observed for O ‐demethylation. The monooxygenase activity and coupling efficiency of sulfoxidation and oxidative demethylation were reduced by similar degrees with the T252A mutant. These observations infer that while Cpd I is required for O ‐dealkylation, another oxidant may contribute to sulfoxidation. Based on the activity of the CYP199A4 mutants it is proposed that this is the Fe(III)‐H 2 O 2 complex which would be more abundant in the peroxide‐driven reactions.
Publisher: Royal Society of Chemistry (RSC)
Date: 2012
DOI: 10.1039/C2DT30783A
Abstract: The crystal structures of the 4-methoxybenzoate bound forms of cytochrome P450 enzymes CYP199A2 and CYP199A4 from the Rhodopseudomonas palustris strains CGA009 and HaA2 have been solved. The structures of these two enzymes, which share 86% sequence identity, are very similar though some differences are found on the proximal surface. In these structures the enzymes have a closed conformation, in contrast to the substrate-free form of CYP199A2 where an obvious substrate access channel is observed. The switch from an open to a closed conformation arises from pronounced residue side-chain movements and alterations of ion pair and hydrogen bonding interactions at the entrance of the access channel. A chloride ion bound just inside the protein surface caps the entrance to the active site and protects the substrate and the heme from the external solvent. In both structures the substrate is held in place via hydrophobic and hydrogen bond interactions. The methoxy group is located over the heme iron, accounting for the high activity and selectivity of these enzymes for oxidative demethylation of the substrate. Mutagenesis studies on CYP199A4 highlight the involvement of hydrophobic (Phe185) and hydrophilic (Arg92, Ser95 and Arg243) amino acid residues in the binding of para-substituted benzoates by these enzymes.
Publisher: Elsevier BV
Date: 08-2007
DOI: 10.1016/J.BBRC.2007.06.119
Abstract: Twelve of the fifteen potential P450 enzymes from the bacterium Novosphingobium aromaticivorans, which is known to degrade a wide range of aromatic hydrocarbons, have been produced via heterologous expression in Escherichia coli. The enzymes were tested for their ability to bind a range of substrates including polyaromatic hydrocarbons. While two of the enzymes were found to bind aromatic compounds (CYP108D1 and CYP203A2), the others show binding with a variety of compounds including linear alkanes (CYP153C1) and mono- and sesqui-terpenoid compounds (CYP101B1, CYP101C1, CYP101D1, CYP101D2, CYP111A1, and CYP219A1). A 2Fe-2S ferredoxin (Arx-A), which is associated with CYP101D2, was also produced. The activity of five of the P450 enzymes (CYP101B1, CYP101C1, CYP101D1, CYP101D2, and CYP111A2) was reconstituted with Arx-A and putidaredoxin reductase (of the P450cam system from Pseudomonas putida) in a Class I type electron transfer system. Preliminary characterisation of the majority of the substrate oxidation products is reported.
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D3CC04626E
Publisher: Wiley
Date: 27-10-2016
Publisher: No publisher found
Date: 2000
Publisher: American Chemical Society (ACS)
Date: 06-01-2022
Publisher: Royal Society of Chemistry (RSC)
Date: 2020
DOI: 10.1039/D0CY01040E
Abstract: The cytochrome P450 enzymes CYP101B1 and CYP101C1, from a Novosphingobium bacterium, can efficiently hydroxylate hydrocarbon derivatives containing a carbonyl moiety. Cyclic ketones (C9 to C15) were oxidised with contrasting yet high selectivity.
Publisher: Royal Society of Chemistry (RSC)
Date: 2001
DOI: 10.1039/B100290M
Publisher: Elsevier BV
Date: 03-2023
Publisher: Elsevier BV
Date: 11-2008
DOI: 10.1016/J.JMB.2008.08.033
Abstract: CYP199A2, a cytochrome P450 enzyme from Rhodopseudomonas palustris, oxidatively demethylates 4-methoxybenzoic acid to 4-hydroxybenzoic acid. 4-Ethylbenzoic acid is converted to a mixture of predominantly 4-(1-hydroxyethyl)-benzoic acid and 4-vinylbenzoic acid, the latter being a rare ex le of CC bond dehydrogenation of an unbranched alkyl group. The crystal structure of CYP199A2 has been determined at 2.0-A resolution. The enzyme has the common P450 fold, but the B' helix is missing and the G helix is broken into two (G and G') by a kink at Pro204. Helices G and G' are bent back from the extended BC loop and the I helix to open up a clearly defined substrate access channel. Channel openings in this region of the P450 fold are rare in bacterial P450 enzymes but more common in eukaryotic P450 enzymes. The channel is hydrophobic except for the basic residue Arg246 at the entrance, which probably plays a role in the specificity of this enzyme for charged benzoates over neutral phenols and benzenes. The substrate binding pocket is hydrophobic, with Ser97 and Ser247 being the only polar residues. Computer docking of 4-ethylbenzoic acid into the active site suggests that the substrate carboxylate oxygens interact with Ser97 and Ser247, and the beta-methyl group is located over the heme iron by Phe185, the side chain of which is only 6.35 A above the iron in the native structure. This binding orientation is consistent with the observed product profile of exclusive attack at the para substituent. Putidaredoxin of the CYP101A1 system from Pseudomonas putida supports substrate oxidation by CYP199A2 at approximately 6% of the activity of the physiological ferredoxin. Comparison of the heme proximal faces of CYP199A2 and CYP101A1 suggests that charge reversal surrounding the surface residue Leu369 in CYP199A2 may be a significant factor in this low cross-activity.
Publisher: Wiley
Date: 12-08-2013
DOI: 10.1002/PRO.2309
Publisher: Royal Society of Chemistry (RSC)
Date: 2019
DOI: 10.1039/C9CC02060H
Abstract: The monooxygenase, CYP101B1, selectively hydroxylates undistinct methylene C–H bonds in medium to large cycloalkyl rings and can generate oxabicycloundecanol derivatives.
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C6RA11025H
Abstract: The cytochrome P450 enzyme, CYP199A4 oxidised para substituted alkyloxy- and alkyl-cinnamic acids, with high product formation activity.
Publisher: American Chemical Society (ACS)
Date: 12-01-2019
DOI: 10.1021/JACS.8B10302
Abstract: Encapsulation of biomacromolecules in metal-organic frameworks (MOFs) can preserve biological functionality in harsh environments. Despite the success of this approach, termed biomimietic mineralization, limited consideration has been given to the chemistry of the MOF coating. Here, we show that enzymes encapsulated within hydrophilic MAF-7 or ZIF-90 retain enzymatic activity upon encapsulation and when exposed to high temperatures, denaturing or proteolytic agents, and organic solvents, whereas hydrophobic ZIF-8 affords inactive catalase and negligible protection to urease.
Publisher: Elsevier BV
Date: 02-2020
DOI: 10.1016/J.JINORGBIO.2019.110913
Abstract: The cytochrome P450 metalloenzyme (CYP) CYP199A4 from Rhodopseudomonas palustris HaA2 catalyzes the highly efficient oxidation of para-substituted benzoic acids. Here we determined crystal structures of CYP199A4, and the binding and turnover parameters, with different meta-substituted benzoic acids in order to establish which criteria are important for efficient catalysis. When compared to the para isomers, the meta-substituted benzoic acids were less efficiently oxidized. For ex le, 3-formylbenzoic acid was oxidized with lower activity than the equivalent para isomer and 3-methoxybenzoic acid did not undergo O-demethylation by CYP199A4. The structural data highlighted that the meta-substituted benzoic acids bound in the enzyme active site in a modified position with incomplete loss of the distal water ligand of the heme moiety. However, for both sets of isomers the meta- or para-substituent pointed towards, and was in close proximity, to the heme iron. The absence of oxidation activity with 3-methoxybenzoic acid was assigned to the observation that the CH bonds of this molecule point away from the heme iron. In contrast, in the para isomer they are in an ideal location for abstraction. These findings were confirmed by using the bulkier 3-ethoxybenzoic acid as a substrate which removed the water ligand and reoriented the meta-substituent so that the methylene hydrogens pointed towards the heme, enabling more efficient oxidation. Overall we show relatively small changes in substrate structure and position in the active site can have a dramatic effect on the activity.
Publisher: International Union of Crystallography (IUCr)
Date: 20-04-2007
Publisher: Springer Science and Business Media LLC
Date: 04-06-2015
DOI: 10.1038/NCOMMS8240
Abstract: Enhancing the robustness of functional biomacromolecules is a critical challenge in biotechnology, which if addressed would enhance their use in pharmaceuticals, chemical processing and biostorage. Here we report a novel method, inspired by natural biomineralization processes, which provides unprecedented protection of biomacromolecules by encapsulating them within a class of porous materials termed metal-organic frameworks. We show that proteins, enzymes and DNA rapidly induce the formation of protective metal-organic framework coatings under physiological conditions by concentrating the framework building blocks and facilitating crystallization around the biomacromolecules. The resulting biocomposite is stable under conditions that would normally decompose many biological macromolecules. For ex le, urease and horseradish peroxidase protected within a metal-organic framework shell are found to retain bioactivity after being treated at 80 °C and boiled in dimethylformamide (153 °C), respectively. This rapid, low-cost biomimetic mineralization process gives rise to new possibilities for the exploitation of biomacromolecules.
Publisher: Elsevier BV
Date: 03-2019
DOI: 10.1016/J.ABB.2018.12.025
Abstract: This study has evaluated the use of the P450 metalloenzymes CYP176A1, CYP101A1 and CYP102A1, together with engineered protein variants of CYP101A1 and CYP102A1, to alter the regioselectivity of 1,8- and 1,4-cineole hydroxylation. CYP176A1 was less selective for 1,4-cineole oxidation when compared to its preferred substrate, 1,8-cineole. The CYP102A1 variants significantly improved the activity over the WT enzyme for oxidation of 1,4- and 1,8-cineole. The CYP102A1 R47L/Y51F/A74G/F87V/L188Q mutant generated predominantly (1S)-6α-hydroxy-1,8-cineole (78% e.e.) from 1,8-cineole. Oxidation of 1,4-cineole by the CYP102A1 R47L/Y51F/F87A/I401P variant generated the 3α product in >90% yield. WT CYP101A1 formed a mixture metabolites with 1,8-cineole and very little product was generated with 1,4-cineole. In contrast the F87W/Y96F/L244A/V247L and F87W/Y96F/L244A variants of CYP101A1 favoured formation of 5α-hydroxy-1,8-cineole (>88%, 1S 86% e.e.) while the F87V/Y96F/L244A variant generated (1S)-6α-hydroxy-1,8-cineole in excess (90% regioselective, >99% e.e.). The CYP101A1 F87W/Y96F/L244A/V247L and F87W/Y96F/L244A mutants improved the oxidation of 1,4-cineole generating an excess of the 3α metabolite (1S > 99% e.e. with the latter). The CYP101A1 F87L/Y96F variant also improved the oxidation of this substrate but shifted the site of oxidation to the isopropyl group, (8-hydroxy-1,4-cineole). When this 8-hydroxy metabolite was generated in significant quantities desaturation of C8C9 to the corresponding alkene was also detected.
Publisher: American Chemical Society (ACS)
Date: 23-04-2005
DOI: 10.1021/AC048151Y
Abstract: The underlying electron-transfer and coupled chemical processes associated with biologically important catalytic reactions can be resolved using a combination of Fourier transform ac voltammetry with an analysis of the separated dc and ac components. This outcome can be achieved because the response associated with generation of the catalytic current is essentially confined to the steady-state dc component, whereas the electron-transfer step is dominant in the fundamental and higher harmonics. For the mediated oxidation of glucose with glucose oxidase, it was found that the underlying reversible redox chemistry of the mediator, ferrocenemonocarboxylic acid, as detected in the third and higher harmonics, was totally unaffected by introduction of the catalytic process. In contrast, for the catalytic reduction of molecular oxygen by cytochrome P450, slight changes in the P450 redox process were detected when the catalytic reaction was present. Simulations of a simple catalytic reaction scheme support the fidelity of this novel FT ac voltammetric approach for examining mechanistic nuances of catalytic forms of electrochemical reaction schemes.
Publisher: Elsevier BV
Date: 08-2010
Publisher: Royal Society of Chemistry (RSC)
Date: 2009
DOI: 10.1039/B913487E
Abstract: CYP199A4 (RPB3613) from Rhodopseudomonas palustris HaA2 is a heme monooxygenase that catalyzes the hydroxylation of para-substituted benzoic acids. Monooxygenase activity of CYP199A4 can be reconstituted in a Class I electron transfer chain with an associated [2Fe-2S] ferredoxin, HaPux, (RPB3614) and the flavin-dependent reductase, HaPuR, (RPB3656) that is not associated with a CYP gene. CYP199A4 and the ferredoxin HaPux are produced in greater quantities using recombinant Escherichia coli expression systems when compared to the equivalent proteins in the closely related CYP199A2-Pux-PuR Class I system from R. palustris CGA009. HaPuR and HaPux can also replace PuR and Pux in supporting the CYP199A2 enzyme turnover with high activity. Whole-cell in vivo substrate oxidation systems for CYP199A4 and CYP199A2 with HaPux and HaPuR as the electron transfer proteins have been constructed. These E. coli systems were capable of selectively demethylating veratric acid at the para position to produce vanillic acid at rates of up to 15.3 microM (g-cdw)(-1) min(-1) and yields of up to 1.2 g L(-1).
Publisher: Royal Society of Chemistry (RSC)
Date: 2012
DOI: 10.1039/C2CP24112A
Abstract: Palustrisredoxin-B (PuxB) from Rhodopseudomonas palustris (CGA009) is a [2Fe-2S] ferredoxin which is able to accept electrons from NADH via the flavin-dependent palustrisredoxin reductase (PuR) these electrons can then be transferred to the P450 enzyme (CYP199A2). This work reports on the paramagnetic state of the [2Fe-2S](+) cluster in PuxB, both alone and in the PuR-PuxB complex. Aided by the X-ray crystal structure of PuxB, the protons nearest to the reduced [2Fe-2S](+) cluster were used as magnetic probes to quantify the g-matrix orientation and anisotropic magnetic moment of the paramagnetic centre. (1)H hyperfine couplings were measured with W-band Davies ENDOR and X-band HYSCORE spectroscopy and fitted to a model in which (1)H dipolar couplings were calculated assuming point magnetic moments located at the Fe ions, and bridging and coordinating cysteine sulfur atoms. The absolute sign of a (1)H hyperfine coupling was measured using a variable mixing time ENDOR experiment to confirm the assignment of the Fe(3+) and Fe(2+) ions. For the anti-ferromagnetically coupled cluster the magnetic moment is described in terms of spin projection factors, and our analysis yields values of K(exp)(A) = +2.33 to +1.85 (ferric site), and K(exp)(B) = -1.33 to -0.85 (ferrous site). These values are discussed in terms of the delocalisation of the spin density and hence the limitations of applying a local site spin coupling model to calculate the spin projection factors in a complex with considerable overlap of the α- and β-spin magnetic oribitals. The accurate description of the g-matrix orientation and magnetic moment of this [2Fe-2S](+) cluster enable it to be utilised as a paramagnetic spin probe, for ex le, to measure electron-electron distances. In the pdb reference frame of PuxB (code ) the g(∥) axis vector is g(∥) = [-0.6524 ± 0.0248, -0.6269 ± 0.0115, 0.4259 ± 0.0405], with the principal g-values of g(⊥) = 1.9328 ± 0.0003, g(∥) = 2.0233 ± 0.0003.
Publisher: Elsevier BV
Date: 03-2023
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C7CY00088J
Abstract: Oxidation of polyaromatic hydrocarbons by P450s can be lowered by redox cycling but CYP101B1 regioselectively hydroxylated substituted naphthalenes and biphenyls.
Publisher: Elsevier BV
Date: 07-2019
DOI: 10.1016/J.ABB.2019.05.007
Abstract: Frankia bacteria are nitrogen fixing species from the Actinobacterium phylum which live on the root nodules of plants. They have been hypothesised to have significant potential for natural product biosynthesis. The cytochrome P450 monooxygenase complement of Frankia sp. EuI1c (Frankia inefficax sp.), which comprises 68 members, was analysed. Several members belonged to previously uncharacterised bacterial P450 families. There was an unusually high number of CYP189 family members (21) suggesting that this family has undergone gene duplication events which are classified as "blooms". The likely electron transfer partners for the P450 enzymes were also identified and analysed. These consisted of predominantly [3Fe-4S] cluster containing ferredoxins (eight), a single [2Fe-2S] ferredoxin and a couple of ferredoxin reductases. Three of these CYP family members were produced and purified, using Escherichia coli as a host, and their substrate range was characterised. CYP1027H1 and CYP150A20 bound a broad range of norisoprenoids and terpenoids. CYP1074A2 was highly specific for certain steroids including testosterone, progesterone, stanolone and 4-androstene-3,17-dione. It is likely that steroids are the physiological substrates of CYP1074A2. These results also give an indication that terpenoids are the likely substrates of CYP1027H1 and CYP150A2. The large number of P450s belonging to distinct families as well as the associated electron transfer partners found in different Frankia strains highlights the importance of this family of enzymes has in the secondary metabolism of these bacteria.
Publisher: Elsevier BV
Date: 02-2017
DOI: 10.1016/J.ABB.2016.12.014
Abstract: A self-sufficient CYP102 family encoding gene (Krac_9955) has been identified from the bacterium Ktedonobacter racemifer DSM44963 which belongs to the Chloroflexi phylum. The characterisation of the substrate range of this enzyme was h ered by low levels of production using E. coli. The yield and purity of the Krac9555 enzyme was improved using a codon optimised gene, the introduction of a tag and modification of the purification protocol. The heme domain was isolated and in vitro analysis of substrate binding and turnover was performed. Krac9955 was found to preferentially bind alkyl- and alkyloxy-benzoic acids (≥95% high spin, K
Publisher: Royal Society of Chemistry (RSC)
Date: 2012
DOI: 10.1039/C1CS15192D
Abstract: P450(BM3) (CYP102A1), a fatty acid hydroxylase from Bacillus megaterium, has been extensively studied over a period of almost forty years. The enzyme has been redesigned to catalyse the oxidation of non-natural substrates as erse as pharmaceuticals, terpenes and gaseous alkanes using a variety of engineering strategies. Crystal structures have provided a basis for several of the catalytic effects brought about by mutagenesis, while changes to reduction potentials, inter-domain electron transfer rates and catalytic parameters have yielded functional insights. Areas of active research interest include drug metabolite production, the development of process-scale techniques, unravelling general mechanistic aspects of P450 chemistry, methane oxidation, and improving selectivity control to allow the synthesis of fine chemicals. This review draws together the disparate research themes and places them in a historical context with the aim of creating a resource that can be used as a gateway to the field.
Publisher: Elsevier BV
Date: 06-2016
DOI: 10.1016/J.BBAGEN.2016.01.023
Abstract: Two self-sufficient CYP102 family encoding genes (Krac_0936 and Krac_9955) from the bacterium Ktedonobacter racemifer DSM44963, which possesses one of the largest bacterial genomes, have been identified. Phylogenetic analysis of both the encoded cytochrome P450 enzymes, Krac0936 and Krac9955. Both enzymes were produced and their turnovers with fatty acid substrates assessed in vitro and using a whole-cell oxidation system. Krac0936 hydroxylated straight chain, saturated fatty acids predominantly at the ω-1 and ω-2 positions using NADPH as the cofactor. Krac0936 was less active towards shorter unsaturated fatty acids but longer unsaturated acids were efficiently oxidised. cis,cis-9,12-Octadecadienoic and pentadecanoic acids were the most active substrates tested with Krac0936. Unusually Krac9955 showed very low levels of NAD(P)H oxidation activity though coupling of the reducing equivalents to product formation was high. The product distribution of tridecanoic, tetradecanoic and pentadecanoic acid oxidation by Krac9955 favoured oxidation at the ω-4, ω-5 and ω-6 positions, respectively. Krac0936 and Krac9955 are self-sufficient P450 monooxygenases. Krac0936 has a preference for pentadecanoic acid over other straight chain fatty acids and showed little or no activity with dodecanoic or octadecanoic acids. Krac9955 preferably oxidised shorter fatty acids compared to Krac0936 with tridecanoic having the highest levels of product formation. Unlike Krac0936 and P450Bm3, Krac9995 showed lower activities with unsaturated fatty acids. In this study of two of the CYP enzymes from K. racemifer we have shown that this bacterium from the Chloroflexi phylum contains genes which encode new proteins with novel activity.
Publisher: Wiley
Date: 06-2017
Publisher: Portland Press Ltd.
Date: 16-02-2018
DOI: 10.1042/BCJ20170946
Abstract: Members of the cytochrome P450 monooxygenase family CYP268 are found across a broad range of Mycobacterium species including the pathogens Mycobacterium avium, M. colombiense, M. kansasii, and M. marinum. CYP268A2, from M. marinum, which is the first member of this family to be studied, was purified and characterised. CYP268A2 was found to bind a variety of substrates with high affinity, including branched and straight chain fatty acids (C10–C12), acetate esters, and aromatic compounds. The enzyme was also found to bind phenylimidazole inhibitors but not larger azoles, such as ketoconazole. The monooxygenase activity of CYP268A2 was efficiently reconstituted using heterologous electron transfer partner proteins. CYP268A2 hydroxylated geranyl acetate and trans-pseudoionone at a terminal methyl group to yield (2E,6E)-8-hydroxy-3,7-dimethylocta-2,6-dien-1-yl acetate and (3E,5E,9E)-11-hydroxy-6,10-dimethylundeca-3,5,9-trien-2-one, respectively. The X-ray crystal structure of CYP268A2 was solved to a resolution of 2.0 Å with trans-pseudoionone bound in the active site. The overall structure was similar to that of the related phytanic acid monooxygenase CYP124A1 enzyme from Mycobacterium tuberculosis, which shares 41% sequence identity. The active site is predominantly hydrophobic, but includes the Ser99 and Gln209 residues which form hydrogen bonds with the terminal carbonyl group of the pseudoionone. The structure provided an explanation on why CYP268A2 shows a preference for shorter substrates over the longer chain fatty acids which bind to CYP124A1 and the selective nature of the catalysed monooxygenase activity.
Publisher: Elsevier BV
Date: 08-2020
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C5RA08730A
Abstract: CYP199A4, a cytochrome P450 enzyme from Rhodopseudomonas palustris HaA2, is able to efficiently demethylate a range of benzoic acids at the para -position. It can also catalyse demethenylation reactions.
Publisher: Elsevier BV
Date: 06-2016
DOI: 10.1016/J.BBAPAP.2016.03.006
Abstract: The cytochrome P450 enzyme CYP199A4 can efficiently demethylate 4-methoxybenzoic acid. The substrate is positioned in the enzyme active site with the methoxy group ideally positioned for demethylation. This occurs through interactions of hydrophobic benzene ring with aromatic phenylalanine residues and the charged carboxylate group with polar and basic amino acids. In vitro substrate binding and kinetic turnover assays coupled with HPLC and GC-MS analysis and whole-cell oxidation turnovers. Modification of the carboxylate group to an amide or aldehyde resulted in substrate binding, as judged by the almost total shift of the spin state to the high-spin form, but binding was three orders of magnitude weaker. Changing the carboxylate to phenol alcohol, ketone, ester and nitro groups and boronic, sulfinic and sulfonic acids resulted in a dramatic reduction in the binding affinity. Even phenylacetic acids were mediocre substrates for CYP199A4, despite maintaining a carboxylate group. The weaker binding of all of these substrates results in lower levels of turnover activity and product formation compared to 4-methoxybenzoic acid. Substrate binding to CYP199A4 is tightly regulated by interactions between the 4-methoxybenzoic acid and the amino acids in the active site. The benzoic acid carboxylate moiety is critical for optimal substrate binding and turnover activity with CYP199A4. An understanding of how the CYP199A4 enzyme has evolved to be highly selective for para-substituted benzoic acids. This provides valuable insight into how other, as yet structurally uncharacterised, monooxygenase enzymes may bind benzoic acid substrates.
Publisher: Wiley
Date: 07-11-2012
Abstract: The cytochrome P450 enzyme CYP199A4, from Rhodopseudomonas palustris HaA2, can efficiently demethylate 4-methoxybenzoic acid. It is also capable of oxidising a range of other related substrates. By investigating substrates with different substituents and ring systems we have been able to show that the carboxylate group and the nature of the ring system and the substituent are all important for optimal substrate binding and activity. The structures of the veratric acid, 2-naphthoic acid and indole-6-carboxylic acid substrate-bound CYP199A4 complexes reveal the substrate binding modes and the side-chain conformational changes of the active site residues to accommodate these larger substrates. They also provide a rationale for the selectivity of product oxidation. The oxidation of alkyl substituted benzoic acids by CYP199A4 is more complex, with desaturation reactions competing with hydroxylation activity. The structure of 4-ethylbenzoic acid-bound CYP199A4 revealed that the substrate is held in a similar position to 4-methoxybenzoic acid, and that the C(β) C-H bonds of the ethyl group are closer to the heme iron than those of the C(α) (3.5 vs. 4.8 Å). This observation, when coupled to the relative energies of the reaction intermediates, indicates that the positioning of the alkyl group relative to the heme iron may be critical in determining the amount of desaturation that is observed. By mutating a single residue in the active site of CYP199A4 (Phe185) we were able to convert the enzyme into a 4-ethylbenzoic acid desaturase.
Publisher: International Union of Crystallography (IUCr)
Date: 27-07-2011
Publisher: Royal Society of Chemistry (RSC)
Date: 2022
DOI: 10.1039/D2CC04698A
Abstract: Selective O -demethylation of the lignin monoaromatics, syringol and guaiacol, using the peroxygenase activity of two distinct cytochrome P450 enzymes.
Publisher: Wiley
Date: 29-12-2009
Abstract: Metallothioneins (MTs) were discovered more than 50 years ago and identified as low-molecular weight, sulfhydryl-rich proteins that were subsequently found to bind zinc predominantly. The binding of seemingly redox inactive zinc ions allows MT to play a central role in oxidoreductive cellular metabolism, cellular zinc distribution and homeostasis. In this interpretive study, we discuss the interaction of MT with physiologically relevant molecules and its effect on zinc-thiolate bonds. These interactions are linked to recent progress in the functional role of MT in cellular zinc transport, energy production, and protection of the organism against oxidative stress and neurodegenerative diseases.
Publisher: Royal Society of Chemistry (RSC)
Date: 2012
DOI: 10.1039/C2CC35968E
Abstract: A ferredoxin associated with biological Fe-S cluster assembly has been remodelled to transfer electrons to a P450 enzyme and support substrate oxidation at 80% of the physiological ferredoxin activity, opening up the possibility of tailoring ferredoxins to reconstitute the activity of P450 enzymes for which the electron transfer partner proteins are not known.
Publisher: Oxford University Press (OUP)
Date: 24-10-2007
Abstract: We had reported engineering of the heme monooxygenase cytochrome P450cam from Pseudomonas putida with the F87W/Y96F/L244A/V247L mutations for the oxidation of pentachlorobenzene (PeCB), a recalcitrant environmental contaminant, to pentachlorophenol. In order to provide further insights into P450 structure, function and substrate recognition, we have determined the crystal structure of this 4-mutant without a substrate and its complex with PeCB. PeCB is bound face-on to the heme, with a weak Fe--Cl interaction. One PeCB chlorine is located in the cavity generated by the L244A mutation, in striking illustration of the role of this mutation in promoting PeCB binding. The structures also show that the P450(cam) oxygen-binding groove between G248 and T252 is flexible and can tolerate significant deviations from their conformations in the wild type without loss of enzyme activity. Analysis of the PeCB binding interactions led to introduction of the T101A mutation to enable the substrate to reorient during the catalytic cycle for more efficient oxidation. The resultant 5-mutant F87W/Y96F/T101A/L244A/V247L is 3-fold more active for PeCB oxidation than the 4-mutant. Polychlorinated benzene binding by the mutants and the partitioning between substrate oxidation and non-productive (uncoupling) side reactions are correlated with the structural data.
Publisher: Wiley
Date: 14-07-2022
Abstract: Cytochrome P450 monooxygenase enzymes are versatile catalysts, which have been adapted for multiple applications in chemical synthesis. Mutation of a highly conserved active site threonine to a glutamate can convert these enzymes into peroxygenases that utilise hydrogen peroxide (H 2 O 2 ). Here, we use the T252E‐CYP199A4 variant to study peroxide‐driven oxidation activity by using H 2 O 2 and urea‐hydrogen peroxide (UHP). We demonstrate that the T252E variant has a higher stability to H 2 O 2 in the presence of substrate that can undergo carbon‐hydrogen abstraction. This peroxygenase variant could efficiently catalyse O ‐demethylation and an enantioselective epoxidation reaction (94 % ee ). Neither the monooxygenase nor peroxygenase pathways of the P450 demonstrated a significant kinetic isotope effect (KIE) for the oxidation of deuterated substrates. These new peroxygenase variants offer the possibility of simpler cytochrome P450 systems for selective oxidations. To demonstrate this, a light driven H 2 O 2 generating system was used to support efficient product formation with this peroxygenase enzyme.
Publisher: Royal Society of Chemistry (RSC)
Date: 2003
DOI: 10.1039/B300869J
Publisher: Portland Press Ltd.
Date: 06-2003
DOI: 10.1042/BST0310558
Abstract: We have a continuing interest in applying the current knowledge of cytochrome P450cam substrate recognition to engineer the enzyme for the biotransformation of unnatural substrates with the long-term aim of applications in the synthesis of fine chemicals and bioremediation of environmental contaminants. Comparisons of the structure of target substrates with that of c hor, the natural substrate, led to the design of active-site mutants with greatly enhanced activity for the oxidation of chlorinated benzenes and selectivity of (+)-α-pinene oxidation. The crystal structures of the F87W/Y96F/V247L mutant with 1,3,5-trichlorobenzene or (+)-α-pinene bound have revealed the enzyme–substrate contacts and provided insights into the activity and selectivity patterns. The structures have also provided a novel basis for further engineering of P450cam for increased activity in the oxidation of the highly inert pentachlorobenzene and hexachlorobenzene, and increased selectivity of (+)-α-pinene oxidation.
Publisher: Elsevier BV
Date: 06-2020
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C5CC07577G
Abstract: Recent studies have demonstrated that metal–organic frameworks can be employed as protective coatings for enzymes.
Publisher: IOP Publishing
Date: 13-03-2009
DOI: 10.1088/0031-9155/54/7/017
Abstract: The development of hypoxia-selective radiopharmaceuticals for use as therapeutic and/or imaging agents is of vital importance for both early identification and treatment of cancer and in the design of new drugs. Radiotracers based on copper for use in positron emission tomography have received great attention due to the successful application of copper(II) bis(thiosemicarbazonato) complexes, such as [(60/62/64)Cu(II)ATSM] and [(60/62/64)Cu(II)PTSM], as markers for tumour hypoxia and blood perfusion, respectively. Recent work has led to the proposal of a revised mechanism of hypoxia-selective cellular uptake and retention of [Cu(II)ATSM]. The work presented here describes non-steady-state kinetic simulations in which the reported pO(2)-dependent in vitro cellular uptake and retention of [(64)Cu(II)ATSM] in EMT6 murine carcinoma cells has been modelled by using the revised mechanistic scheme. Non-steady-state (NSS) kinetic analysis reveals that the model is in very good agreement with the reported experimental data with a root-mean-squared error of less than 6% between the simulated and experimental cellular uptake profiles. Estimated rate constants are derived for the cellular uptake and washout (k(1) = 9.8 +/- 0.59 x 10(-4) s(-1) and k(2) = 2.9 +/- 0.17 x 10(-3) s(-1)), intracellular reduction (k(3) = 5.2 +/- 0.31 x 10(-2) s(-1)), reoxidation (k(4) = 2.2 +/- 0.13 mol(-1) dm(3) s(-1)) and proton-mediated ligand dissociation (k(5) = 9.0 +/- 0.54 x 10(-5) s(-1)). Previous mechanisms focused on the reduction and reoxidation steps. However, the data suggest that the origins of hypoxia-selective retention may reside with the stability of the copper(I) anion with respect to protonation and ligand dissociation. In vitro kinetic studies using the nicotimamide adenine dinucleotide (NADH)-dependent ferredoxin reductase enzyme PuR isolated from the bacterium Rhodopseudomonas palustris have also been conducted. NADH turnover frequencies are found to be dependent on the structure of the ligand and the results confirm that the proposed reduction step in the mechanism of hypoxia selectivity is likely to be mediated by NADH-dependent enzymes. Further understanding of the mechanism of hypoxia selectivity may facilitate the development of new imaging and radiotherapeutic agents with increased specificity for tumour hypoxia.
Publisher: Royal Society of Chemistry (RSC)
Date: 2009
DOI: 10.1039/B907010A
Abstract: Double electron-electron resonance (DEER) spectroscopy can determine, from measurement of the dipolar interaction, the distance and orientation between two paramagnetic centres in systems lacking long-range order such as powders or frozen solution s les. In spin systems with considerable anisotropy, the microwave pulses excite only a fraction of the electron paramagnetic resonance (EPR) spectrum and the resulting orientation selection needs to be explicitly taken into account if a meaningful distance and orientation is to be determined. Here, a general method is presented to analyze the dipolar interaction between two paramagnetic spin centres from a series of DEER traces recorded so that different orientations of the spin-spin vector are s led. Delocalised spin density distributions and spin projection factors (as for ex le in iron-sulfur clusters), are explicitly included. Application of the analysis to a spin-labelled flavoprotein reductase/reduced iron-sulfur ferredoxin protein complex and a bi-radical with two Cu(ii) ions provides distance and orientation information between the radical centres. In the protein complex this enables the protein-protein binding geometry to be defined. Experimentally, orientationally selective DEER measurements are possible on paramagnetic systems where the resonator bandwidth allows the frequencies of pump and detection pulses to be separated sufficiently to excite enough orientations to define adequately the spin-spin vector.
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C8SC00825F
Abstract: The surface charge and chemistry of a protein determines its ability to facilitate biomimetic mineralisation.
Publisher: American Chemical Society (ACS)
Date: 02-2021
Publisher: Portland Press Ltd.
Date: 15-12-2010
DOI: 10.1042/BJ20101017
Abstract: The cytochrome P450 CYP101D2 from Novosphingobium aromaticivorans DSM12444 is closely related to CYP101D1 from the same bacterium and to P450cam (CYP101A1) from Pseudomonas putida. All three are capable of oxidizing c hor stereoselectively to 5-exo-hydroxyc hor. The crystal structure of CYP101D2 revealed that the likely ferredoxin-binding site on the proximal face is largely positively charged, similar to that of CYP101D1. However, both the native and c hor-soaked forms of CYP101D2 had open conformations with an access channel. In the active site of the c hor-soaked form, the c hor carbonyl interacted with the haem-iron-bound water. Two other potential c hor-binding sites were also identified from electron densities in the c hor-soaked structure: one located in the access channel, flanked by the B/C and F/G loops and the I helix, and the other in a cavity on the surface of the enzyme near the F helix side of the F/G loop. The observed open structures may be conformers of the CYP101D2 enzyme that enable the substrate to enter the buried active site via a conformational selection mechanism. The second and third binding sites may be intermediate locations of substrate entry and translocation into the active site, and provide insight into a multi-step substrate-binding mechanism.
Publisher: American Chemical Society (ACS)
Date: 06-08-2010
DOI: 10.1021/BI100910Y
Abstract: Cytochrome P450 CypX (CYP134A1), isolated from Bacillus subtilis, has previously been implicated in the three-step oxidative transformation of the diketopiperazine cyclo-l-leucyl-l-leucyl into pulcherriminic acid, a precursor of the extracellular iron chelate pulcherrimin. In this study, we present the first experimental data relating to CYP134A1, where we show that CYP134A1 binds cyclo-l-leucyl-l-leucyl with an affinity of 24.5 +/- 0.5 muM. Structurally related diketopiperazines sharing similar alkyl side chains to cyclo-l-leucyl-l-leucyl also bind to CYP134A1 with comparable affinity. CYP134A1 is capable of catalyzing the in vitro oxidation of diketopiperazine substrates when supported with several alternate electron transfer partner systems. Products containing one additional oxygen atom and which are intermediate products of the expected pulcherriminic acid were identified by GCMS. The oxidation of related diketopiperazines reveals that different oxidative pathways exist for CYP134A1-catalyzed diketopiperazine oxidation. The crystal structure of CYP134A1 has been determined to 2.7 A resolution in the absence of substrate and in the presence of bound phenylimidazole ligands to 3.1 and 3.2 A resolution. The active site is dominated by hydrophobic residues and contains an unusual proline residue in place of the normally conserved alcohol residue that typically plays an important role in oxygen activation. The B-B(2) substrate recognition loop, which forms part of the active site, shows considerable flexibility and was found in both open and closed conformations in different structures. These results represent the first insights into the structural and biochemical basis underlying the multistep oxidation catalyzed by CYP134A1.
Publisher: Wiley
Date: 06-10-2022
Abstract: The cytochrome P450 (CYP) family of heme monooxygenases catalyse the selective oxidation of C-H bonds under ambient conditions. The CYP199A4 enzyme from Rhodopseudomonas palustris catalyses aliphatic oxidation of 4-cyclohexylbenzoic acid but not the aromatic oxidation of 4-phenylbenzoic acid, due to the distinct mechanisms of aliphatic and aromatic oxidation. The aromatic substrates 4-benzyl-, 4-phenoxy- and 4-benzoyl-benzoic acid and methoxy-substituted phenylbenzoic acids were assessed to see if they could achieve an orientation more amenable to aromatic oxidation. CYP199A4 could catalyse the efficient benzylic oxidation of 4-benzylbenzoic acid. The methoxy-substituted phenylbenzoic acids were oxidatively demethylated with low activity. However, no aromatic oxidation was observed with any of these substrates. Crystal structures of CYP199A4 with 4-(3'-methoxyphenyl)benzoic acid demonstrated that the substrate binding mode was like that of 4-phenylbenzoic acid. 4-Phenoxy- and 4-benzoyl-benzoic acid bound with the ether or ketone oxygen atom hydrogen-bonded to the heme aqua ligand. We also investigated whether the substitution of phenylalanine residues in the active site could permit aromatic hydroxylation. Mutagenesis of the F298 residue to a valine did not significantly alter the substrate binding position or enable the aromatic oxidation of 4-phenylbenzoic acid however the F182L mutant was able to catalyse 4-phenylbenzoic acid oxidation generating 2'-hydroxy-, 3'-hydroxy- and 4'-hydroxy metabolites in a 83 : 9 : 8 ratio, respectively. Molecular dynamics simulations, in which the distance and angle of attack were considered, demonstrated that in the F182L variant, in contrast to the wild-type enzyme, the phenyl ring of 4-phenylbenzoic acid attained a productive geometry for aromatic oxidation to occur.
Publisher: Elsevier BV
Date: 05-2023
Publisher: American Chemical Society (ACS)
Date: 06-02-2018
DOI: 10.1021/JACS.7B11056
Abstract: Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of chemically inert carbon-hydrogen bonds in erse endogenous and exogenous organic compounds by atmospheric oxygen. This C-H bond oxy-functionalization activity has huge potential in biotechnological applications. Class I CYPs receive the two electrons required for oxygen activation from NAD(P)H via a ferredoxin reductase and ferredoxin. The interaction of Class I CYPs with their cognate ferredoxin is specific. In order to reconstitute the activity of erse CYPs, structural characterization of CYP-ferredoxin complexes is necessary, but little structural information is available. Here we report a structural model of such a complex (CYP199A2-HaPux) in frozen solution derived from distance and orientation restraints gathered by the EPR technique of orientation-selective double electron-electron resonance (os-DEER). The long-lived oscillations in the os-DEER spectra were well modeled by a single orientation of the CYP199A2-HaPux complex. The structure is different from the two known Class I CYP-Fdx structures: CYP11A1-Adx and CYP101A1-Pdx. At the protein interface, HaPux residues in the [Fe
Publisher: Wiley
Date: 09-10-2017
Abstract: The cytochrome P450 monooxygenase CYP101B1, from a Novosphingobium bacterium is able to bind and oxidise aromatic substrates but at a lower activity and efficiency than norisoprenoids and monoterpenoid esters. Histidine 85 of CYP101B1 aligns with tyrosine 96 of CYP101A1, which, in the latter enzyme forms the only hydrophilic interaction with its substrate, c hor. The histidine residue of CYP101B1 was mutated to phenylalanine with the aim of improving the activity of the enzyme for hydrophobic substrates. The H85F mutant lowered the binding affinity and activity of the enzyme for β-ionone and altered the oxidation selectivity. This variant also showed enhanced affinity and activity towards alkylbenzenes, styrenes and methylnaphthalenes. For ex le the rate of product formation for acenaphthene oxidation was improved sixfold to 245 nmol per nmol CYP per min. Certain disubstituted naphthalenes and substrates, such as phenylcyclohexane and biphenyls, were oxidised with lower activity by the H85F variant. Variants at H85 (A and G) designed to introduce additional space into the active site so as to accommodate these larger substrates did not improve the oxidation activity. As the H85F mutant of CYP101B1 improved the oxidation of hydrophobic substrates, this residue is likely to be in the substrate binding pocket or the access channel of the enzyme. The side chain of the histidine might interact with the carbonyl groups of the favoured norisoprenoid substrates of CYP101B1.
Publisher: American Chemical Society (ACS)
Date: 12-04-2023
DOI: 10.1021/JACS.3C01456
Publisher: Elsevier BV
Date: 07-2023
Publisher: Wiley
Date: 11-11-2022
Abstract: The cytochrome P450 family of monooxygenase enzymes have essential biological roles involving the selective oxidation of carbon‐hydrogen bonds. They can also catalyze other important metabolic reactions including desaturation to form alkenes. Currently the factors that control the partition between P450 hydroxylation and desaturation pathways are poorly defined. The CYP199A4 enzyme from the bacterium Rhodopseudomonas palustris HaA2 catalyzes the oxidation of 4‐ethyl‐ and 4‐isopropyl‐ benzoic acids with hydroxylation and desaturation occurring in significant quantities. Here we demonstrate that 4‐cyclopropylbenzoic acid is regioselectively hydroxylated by CYP199A4 at the benzylic carbon. In contrast, the oxidation of 4‐ n ‐propylbenzoic acid by CYP199A4 results in three major metabolites: an alkene from desaturation and two hydroxylation products at the benzylic (Cα) and Cβ carbons in similar quantities. Extending the length of the alkyl substituent resulted in 4‐ n ‐butylbenzoic acid being oxidized at the benzylic position (45%) and desaturated (55%). In contrast, 4‐isobutylbenzoic generated very little alkene (5%) but was hydroxylated at the benzylic position (54%) and at the tertiary Cβ position (41%). The oxidation of 4‐ n ‐propylbenzoic acid by the F298 V mutant of CYP199A4 occurred with no hydroxylation at Cβ and a significant increase in metabolites arising from desaturation (73%). The X‐ray crystal structures of CYP199A4 with each substrate revealed that they bind in the active site with the alkyl substituent positioned over the heme. However, the longer alkylbenzoic acids were bound in a different conformation as was 4‐ n ‐propylbenzoic acid in the F298 V mutant. Overall, the changes in metabolite distribution could be ascribed to bond strength differences and the position of the alkyl group relative to the heme.
Publisher: American Chemical Society (ACS)
Date: 07-05-2021
Publisher: Wiley
Date: 16-12-2016
Publisher: Wiley
Date: 17-06-2005
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D3CC02312E
Abstract: Cholesterol catabolism is an important survival mechanism for the pathogenic Mycobacterium tuberculosis . Here we demonstrate that the CYP125 family of cytochrome P450 enzymes can catalyse the oxidation of phytosterols as well as cholesterol.
Publisher: Royal Society of Chemistry (RSC)
Date: 2005
DOI: 10.1039/B413068E
Abstract: The sesquiterpenoids are a large class of naturally occurring compounds with biological functions and desirable properties. Oxidation of the sesquiterpene (+)-valencene by wild type and mutants of P450cam from Pseudomonas putida, and of P450BM-3 from Bacillus megaterium, have been investigated as a potential route to (+)-nootkatone, a fine fragrance. Wild type P450cam did not oxidise (+)-valencene but the mutants showed activities up to 9.8 nmol (nmol P450)(-1) min(-1), with (+)-trans-nootkatol and (+)-nootkatone constituting >85% of the products. Wild type P450BM-3 and mutants had higher activities (up to 43 min(-1)) than P450cam but were much less selective. Of the many products, cis- and trans-(+)-nootkatol, (+)-nootkatone, cis-(+)-valencene-1,10-epoxide, trans-(+)-nootkaton-9-ol, and (+)-nootkatone-13S,14-epoxide were isolated from whole-cell reactions and characterised. The selectivity patterns suggest that (+)-valencene has one binding orientation in P450cam but multiple orientations in P450BM-3.
Publisher: Elsevier BV
Date: 02-2019
DOI: 10.1016/J.BBAGEN.2018.11.013
Abstract: Cyp147G1 is one of 47 cytochrome P450 encoding genes in Mycobacterium marinum M, a pathogenic bacterium with a high degree of sequence similarity to Mycobacterium tuberculosis and Mycobacterium ulcerans. Cyp147G1 is one of only two of these cyp genes which are closely associated with a complete electron transfer system. The substrate range of the enzyme was tested in vitro and the activity of CYP147G1 was reconstituted in vivo by co-producing the P450 with the ferredoxin and ferredoxin reductase. Substrates of CYP147G1 include fatty acids ranging from octanoic to hexadecanoic acid. CYP147G1 catalysed the selective hydroxylation of linear and ω-2 methyl branched fatty acids at the ω-1 position (≥ 98%). Oxidation of ω-1 methyl branched fatty acids generated the ω and ω-1 hydroxylation products in almost equal proportions, indicating altered position of hydrogen abstraction. This selectivity of fatty acid hydroxylation inferred that linear species must bind in the active site of the enzyme with the terminal methyl group sequestered so that abstraction at the CH bonds of the ω-1 position is favoured. With branched substrates, one of the methyl groups must be close to the compound I oxygen atom and enable hydroxylation at the terminal methyl group to compete with the reaction at the ω-1CH bond. Hydroxy fatty acids are widely used for industrial, food and medical purposes. CYP147G1 demonstrates high regioselectivity for hydroxylation at a sub-terminal position on a broad range of linear fatty acids, not seen in other CYP enzymes.
Publisher: American Chemical Society (ACS)
Date: 26-07-2022
DOI: 10.1021/ACSINFECDIS.2C00215
Abstract: The steroid binding CYP142 cytochrome P450 enzymes of
Publisher: Elsevier BV
Date: 05-2019
DOI: 10.1016/J.BBAGEN.2019.02.016
Abstract: Actinobacteria, including the Mycobacteria, have a large component of cytochrome P450 family monooxygenases. This includes Mycobacterium tuberculosis, M. ulcerans and M. marinum, and M. vanbaalenii. These enzymes can abstract CH bonds and have important roles in natural product biosynthesis. Two members of the bacterial CYP150 family, CYP150A5 and CYP150A6 from M. marinum, were produced, purified and characterised. The potential substrate ranges of both enzymes were analysed and the monooxygenase activity of CYP150A5 was reconstituted using a physiological electron transfer partner system. CYP150A6 was structurally characterised by X-ray crystallography. CYP150A5 was shown to bind various norisoprenoids and terpenoids. It could regioselectively hydroxylate β-ionol. The X-ray crystal structure of substrate-free CYP150A6 was solved to 1.5 Å. This displayed an open conformation with short F and G helices, an unresolved F-G loop region and exposed active site pocket. The active site residues could be identified and important variations were found among the CYP150A enzymes. Haem-binding azole inhibitors were identified for both enzymes. The structure of CYP150A6 will facilitate the identification of physiological substrates and the design of better inhibitors for members of this P450 family. Based on the observed differences in substrate binding preference and sequence variations among the active site residues, their roles are predicted to be different. Multiple CYP150 family members were found in many bacteria and are prevalent in the Mycobacteria including several human pathogens. Inhibition and structural data are reported here for these enzymes for the first time.
Publisher: Royal Society of Chemistry (RSC)
Date: 1996
DOI: 10.1039/CC9960000357
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2015
End Date: 12-2019
Amount: $772,104.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2017
End Date: 12-2019
Amount: $412,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2020
End Date: 12-2023
Amount: $550,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 03-2021
End Date: 02-2024
Amount: $498,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2014
End Date: 12-2016
Amount: $330,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 03-2023
End Date: 03-2024
Amount: $570,702.00
Funder: Australian Research Council
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