ORCID Profile
0000-0002-0731-2950
Current Organisation
Macquarie University
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Biochemistry and Cell Biology | Genetics | Biochemistry and Cell Biology not elsewhere classified | Systems Biology | Genomics | Medical Biotechnology | Medical Biotechnology Diagnostics (incl. Biosensors) | Food Processing | Biochemistry And Cell Biology Not Elsewhere Classified | Biomedical Instrumentation | Global Change Biology | Biotechnology Not Elsewhere Classified | Agricultural Biotechnology not elsewhere classified | Agricultural Biotechnology | Microbial Genetics | Gene Expression (incl. Microarray and other genome-wide approaches) | Proteomics and Intermolecular Interactions (excl. Medical Proteomics) | Plant Physiology | Analytical Biochemistry | Plant Physiology | Bioprocessing, Bioproduction and Bioproducts | Photonics, Optoelectronics and Optical Communications | Analytical Biochemistry | Bioinformatics | Nanobiotechnology
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Chemical Sciences | Plant Production and Plant Primary Products not elsewhere classified | Biological sciences | Cancer and Related Disorders | Infectious Diseases | Primary products from plants | Barley | Aquaculture Oysters | Nutraceuticals and Functional foods | Clinical Health (Organs, Diseases and Abnormal Conditions) not elsewhere classified | Chemical sciences | Expanding Knowledge in the Medical and Health Sciences | Expanding Knowledge in Technology | Integrated (ecosystem) assessment and management | Solar-Photovoltaic Energy | Diagnostic Methods | Medical Instruments | Marine Flora, Fauna and Biodiversity |
Related Links
Publications
Inhibition of indoleamine 2,3 dioxygenase activity by H2O2
Publisher: Elsevier BV
Date: 06-2006
DOI: 10.1016/J.ABB.2006.03.003
Abstract: Indoleamine 2,3-dioxygenase is the first and rate limiting enzyme of the kynurenine pathway of tryptophan metabolism, has potent effects on cell proliferation and mediates antimicrobial, antitumorogenic, and immunosuppressive effects. As a potent cytotoxic effector, the mechanisms of indoleamine 2,3-dioxygenase inhibition deserve greater attention. The work presented here represents the first systematic study exploring the mechanisms by which low levels of hydrogen peroxide (10-100 microM) inhibit indoleamine 2,3-dioxygenase in vitro. Following brief peroxide exposure both enzyme inhibition and structural changes were observed. Loss of catalysis was accompanied by oxidation of several cysteine residues to sulfinic and sulfonic acids, observed by electrospray and MALDI mass spectrometry. Enzyme activity could in part be preserved in the presence of sulfhydryl containing compounds, particularly DTT and methionine. However, these structural alterations did not prevent substrate (l-tryptophan) binding. Some enzyme activity could be recovered in the presence of thioredoxin, indicating that the inhibitory effect of H(2)O(2) is at least partially reversible in vitro. We present evidence that cysteine oxidation represents one mechanism of indoleamine 2,3-dioxygenase inhibition.
ATP-Induced Conformational Dynamics in the AAA+ Motor Unit of Magnesium Chelatase
Publisher: Elsevier BV
Date: 03-2010
DOI: 10.1016/J.STR.2010.01.001
Abstract: Mg-chelatase catalyzes the first committed step of the chlorophyll biosynthetic pathway, the ATP-dependent insertion of Mg(2+) into protoporphyrin IX (PPIX). Here we report the reconstruction using single-particle cryo-electron microscopy of the complex between subunits BchD and BchI of Rhodobacter capsulatus Mg-chelatase in the presence of ADP, the nonhydrolyzable ATP analog AMPPNP, and ATP at 7.5 A, 14 A, and 13 A resolution, respectively. We show that the two AAA+ modules of the subunits form a unique complex of 3 dimers related by a three-fold axis. The reconstructions demonstrate substantial differences between the conformations of the complex in the presence of ATP and ADP, and suggest that the C-terminal integrin-I domains of the BchD subunits play a central role in transmitting conformational changes of BchI to BchD. Based on these data a model for the function of magnesium chelatase is proposed.
The Mg branch of chlorophyll synthesis
Publisher: Elsevier
Date: 2019
New enzymes from environmental cassette arrays
Publisher: Wiley
Date: 06-2004
DOI: 10.1110/PS.04638704
Construction of a cell-free synthetic pathway for the production of lactic acid from spent coffee grounds
Publisher: Elsevier BV
Date: 10-2018
International comparability in spectroscopic measurements of protein structure by circular dichroism: CCQM-P59.1
Publisher: IOP Publishing
Date: 04-10-2010
The Chlamydomonas reinhardtii gtr gene encoding the tetrapyrrole biosynthetic enzyme glutamyl-tRNA reductase
Publisher: Springer Science and Business Media LLC
Date: 07-2005
DOI: 10.1007/S11103-005-6803-X
Abstract: Plants, algae, cyanobacteria and many other bacteria synthesize the tetrapyrrole precursor, delta-aminolevulinic acid (ALA), from glutamate by means of a tRNAGlu-mediated pathway. The enzyme glutamyl-tRNA reductase (GTR) catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate 1-semialdehyde. Chlamydomonas reinhardtii mRNA encoding gtr was sequenced from a cDNA and genomic libraries. The 3179-bp gtr cDNA contains a 1566-bp open reading frame that encodes a 522-amino acid polypeptide. After removal of the predicted transit peptide, the mature 480-residue GTR has a calculated molecular weight of 52,502. The deduced C. reinhardtii mature GTR amino acid sequence has more than 55% identity to a GTR sequence of Arabidopsis thaliana, and significant similarity to GTR proteins of other plants and prokaryotes. Southern blot analysis of C. reinhardtii genomic DNA indicates that C. reinhardtii has only one gtr gene. Genomic DNA sequencing revealed the presence of a small intron near the putative transit peptide cleavage site. Expression constructs for the full-length initial gtr translation product, the mature protein after transit peptide removal, and the coding sequence of the second exon were cloned into expression vector that also introduced a C-terminal His6 tag. All of these constructs were expressed in E. coli, and both the mature protein and the exon 2 translation product complemented a hemA mutation. The expressed proteins were purified by Ni-affinity column chromatography to yield active GTR. Purified mature GTR was not inhibited by heme, but heme inhibition was restored upon addition of C. reinhardtii soluble proteins.
Biosynthesis of bacteriochlorophylls in purple bacteria
Publisher: Springer Netherlands
Date: 2008
Phytobilin biosynthesis
Publisher: Springer Science and Business Media LLC
Date: 2000
Abstract: The phytobilin chromophores of phycobiliproteins and phytochromes are biosynthesized from heme in a pathway that begins with the opening of the tetrapyrrole macrocycle of protoheme to form biliverdin IXalpha, in a reaction catalyzed by heme oxygenase. An Arabidopsis thaliana hy1 mutant was previously shown to be deficient in phytochrome responses, and these responses were regained when the plants were administered biliverdin IXalpha. A heme oxygenase-encoding gene, ho1, was recently cloned from the cyanobacterium Synechocystis sp. PCC 6803. When ho1 was expressed in Escherichia coli, the cells produced active ferredoxin-dependent soluble heme oxygenase. The open reading frame of ho1 was fused in frame with a chloroplast transit peptide-encoding sequence from the oli gene of Antirrhinum majus. This construct was placed in a binary plasmid vectorcontaining a kanamycin resistance marker and a cauliflower mosaic virus 35S promoter to control expression of the chimeric oli-ho1 gene and used to transform A. thaliana hy1 plants. Two independent transformed lines were obtained that had the phenotype of the parental Landsberg erecta line and expressed the chimeric gene, as indicated by detection of its mRNA by reverse transcriptase-polymerase chain reaction. The results indicate that Synechocystis sp. PCC 6803 heme oxygenase encoded by ho1 can substitute for the defective HY1 gene product and that the only required enzyme activity of the HY1 gene product is heme oxygenase.
Stable Upconversion Nanohybrid Particles for Specific Prostate Cancer Cell Immunodetection
Publisher: Springer Science and Business Media LLC
Date: 22-11-2016
DOI: 10.1038/SREP37533
Abstract: Prostate cancer is one of the male killing diseases and early detection of prostate cancer is the key for better treatment and lower cost. However, the number of prostate cancer cells is low at the early stage, so it is very challenging to detect. In this study, we successfully designed and developed upconversion immune-nanohybrids (UINBs) with sustainable stability in a physiological environment, stable optical properties and highly specific targeting capability for early-stage prostate cancer cell detection. The developed UINBs were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) and luminescence spectroscopy. The targeting function of the biotinylated antibody nanohybrids were confirmed by immunofluorescence assay and western blot analysis. The UINB system is able to specifically detect prostate cancer cells with stable and background-free luminescent signals for highly sensitive prostate cancer cell detection. This work demonstrates a versatile strategy to develop UCNPs based sustainably stable UINBs for sensitive diseased cell detection.
Mammalian forebrain ketimine reductase identified as μ‐crystallin; potential regulation by thyroid hormones
Publisher: Wiley
Date: 15-03-2011
DOI: 10.1111/J.1471-4159.2011.07220.X
Abstract: Ketimine reductase (E.C. 1.5.1.25) was purified to apparent homogeneity from lamb forebrain by means of a rapid multi-step chromatography protocol. The purified enzyme was identified by MS/MS (mass spectrometry) as μ-crystallin. The identity was confirmed by heterologously expressing human μ-crystallin in Escherichia coli and subsequent chromatographic purification of the protein. The purified human μ-crystallin was confirmed to have ketimine reductase activity with a maximum specific activity similar to that of native ovine ketimine reductase, and was found to catalyse a sequential reaction. The enzyme substrates are putative neuromodulator/transmitters. The thyroid hormone 3,5,3'-l-triiodothyronine (T3) was found to be a strong reversible competitive inhibitor, and may have a novel role in regulating their concentrations. μ-Crystallin is also involved in intracellular T3 storage and transport. This research is the first to demonstrate an enzyme function for μ-crystallin. This newly demonstrated enzymatic activity identifies a new role for thyroid hormones in regulating mammalian amino acid metabolism, and a possible reciprocal role of enzyme activity regulating bioavailability of intracellular T3.
Synthesis of stably deuteriated abscisic acid, phaseic acid and related compounds
Publisher: Elsevier BV
Date: 1991
Hydroxylation of the C132 and C18 carbons of chlorophylls by heme and molecular oxygen
Publisher: World Scientific Pub Co Pte Lt
Date: 09-2015
DOI: 10.1142/S1088424615500571
Abstract: Following extraction from photosynthetic organisms, chlorophylls are prone to reactions including demetalation, dephytylation and specific oxidations of the exocyclic ring E, termed allomerizations. Allomerization of chlorophylls has been well-characterized in methanol and to a lesser extent in aqueous solution. Here we detail novel allomerization-like reactions of chlorophyll a and chlorophyll b. In the presence of heme, detergent-solubilized chlorophyll a is hydroxylated at its C 13 2 position in ring E and, surprisingly, the C 18 position in ring D. Two major oxidation products are synthesized — a C 13 2 - OH and a C 13 2 - OH , C 18- OH derivative of chlorophyll a. We track the origin of the oxygen atoms added in these hydroxylated chlorophylls using 18 O 2 labeling and demonstrate that the additional oxygen atoms are derived from molecular oxygen. A similar heme-catalyzed reaction is also observed using chlorophyll b as a substrate. These results highlight the need for care when dealing with extracted chlorophylls and demonstrate an unusual hydroxylation of the C 18 position of chlorophylls in the presence of heme.
In vitro Conversion of Vinyl to Formyl Groups in Naturally Occurring Chlorophylls
Publisher: Springer Science and Business Media LLC
Date: 14-08-2014
DOI: 10.1038/SREP06069
A bacterial genome assembly and annotation laboratory using a virtual machine
Publisher: Wiley
Date: 03-03-2023
DOI: 10.1002/BMB.21720
Abstract: With the global increase of infections caused by antibiotic‐resistant bacterial strains, there is an urgent need for new methods of tackling the issue. Genomic analysis of bacterial strains can help to understand their virulence and antibiotic resistance profile. Bioinformatic skills are in great demand across the biological sciences. We designed a workshop that allows university students to learn the process of genome assembly using command‐line tools within a virtual machine on a Linux operating system. We use Illumina and Nanopore short and long‐read raw sequences to reveal the advantages and disadvantages of short, long, and hybrid assembly methods. The workshop teaches how to assess read and assembly quality, perform genome annotation, and analyze pathogenicity, antibiotic and phage resistance. The workshop is intended for a five‐week teaching period and is concluded by a student poster presentation assessment.
Rapid extraction of RNA and analysis of transcript levels in Chlamydomonas reinhardtii using real-time RT-PCR
Publisher: Springer Science and Business Media LLC
Date: 2003
Barley Grain Proteomics
Publisher: Elsevier
Date: 2017
The Evolution of Far-Red Light Perception in Acaryochloris Marina, a Chlorophyll d-Containing Cyanobacterium
Publisher: Springer Berlin Heidelberg
Date: 2013
Making light of a dark situation
Publisher: Springer Science and Business Media LLC
Date: 1999
DOI: 10.1038/16161
Characterisation of the first archaeal mannonate dehydratase from Thermoplasma acidophilum and its potential role in the catabolism of D-Mannose
Publisher: MDPI AG
Date: 03-03-2019
DOI: 10.3390/CATAL9030234
Abstract: Mannonate dehydratases catalyse the dehydration reaction from mannonate to 2-keto-3-deoxygluconate as part of the hexuronic acid metabolism in bacteria. Bacterial mannonate dehydratases present in this gene cluster usually belong to the xylose isomerase-like superfamily, which have been the focus of structural, biochemical and physiological studies. Mannonate dehydratases from archaea have not been studied in detail. Here, we identified and characterised the first archaeal mannonate dehydratase (TaManD) from the thermoacidophilic archaeon Thermoplasma acidophilum. The recombinant TaManD enzyme was optimally active at 65 °C and showed high specificity towards D-mannonate and its lactone, D-mannono-1,4-lactone. The gene encoding for TaManD is located adjacent to a previously studied mannose-specific aldohexose dehydrogenase (AldT) in the genome of T. acidophilum. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that the mannose-specific AldT produces the substrates for TaManD, demonstrating the possibility for an oxidative metabolism of mannose in T. acidophilum. Among previously studied mannonate dehydratases, TaManD showed closest homology to enzymes belonging to the xylose isomerase-like superfamily. Genetic analysis revealed that closely related mannonate dehydratases among archaea are not located in a hexuronate gene cluster like in bacteria, but next to putative aldohexose dehydrogenases, implying a different physiological role of mannonate dehydratases in those archaeal species.
18 O Labeling of Chlorophyll d in Acaryochloris marina Reveals That Chlorophyll a and Molecular Oxygen Are Precursors
Publisher: Elsevier BV
Date: 09-2010
Configurations and conformations of abscisic acid
Publisher: Elsevier BV
Date: 08-1993
A proteomic approach to the identification and characterisation of protein composition in wheat germ
Publisher: Springer Science and Business Media LLC
Date: 12-01-2006
DOI: 10.1007/S10142-005-0018-8
Abstract: Proteome analyses were carried out on commercial wheat germ of mature grain from the biscuit-making wheat cultivar, Rosella. Wheat germ protein extracts were fractionated by two-dimensional gel electrophoresis across two different immobilised pH gradients: pH 4.0-7.0 and 6.0-9.0. A total of 612 in idual protein spots were excised from the gels and characterised by peptide mass fingerprinting. From these analyses, 347 in idual proteins were identified from protein sequence database interrogation, and 301 different types of protein were catalogued according to protein function. The remaining 265 protein spots gave poor or no matches to proteins in the databases and were not identified in this study. Six different classes of enzymes were identified in the germ, many of them having roles in the mobilisation of energy reserves for germination. Abundantly expressed enzyme classes include the oxidoreductases, transferases and hydrolases. A comparison was also made between the major protein classes expressed in the germ and protein classes expressed in the endosperm from previous proteomic work. This study contributes significantly to our knowledge of protein expression and heterogeneity in the germ of wheat grain and forms the basis for future studies in regard to the characterisation of proteins during the initial stages of germination.
ATPase activity of magnesium chelatase subunit I is required to maintain subunit D in vivo
Publisher: Wiley
Date: 10-05-2004
Kinetic analyses of the magnesium chelatase provide insights into the mechanism, structure, and formation of the complex
Publisher: Elsevier BV
Date: 11-2008
Extinction coefficient for red-shifted chlorophylls: Chlorophyll d and chlorophyll f
Publisher: Elsevier BV
Date: 08-2012
DOI: 10.1016/J.BBABIO.2012.02.026
Abstract: Both chlorophyll f and chlorophyll d are red-shifted chlorophylls in oxygenic photosynthetic organisms, which extend photon absorbance into the near infrared region. This expands the range of light that can be used to drive photosynthesis. Quantitative determination of chlorophylls is a crucial step in the investigation of chlorophyll-photosynthetic reactions in the field of photobiology and photochemistry. No methods have yet been worked out for the quantitative determination of chlorophyll f. There is also no method available for the precise quantitative determination of chlorophyll d although it was discovered in 1943. In order to obtain the extinction coefficients (ε) of chlorophyll f and chlorophyll d, the concentrations of chlorophylls were determined by Inductive Coupled Plasma Mass Spectrometry according to the fact that each chlorophyll molecule contains one magnesium (Mg) atom. Molar extinction coefficient ε(chl f) is 71.11×10(3)Lmol(-1)A(707nm)cm(-1) and ε(chl d) is 63.68×10(3)Lmol(-1)A(697nm)cm(-1) in 100% methanol. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.
Magnesium-protoporphyrin chelatase of Rhodobacter sphaeroides
Publisher: Proceedings of the National Academy of Sciences
Date: 14-03-1995
Abstract: Magnesium-protoporphyrin chelatase lies at the branch point of the heme and (bacterio)chlorophyll biosynthetic pathways. In this work, the photosynthetic bacterium Rhodobacter sphaeroides has been used as a model system for the study of this reaction. The bchH and the bchI and -D genes from R. sphaeroides were expressed in Escherichia coli. When cell-free extracts from strains expressing BchH, BchI, and BchD were combined, the mixture was able to catalyze the insertion of Mg into protoporphyrin IX in an ATP-dependent manner. This was possible only when all three genes were expressed. The bchH, -I, and -D gene products are therefore assigned to the Mg chelatase step in bacteriochlorophyll biosynthesis. The mechanism of the Mg chelation reaction and the implications for chlorophyll biosynthesis in plants are discussed.
Abscisic aldehyde
Publisher: Elsevier BV
Date: 08-1992
The C21-formyl group in chlorophyll f originates from molecular oxygen
Publisher: Elsevier BV
Date: 11-2017
C-terminal residues of Oryza sativa GUN4 are required for the activation of the ChlH subunit of magnesium chelatase in chlorophyll synthesis
Publisher: Wiley
Date: 02-01-2012
DOI: 10.1016/J.FEBSLET.2011.12.026
Abstract: Oryza sativa GUN4 together with the magnesium chelatase subunits ChlI, ChlD, and ChlH have been heterologously expressed and purified to reconstitute magnesium chelatase activity in vitro. Maximum magnesium chelatase activity requires pre-activation of OsChlH with OsGUN4, Mg(2+) and protoporphyrin-IX. OsGUN4 and OsChlH preincubated without protoporphyrin-IX yields magnesium chelatase activity similar to assays without OsGUN4, suggesting formation of a dead-end complex. Either 9 or 10 C-terminal amino acids of OsGUN4 are slowly hydrolyzed to yield a truncated OsGUN4. These truncated OsGUN4 still bind protoporphyrin-IX and Mg-protoporphyrin-IX but are unable to activate OsChlH. This suggests the mechanism of GUN4 activation of magnesium chelatase is different in eukaryotes compared to cyanobacteria as the orthologous cyanobacterial GUN4 proteins lack this C-terminal extension.
Chlorophyll synthesis
Publisher: Springer Netherlands
Date: 2006
The stereochemistry of the hydrogen atoms at C-5′of abscisic acid
Publisher: Elsevier BV
Date: 1989
Genome and proteome of the chlorophyll f-producing cyanobacterium Halomicronema hongdechloris: adaptative proteomic shifts under different light conditions.
Publisher: Springer Science and Business Media LLC
Date: 12-03-2019
Strategic Distribution of Protective Proteins within Bran Layers of Wheat Protects the Nutrient-Rich Endosperm
Publisher: Oxford University Press (OUP)
Date: 08-01-2010
Abstract: Bran from bread wheat (Triticum aestivum ‘Babbler’) grain is composed of many outer layers of dead maternal tissues that overlie living aleurone cells. The dead cell layers function as a barrier resistant to degradation, whereas the aleurone layer is involved in mobilizing organic substrates in the endosperm during germination. We microdissected three defined bran fractions, outer layers (epidermis and hypodermis), intermediate fraction (cross cells, tube cells, testa, and nucellar tissue), and inner layer (aleurone cells), and used proteomics to identify their in idual protein complements. All proteins of the outer layers were enzymes, whose function is to provide direct protection against pathogens or improve tissue strength. The more complex proteome of the intermediate layers suggests a greater ersity of function, including the inhibition of enzymes secreted by pathogens. The inner layer contains proteins involved in metabolism, as would be expected from live aleurone cells, but this layer also includes defense enzymes and inhibitors as well as 7S globulin (specific to this layer). Using immunofluorescence microscopy, oxalate oxidase was localized predominantly to the outer layers, xylanase inhibitor protein I to the xylan-rich nucellar layer of the intermediate fraction and pathogenesis-related protein 4 mainly to the aleurone. Activities of the water-extractable enzymes oxalate oxidase, peroxidase, and polyphenol oxidase were highest in the outer layers, whereas chitinase activity was found only in assays of whole grains. We conclude that the differential protein complements of each bran layer in wheat provide distinct lines of defense in protecting the embryo and nutrient-rich endosperm.
Inactivation of Mg chelatase during transition from anaerobic to aerobic growth in Rhodobacter capsulatus
Publisher: American Society for Microbiology
Date: 06-2003
DOI: 10.1128/JB.185.11.3249-3258.2003
Abstract: The facultative photosynthetic bacterium Rhodobacter capsulatus can adapt from an anaerobic photosynthetic mode of growth to aerobic heterotrophic metabolism. As this adaptation occurs, the cells must rapidly halt bacteriochlorophyll synthesis to prevent phototoxic tetrapyrroles from accumulating, while still allowing heme synthesis to continue. A likely control point is Mg chelatase, the enzyme that erts protoporphyrin IX from heme biosynthesis toward the bacteriochlorophyll biosynthetic pathway by inserting Mg 2+ to form Mg-protoporphyrin IX. Mg chelatase is composed of three subunits that are encoded by the bchI , bchD , and bchH genes in R . capsulatus . We report that BchH is the rate-limiting component of Mg chelatase activity in cell extracts. BchH binds protoporphyrin IX, and BchH that has been expressed and purified from Escherichia coli is red in color due to the bound protoporphyrin IX. Recombinant BchH is rapidly inactivated by light in the presence of O 2 , and the inactivation results in the formation of a covalent adduct between the protein and the bound protoporphyrin IX. When photosynthetically growing R . capsulatus cells are transferred to aerobic conditions, Mg chelatase is rapidly inactivated, and BchH is the component that is most rapidly inactivated in vivo when cells are exposed to aerobic conditions. The light- and O 2 -stimulated inactivation of BchH could account for the rapid inactivation of Mg chelatase in vivo and provide a mechanism for inhibiting the synthesis of bacteriochlorophyll during adaptation of photosynthetically grown cells to aerobic conditions while still allowing heme synthesis to occur for aerobic respiration.
Characterisation of protein isolates prepared from processed mungbean (Vigna radiata) flours
Publisher: Canadian Center of Science and Education
Date: 15-11-2017
DOI: 10.5539/JAS.V9N12P1
Abstract: Pulses are normally processed prior to consumption, are high in protein, providing opportunities for improving nutritional qualities of food. Processing methods can modify nutritional properties, affecting protein content, composition and functionality. In this study, we investigated the effect of processing methods on protein concentration and yield from protein isolates prepared by alkaline solubilisation and isoelectric precipitation, from whole seed flour, raw dahl flour, roasted dahl flour and germinated flour of mungbean. Protein isolates contained protein contents ranging from 87.3 to 90.4 g 100 g-1. The effect of processing methods on protein solubility characteristics was evident, as protein yields ranged from 75.7 (whole seed flour), 80.8 (raw dahl flour), 27.1 (roasted dahl flour) and 65.9 g 100 g-1 (germinated seed flour). Essential amino acid content was higher in protein isolates prepared from germinated flour, at 30.03 g 100 g-1 (39.1% of amino acids), compared to raw dahl flour, at 27.08 g 100 g-1 (38.3% of amino acids). A comparative proteomic analysis of protein isolates, prepared from raw dahl and germinated flour, resulted in the inferred identification of 214 proteins from protein sequence databases, in which, 72 proteins were classified as being common, 42 proteins specific to raw dahl flour and 28 proteins specific to germinated flour. Processing methods such as roasting and germination can significantly alter flour protein solubility, consequently, impacting on overall efficiency of protein extraction. The comparative proteomic analysis used in this study proved to be useful for investigating changes in protein composition and relative abundance, highlighting the potential in applying this technology for further characterisation of modified protein fractions for food applications.
BchJ and BchM interact in a 1:1 ratio with the magnesium chelatase BchH subunit of Rhodobacter capsulatus
Publisher: Wiley
Date: 19-10-2010
DOI: 10.1111/J.1742-4658.2010.07877.X
Abstract: Substrate channeling between the enzymatic steps in the (bacterio)chlorophyll biosynthetic pathway catalyzed by magnesium chelatase (BchI/ChlI, BchD/ChlD and BchH/ChlH subunits) and S-adenosyl-L-methionine:magnesium-protoporphyrin IX O-methyltransferase (BchM/ChlM) has been suggested. This involves delivery of magnesium-protoporphyrin IX from the BchH/ChlH subunit of magnesium chelatase to BchM/ChlM. Stimulation of BchM/ChlM activity by BchH/ChlH has previously been shown, and physical interaction of the two proteins has been demonstrated. In plants and cyanobacteria, there is an added layer of complexity, as Gun4 serves as a porphyrin (protoporphyrin IX and magnesium-protoporphyrin IX) carrier, but this protein does not exist in anoxygenic photosynthetic bacteria. BchJ may play a similar role to Gun4 in Rhodobacter, as it has no currently assigned function in the established pathway. Purified recombinant Rhodobacter capsulatus BchJ and BchM were found to cause a shift in the equilibrium amount of Mg-protoporphyrin IX formed in a magnesium chelatase assay. Analysis of this shift revealed that it was always in a 1 : 1 ratio with either of these proteins and the BchH subunit of the magnesium chelatase. The establishment of the new equilibrium was faster with BchM than with BchJ in a coupled magnesium chelatase assay. BchJ bound magnesium-protoporphyrin IX or formed a ternary complex with BchH and magnesium-protoporphyrin IX. These results suggest that BchJ may play a role as a general magnesium porphyrin carrier, similar to one of the roles of GUN4 in oxygenic organisms.
Recessiveness and Dominance in Barley Mutants Deficient in Mg-Chelatase Subunit D, an AAA Protein Involved in Chlorophyll Biosynthesis
Publisher: Oxford University Press (OUP)
Date: 12-2006
Abstract: Mg-chelatase catalyzes the insertion of Mg2+ into protoporphyrin IX at the first committed step of the chlorophyll biosynthetic pathway. It consists of three subunits: I, D, and H. The I subunit belongs to the AAA protein superfamily (ATPases associated with various cellular activities) that is known to form hexameric ring structures in an ATP-dependant fashion. Dominant mutations in the I subunit revealed that it functions in a cooperative manner. We demonstrated that the D subunit forms ATP-independent oligomeric structures and should also be classified as an AAA protein. Furthermore, we addressed the question of cooperativity of the D subunit with barley (Hordeum vulgare) mutant analyses. The recessive behavior in vivo was explained by the absence of mutant proteins in the barley cell. Analogous mutations in Rhodobacter capsulatus and the resulting D proteins were studied in vitro. Mixtures of wild-type and mutant R. capsulatus D subunits showed a lower activity compared with wild-type subunits alone. Thus, the mutant D subunits displayed dominant behavior in vitro, revealing cooperativity between the D subunits in the oligomeric state. We propose a model where the D oligomer forms a platform for the stepwise assembly of the I subunits. The cooperative behavior suggests that the D oligomer takes an active part in the conformational dynamics between the subunits of the enzyme.
Mutation of cysteine residues alters the heme-binding pocket of indoleamine 2,3-dioxygenase-1
Publisher: Elsevier BV
Date: 07-2013
DOI: 10.1016/J.BBRC.2013.05.119
Abstract: The hemoprotein indoleamine 2,3-dioxygenase-1 (IDO1) is the first and rate-limiting enzyme in mammalian tryptophan metabolism. Interest in IDO1 continues to grow, due to the ever expanding influence IDO1 plays in the immune response. This study examined the contribution of all in idual cysteine residues towards the overall catalytic properties and stability of recombinant human IDO1 via mutagenesis studies using a range of biochemical and spectroscopic techniques, including in vitro kinetic assessment, secondary structure identification via circular dichroism spectroscopy and thermal stability assessment. Upon mutation of cysteine residues we observed changes in secondary structure (principally, shifting from α-helix/β-sheet features to random coil structures) that produced out of plane heme torsion and puckering, changes to thermal stability (including gains in stability for one mutant protein) and differences in enzymatic activity (such as, increased ability to convert non-natural substrates, e.g.d-tryptophan) from wild type IDO1 enzyme.
Synthesis and properties of C-1-azido-aba
Publisher: Elsevier BV
Date: 03-1993
Crystallization and preliminary X-ray analysis of theRhodobacter capsulatusmagnesium chelatase BchI subunit
Publisher: International Union of Crystallography (IUCr)
Date: 03-1999
DOI: 10.1107/S0907444998014759
Abstract: The Rhodobacter capsulatus BchI protein is one of three subunits of Mg chelatase, the enzyme which catalyzes the first committed step of chlorophyll and bacteriochlorophyll biosynthesis. The BchI protein was produced with an inducible T7 RNA polymerase expression system in Escherichia coli. The protein was purified from the soluble cell-extract fraction and crystallized from polyethylene glycol solution. The crystals diffract to a minimum Bragg spacing of 2.1 A. The space group is P63 with unit-cell dimensions a = b = 90.6, c = 84.1 A.
Structure of GUN4 from Chlamydomonas reinhardtii
Publisher: International Union of Crystallography (IUCr)
Date: 29-07-2015
DOI: 10.1107/S2053230X15012248
Abstract: The genomes uncoupled 4 (GUN4) protein stimulates chlorophyll biosynthesis by increasing the activity of Mg-chelatase, the enzyme that inserts magnesium into protoporphyrin IX (PPIX) in the chlorophyll biosynthesis pathway. One of the roles of GUN4 is in binding PPIX and Mg-PPIX. In eukaryotes, GUN4 also participates in plastid-to-nucleus signalling, although the mechanism for this is unclear. Here, the first crystal structure of a eukaryotic GUN4, from Chlamydomonas reinhardtii , is presented. The structure is in broad agreement with those of previously solved cyanobacterial structures. Most interestingly, conformational ergence is restricted to several loops which cover the porphyrin-binding cleft. The conformational dynamics suggested by this ensemble of structures lend support to the understanding of how GUN4 binds PPIX or Mg-PPIX.
The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions
Publisher: American Association for the Advancement of Science (AAAS)
Date: 12-10-2007
Abstract: Chlamydomonas reinhardtii is a unicellular green alga whose lineage erged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.
Black Point is associated with reduced levels of stress, disease- and defence-related proteins in wheat grain
Publisher: Wiley
Date: 07-04-2006
DOI: 10.1111/J.1364-3703.2006.00330.X
Abstract: SUMMARY Black Point in wheat is a dark discoloration at the embryo end of the grain, which causes substantial financial losses to wheat growers due to down-grading of otherwise high-grade wheat. There does not appear to be a single cause for Black Point, although evidence suggests that fungal infection is the main link to Black Point symptoms. We sought to identify grain proteins from Black Point-affected and Black Point-free wheat cultivar SUN239V, which is known to be very susceptible to Black Point. The proteomes of both the germ and endosperm-bran components of Black Point-affected and Black Point-free grain were compared using two-dimensional gel electrophoresis (2-DE) with six replicate gels run for each protein s le. Approximately 1478 discrete protein spots were found in 2-DE gels from the germ fraction of the grain, of which 354 were identified by mass spectrometry (MS). Similarly, 1360 discrete protein spots were found from the endosperm-bran fraction, of which 303 were identified by MS. No proteins of fungal or bacterial origin were positively identified, suggesting that, at least in some cases, Black Point is not associated with microbial activity. Of the germ proteins, 252 were differentially expressed in Black Point-affected tissue, with 67 of these proteins identified by MS. Of the endosperm-bran proteins, 317 were differentially expressed in Black Point-affected tissue, with 86 identified. The largest of 12 functional classes to which the differentially abundant proteins were assigned was the 'stress' class, i.e. products of genes associated with stress, disease and defence. Higher levels of these proteins were found in Black Point-free grain, suggesting that protection from the disease might be afforded by increased levels of the 'stress' proteins.
Effect of processing on Mungbean (Vigna radiata) flour nutritional properties and protein composition
Publisher: Canadian Center of Science and Education
Date: 15-10-2018
Abstract: Pulses are traditionally processed prior to consumption, providing opportunities for modifying nutritional composition, dependant on the type of pulse and method used. In this study, we investigated the effect of whole seed, dehulling (dahl), germination and roasting on changes in mungbean flour nutritional properties, protein composition and relative protein abundance. Processed flours were analysed and compared for protein content, moisture, fat, ash, dietary fibre, total starch and amylose. Significant differences were imparted on dietary fibre content, with roasting and germination increasing the ratio of insoluble/soluble fibre as well as resistant starch. Comparative proteomic analysis resulted in a combined total of 539 protein identifications, searching against the Mungbean reference genome (NCBI Vigna radiata Annotation Release 100). Normalised spectral abundance factors were used as a measure of relative abundance and statistical analysis was applied (Students& rsquo T-Test), where proteins with a p-value of & 0.05 considered significantly different. Processing imparted considerable changes to nutritional composition and should be further exploited for food applications. The comparative proteomic analyses carried out in this study proved useful for investigating the effect of processing on subsequent changes in protein composition and relative abundance.
The isolation and identification of the prosthetic group released from a bound form of abscisic acid
Publisher: Springer Science and Business Media LLC
Date: 08-1992
DOI: 10.1007/BF00024572
A cyanobacterium that contains chlorophyllf- a red-absorbing photopigment
Publisher: Wiley
Date: 13-07-2012
DOI: 10.1016/J.FEBSLET.2012.06.045
Abstract: A Chl f-containing filamentous cyanobacterium was purified from stromatolites and named as Halomicronema hongdechloris gen., sp. nov. after its phylogenetic classification and the morphological characteristics. Hongdechloris contains four main carotenoids and two chlorophylls, a and f. The ratio of Chl f to Chl a is reversibly changed from 1:8 under red light to an undetectable level of Chl f under white-light culture conditions. Phycobiliproteins were induced under white light growth conditions. A fluorescence emission peak of 748 nm was identified as due to Chl f. The results suggest that Chl f is a red-light inducible chlorophyll.
Bilin-dependent regulation of chlorophyll biosynthesis by GUN4
Publisher: Proceedings of the National Academy of Sciences
Date: 11-05-2021
Abstract: Enzymes of the chlorophyll biosynthetic pathway, which bind protoporphyrin and Mg-porphyrins, are susceptible to damage by singlet oxygen production in the presence of light and oxygen. These studies show that heme-derived linear tetrapyrroles (bilins) both stimulate and protect the protoporphyrin-binding CHLH subunit of Mg chelatase, the first committed enzyme of the chlorophyll synthesis, from self-sensitized photodamage and turnover via formation of nonphotosensitizing GENOMES UNCOUPLED 4 (GUN4):bilin:porphyrin adducts, which deliver protoporphyrin to CHLH. GUN4:bilin adducts likely evolved to sustain chlorophyll biosynthesis in an oxic world, accounting for retention of bilin synthesis in nearly all oxygenic photosynthetic species on Earth.
Sensitive Time-Gated Immunoluminescence Detection of Prostate Cancer Cells Using a TEGylated Europium Ligand
Publisher: American Chemical Society (ACS)
Date: 15-09-2016
DOI: 10.1021/ACS.ANALCHEM.6B02191
Abstract: We describe the application of a synthetically developed tetradentate β-diketonate-europium chelate with high quantum yield (39%), for sensitive immunodetection of prostate cancer cells (DU145). MIL38 antibody, a mouse monoclonal antibody against Glypican 1, conjugated directly to the chelate via lysine residues, resulted in soluble (hydrophilic) and stable immunoconjugates. Indirect labeling of the antibody by a europium chelated secondary polyclonal antibody and a streptavidin/biotin pair was also performed. All of these bright luminescent conjugates were used to stain DU145 cells, a prostate cancer cell line, using time gated luminescence microscopy for imaging, and their performances were compared to conventional FITC labeling. For all prepared conjugates, the europium chelate in conjunction with a gated autosynchronous luminescence detector (GALD) completely suppressed the cellular autofluorescence background to allow capture of vivid, high contrast images of immune-stained cancer cells.
Inducing the oxidative stress response in Escherichia coli improves the quality of a recombinant protein
Publisher: Elsevier BV
Date: 09-2014
DOI: 10.1016/J.PEP.2014.06.004
Abstract: The ∼150kDa ChlH subunit of magnesium chelatase from Oryza sativa, Hordeum vulgare and Chlamydomonas reinhardtii have been heterologously expressed in Escherichiacoli. The active soluble protein is found as both a multimeric and a monomeric form. The multimeric ChlH appears to be oxidatively damaged but monomer production is favoured in growth conditions that are known to cause an oxidative stress response in E.coli. Inducing an oxidative stress response may be of general utility to improve the quality of proteins expressed in E. coli. The similar responses of ChlH's from the three different species suggest that oligomerization of oxidatively damaged ChlH may have a functional role in the chloroplast, possibly as a signal of oxidative stress or damage.
Nucleotides of tRNA (Glu) involved in recognition by barley chloroplast glutamyl-tRNA synthetase and glutamyl-tRNA reductase
Publisher: Elsevier BV
Date: 09-1995
DOI: 10.1016/0167-4781(95)00105-P
Abstract: The biosynthesis of delta-aminolevulinate (ALA), via the C-5 pathway, requires tRNA(Glu) as a cofactor for the glutamyl tRNA(Glu) synthetase and the glutamyl tRNA(Glu) reductase which are the first two enzymes in this three step pathway. These two enzymes form a ternary complex with the tRNA(Glu) in Chlamydomonas reinhardtii suggesting that the recognition elements on the tRNA cofactor are different for each enzyme. Chemical modification and comparative studies with tRNA(Glu)s from a number of species were used to determine the nucleotides involved in the recognition of the barley chloroplast tRNA(Glu) by the barley enzymes. The barley chloroplast tRNA(Glu) is chemically modified both before and after ligation to glutamate with monobromobimane or CNBr. The chemically modified tRNA(Glu) is a poor substrate for the glutamyl-tRNA synthetase and the chemically modified glutamyl-tRNA(Glu) is used as a substrate for glutamyl-tRNA(Glu) reductase. The tRNA(Glu) from the chloroplasts if barley, Chlamydomonas reinhardtii, tobacco, cucumber, wheat and spinach and tRNA(Glu) from Synechocystis PCC6803, Escherichia coli, barley germ and bakers yeast and the barley chloroplast tRNA(Gln) are all effective substrates for the barley chloroplast glutamyl-tRNA synthetase. A comparison of the sequences of these tRNAs shows 19 conserved bases and five of these bases, G10, A26, U34, U35 and A37 are suggested as recognition elements of barley glutamyl tRNA(Glu) synthetase by assuming a similar binding orientation as in the crystal structure of the E. coli tRNA(Gln) GlnRS complex. The glutamyl-tRNA(Glu) from E. coli, bakers yeast and barley germ and the barley chloroplast glutamyl-tRNA(Gln) are not effective substrates for the barley chloroplast glutamyl-tRNA(Glu) reductase. A comparison of the sequences of these four tRNA species with the sequences of the tRNA(Glu) species that can be used as substrate by the glutamyl-tRNA(Glu) reductase yields seven common differences in the primary sequence. These 7 nucleotides, A7-U66, U29-A41, A53-U61, and U72 are expected to be required for recognition by the barley chloroplast glutamyl-tRNA(Glu) reductase.
Novel indoleamine 2,3-dioxygenase-1 inhibitors from a multistep in silico screen
Publisher: Elsevier BV
Date: 02-2012
DOI: 10.1016/J.BMC.2011.10.068
Abstract: Indoleamine 2,3-dioxygenase-1 (IDO-1) is a heme containing enzyme that catalyses the initial step in the major pathway of l-tryptophan catabolism the kynurenine pathway. A large body of evidence has been accumulating for its immunosuppressive and tumoural escape roles and its applicability as a therapeutic target. Of particular interest is the possibility that IDO-1 inhibition may arrest, and sometimes revert, tumour growth. There exists a continuing need for the development of new and specific inhibitors for IDO-1, and we have created three pharmacophores designed to aid in this search. Initial database hits were further screened using Kier flexibility and a 'What-If' docking technique, designed to overcome the inherent limitations of today's forcefields with regards to heme chemistry. Eighteen compounds were tested in vitro, yielding four novel inhibitors with low micromolar IC(50) values, comparable with current inhibitors.
Optimised expression and purification of recombinant human indoleamine 2,3-dioxygenase
Publisher: Elsevier BV
Date: 10-2004
Chlorophyll-deficient mutants of Chlamydomonas reinhardtii that accumulate magnesium protoporphyrin IX
Publisher: Springer Science and Business Media LLC
Date: 03-02-2010
Cell-free enzymatic conversion of spent coffee grounds into the platform chemical lactic acid
Publisher: Frontiers Media SA
Date: 03-12-2019
Phytobilin biosynthesis
Publisher: Wiley
Date: 07-1998
DOI: 10.1046/J.1365-313X.1998.00186.X
Abstract: The phytobilin chromophores of phycobiliproteins and phytochromes are biosynthesized from heme in a pathway that begins with the opening of the tetrapyrrole macrocycle of protoheme to form biliverdin IX alpha, in a reaction catalyzed by heme oxygenase. A gene containing an open reading frame with a predicted polypeptide that has a sequence similar to that of a conserved region of animal microsomal heme oxygenases was identified in the published genomic sequence of Synechocystis sp. PCC 6803. This gene, named ho1, was cloned and expressed in Escherichia coli under the control of the lacZ promoter. Cells expressing the gene became green colored due to the accumulation of biliverdin IX alpha. The size of the expressed protein was equal to the predicted size of the Synechocystis gene product, named HO1. Heme oxygenase activity was assayed in incubations containing extract of transformed E. coli cells. Incubations containing extract of induced cells, but not those containing extract of uninduced cells, had ferredoxin-dependent heme oxygenase activity. With mesoheme as the substrate, the reaction product was identified as mesobiliverdin IX alpha by spectrophotometry and reverse-phase HPLC. Heme oxygenase activity was not sedimented by centrifugation at 100, 000 g. Expression of HO1 increased several-fold during incubation of the cells for 72 h in iron-deficient medium.
Mechanism, structure, and regulation of magnesium chelatase
Publisher: Elsevier
Date: 2003
Tetrapyrrole biosynthesis and signaling (chlorophyll, heme, and bilins)
Publisher: Elsevier
Date: 2023
Substrate-binding Model of the Chlorophyll Biosynthetic Magnesium Chelatase BchH Subunit
Publisher: Elsevier BV
Date: 04-2008
1-N-histidine phosphorylation of ChlD by the AAA⁺ ChlI2 stimulates magnesium chelatase activity in chlorophyll synthesis
Publisher: Portland Press Ltd.
Date: 09-06-2017
DOI: 10.1042/BCJ20161094
Abstract: Magnesium chelatase (Mg-chelatase) inserts magnesium into protoporphyrin during the biosynthesis of chlorophyll and bacteriochlorophyll. Enzyme activity is reconstituted by forming two separate preactivated complexes consisting of a GUN4/ChlH rotoporphyrin IX substrate complex and a ChlI/ChlD enzyme ‘motor’ complex. Formation of the ChlI/ChlD complex in both Chlamydomonas reinhardtii and Oryza sativa is accompanied by phosphorylation of ChlD by ChlI, but the orthologous protein complex from Rhodobacter capsulatus, BchI/BchD, gives no detectable phosphorylation of BchD. Phosphorylation produces a 1-N-phospho-histidine within ChlD. Proteomic analysis indicates that phosphorylation occurs at a conserved His residue in the C-terminal integrin I domain of ChlD. Comparative analysis of the ChlD phosphorylation with enzyme activities of various ChlI/ChlD complexes correlates the phosphorylation by ChlI2 with stimulation of Mg-chelatase activity. Mutation of the H641 of CrChlD to E641 prevents both phosphorylation and stimulation of Mg-chelatase activity, confirming that phosphorylation at H641 stimulates Mg-chelatase. The properties of ChlI2 compared with ChlI1 of Chlamydomonas and with ChlI of Oryza, shows that ChlI2 has a regulatory role in Chlamydomonas.
Biosynthesis of chlorophylls from protoporphyrin IX
Publisher: Royal Society of Chemistry (RSC)
Date: 2003
DOI: 10.1039/B110549N
Abstract: A review of the biosynthesis of chlorophylls and bacteriochlorophylls from protoporphyrin IX with 235 references. The literature on the enzymes magnesium chelatase, S-adenosyl-L-methionine:magnesium protoporphyrin IX O-methyltransferase, magnesium-protoporphyrin IX monomethyl ester oxidative cyclase, protochlorophyllide oxidoreductase, chlorophyll synthase, bacteriochlorophyll synthase, protochlorophyllide 8-vinyl reductase and chlorophyll a oxidase from 1989 is discussed.
Spectral properties of bacteriophytochrome AM1_5894 in the chlorophyll dcontaining cyanobacterium Acaryochloris marina.
Publisher: Springer Science and Business Media LLC
Date: 10-06-2016
DOI: 10.1038/SREP27547
Abstract: Acaryochloris marina , a unicellular oxygenic photosynthetic cyanobacterium, has uniquely adapted to far-red light-enriched environments using red-shifted chlorophyll d . To understand red-light use in Acaryochloris , the genome of this cyanobacterium was searched for red/far-red light photoreceptors from the phytochrome family, resulting in identification of a putative bacteriophytochrome AM1_5894 . AM1_5894 contains three standard domains of photosensory components as well as a putative C-terminal signal transduction component consisting of a histidine kinase and receiver domain. The photosensory domains of AM1_5894 autocatalytically assemble with biliverdin in a covalent fashion. This assembled AM1_5894 shows the typical photoreversible conversion of bacterial phytochromes with a ground-state red-light absorbing (Pr) form with λ BV max [Pr] 705 nm, and a red-light inducible far-red light absorbing (Pfr) form with λ BV max [Pfr] 758 nm. Surprisingly, AM1_5894 also autocatalytically assembles with phycocyanobilin, involving photoreversible conversion of λ PCB max [Pr] 682 nm and λ PCB max [Pfr] 734 nm, respectively. Our results suggest phycocyanobilin is also covalently bound to AM1_5894, while mutation of a cysteine residue (Cys11Ser) abolishes this covalent binding. The physiological function of AM1_5894 in cyanobacteria containing red-shifted chlorophylls is discussed.
ATPase activity associated with the magnesium chelatase H-subunit of the chlorophyll biosynthetic pathway is an artefact
Publisher: Portland Press Ltd.
Date: 28-11-2006
DOI: 10.1042/BJ20061103
Abstract: Magnesium chelatase inserts Mg2+ into protoporphyrin IX and is the first unique enzyme of the chlorophyll biosynthetic pathway. It is a heterotrimeric enzyme, composed of I- (40 kDa), D- (70 kDa) and H- (140 kDa) subunits. The I- and D-proteins belong to the family of AAA+ (ATPases associated with various cellular activities), but only I-subunit hydrolyses ATP to ADP. The D-subunits provide a platform for the assembly of the I-subunits, which results in a two-tiered hexameric ring complex. However, the D-subunits are unstable in the chloroplast unless ATPase active I-subunits are present. The H-subunit binds protoporphyrin and is suggested to be the catalytic subunit. Previous studies have indicated that the H-subunit also has ATPase activity, which is in accordance with an earlier suggested two-stage mechanism of the reaction. In the present study, we demonstrate that gel filtration chromatography of affinity-purified Rhodobacter capsulatus H-subunit produced in Escherichia coli generates a high- and a low-molecular-mass fraction. Both fractions were dominated by the H-subunit, but the ATPase activity was only found in the high-molecular-mass fraction and magnesium chelatase activity was only associated with the low-molecular-mass fraction. We demonstrated that light converted monomeric low-molecular-mass H-subunit into high-molecular-mass aggregates. We conclude that ATP utilization by magnesium chelatase is solely connected to the I-subunit and suggest that a contaminating E. coli protein, which binds to aggregates of the H-subunit, caused the previously reported ATPase activity of the H-subunit.
Structure of Chlorophyll f
Publisher: American Chemical Society (ACS)
Date: 15-03-2013
DOI: 10.1021/OL400327J
Abstract: Chlorophyll f (1) is the most red-shifted absorbing natural chlorophyll reported, and it is assigned the structure [2-formyl]-chlorophyll a (C55H70O6N4Mg). This structural assignment is confirmed based on the relative retention time on HPLC, mass spectroscopy, UV/vis absorption, and CD spectroscopy, and proton and carbon NMR of chlorophyll f purified from Halomicronema hongdechloris.
Three semidominant barley mutants with single amino acid substitutions in the smallest magnesium chelatase subunit form defective AAA + hexamers
Publisher: Proceedings of the National Academy of Sciences
Date: 30-09-2002
Abstract: Many enzymes of the bacteriochlorophyll and chlorophyll biosynthesis pathways have been conserved throughout evolution, but the molecular mechanisms of the key steps remain unclear. The magnesium chelatase reaction is one of these steps, and it requires the proteins BchI, BchD, and BchH to catalyze the insertion of Mg 2+ into protoporphyrin IX upon ATP hydrolysis. Structural analyses have shown that BchI forms hexamers and belongs to the ATPases associated with various cellular activities (AAA + ) family of proteins. AAA + proteins are Mg 2+ -dependent ATPases that normally form oligomeric ring structures in the presence of ATP. By using ATPase-deficient BchI subunits, we demonstrate that binding of ATP is sufficient to form BchI oligomers. Further, ATPase-deficient BchI proteins can form mixed oligomers with WT BchI. The formation of BchI oligomers is not sufficient for magnesium chelatase activity when combined with BchD and BchH. Combining WT BchI with ATPase-deficient BchI in an assay disrupts the chelatase reaction, but the presence of deficient BchI does not inhibit ATPase activity of the WT BchI. Thus, the ATPase of every WT segment of the hexamer is autonomous, but all segments of the hexamer must be capable of ATP hydrolysis for magnesium chelatase activity. We suggest that ATP hydrolysis of each BchI within the hexamer causes a conformational change of the hexamer as a whole. However, hexamers containing ATPase-deficient BchI are unable to perform this ATP-dependent conformational change, and the magnesium chelatase reaction is stalled in an early stage.
Spectral signatures of five hydroxymethyl chlorophyll a derivatives chemically derived from chlorophyll b or chlorophyll f
Publisher: Springer Science and Business Media LLC
Date: 02-01-2019
DOI: 10.1007/S11120-018-00611-8
Abstract: Chlorophylls (Chls) are pigments involved in light capture and light reactions in photosynthesis. Chl a, Chl b, Chl d, and Chl f are characterized by unique absorbance maxima in the blue (Soret) and red (Q
EM single particle analysis of the ATP-dependent BchI complex of magnesium chelatase: an AAA+ hexamer
Publisher: Elsevier BV
Date: 04-2004
Applications of Quantitative Proteomics in Plant Research
Publisher: Springer International Publishing
Date: 2016
Structural genes for Mg-chelatase subunits in barley:
Publisher: Springer Science and Business Media LLC
Date: 1996
S-adenosyl-L-methionine
Publisher: Portland Press Ltd.
Date: 29-08-2007
DOI: 10.1042/BJ20070284
Abstract: The enzyme BchM (S-adenosyl-L-methionine:magnesium-protoporphyrin IX O-methyltransferase) from Rhodobacter capsulatus catalyses an intermediate reaction in the bacteriochlorophyll biosynthetic pathway. Overexpression of His6-tagged protein in Escherichia coli resulted in the majority of polypeptide existing as inclusion bodies. Purification from inclusion bodies was performed using metal-affinity chromatography after an elaborate wash step involving surfactant polysorbate-20. Initial enzymatic assays involved an in situ generation of S-adenosyl-L-methionine substrate using a crude preparation of S-adenosyl-L-methionine synthetase and this resulted in higher enzymatic activity compared with commercial S-adenosyl-L-methionine. A heat-stable stimulatory component present in the S-adenosyl-L-methionine synthetase was found to be a phospholipid, which increased enzymatic activity 3–4-fold. Purified phospholipids also stabilized enzymatic activity and caused a disaggregation of the protein to lower molecular mass forms, which ranged from monomeric to multimeric species as determined by size-exclusion chromatography. There was no stimulatory effect observed with magnesium–chelatase subunits on methyltransferase activity using His–BchM that had been stabilized with phospholipids. Substrate specificity of the enzyme was limited to 5-co-ordinate square-pyramidal metalloporphyrins, with magnesium-protoporphyrin IX being the superior substrate followed by zinc-protoporphyrin IX and magnesium-deuteroporphyrin. Kinetic analysis indicated a random sequential reaction mechanism. Three non-substrate metalloporphyrins acted as inhibitors with different modes of inhibition exhibited with manganese III-protoporphyrin IX (non-competitive or uncompetitive) compared with cobalt II-protoporphyrin IX (competitive).
Subsurface Stappia: Success Through Defence, Specialisation and Putative Pressure-Dependent Carbon Fixation
Publisher: Springer Science and Business Media LLC
Date: 11-12-2019
DOI: 10.1007/S00248-019-01471-Y
Abstract: Diverse microbial communities living in subsurface coal seams are responsible for important geochemical processes including the movement of carbon between the geosphere, biosphere and atmosphere. Microbial conversion of the organic matter in coal to methane involves a complex assemblage of bacteria and archaea working in syntrophic relationships. Despite the importance and value of this microbial process, very few of the microbial taxa have defined metabolic or ecological roles in these environments. Additionally, the genomic features mediating life in this chemically reduced, energy poor, deep subsurface environment are not well characterised. Here we describe the isolation and genomic and catabolic characterisation of three alphaproteobacterial Stappia indica species from three coal basins across Australia. S. indica genomes from coal seams were compared with those from closely related S. indica isolated from erse surface waters, revealing a coal seam-specific suite of genes associated with life in the subsurface. These genes are linked to processes including viral defence, secondary metabolite production, polyamine metabolism, polypeptide uptake membrane transporters and putative energy neutral pressure-dependent CO
Rapid isolation of novel FK506 binding proteins from multiple organisms using gDNA and cDNA T7 phage display
Publisher: Elsevier BV
Date: 10-2009
DOI: 10.1016/J.BMC.2009.08.039
Abstract: Reverse chemical proteomics using T7 phage display is a powerful technique for identifying cellular receptors of biologically active small molecules. However, to date this method has generally been limited to cDNA libraries constructed from mRNA isolated from eukaryotes. In this paper, we describe the construction of the first prokaryotic T7 phage display libraries from randomly digested Pseudomonas stutzeri and Vibrio fischeri gDNA, as well as a plant cDNA library from Arabidopsis thaliana. We also describe the use of T7 phage display to identify novel proteins from environmental DNA s les using biotinylated FK506 as a model affinity probe.
Synthetic biology for improved hydrogen production in Chlamydomonas reinhardtii
Publisher: Wiley
Date: 26-03-2022
Abstract: Hydrogen is a clean alternative to fossil fuels. It has applications for electricity generation and transportation and is used for the manufacturing of ammonia and steel. However, today, H 2 is almost exclusively produced from coal and natural gas. As such, methods to produce H 2 that do not use fossil fuels need to be developed and adopted. The biological manufacturing of H 2 may be one promising solution as this process is clean and renewable. Hydrogen is produced biologically via enzymes called hydrogenases. There are three classes of hydrogenases namely [FeFe], [NiFe] and [Fe] hydrogenases. The [FeFe] hydrogenase HydA1 from the model unicellular algae Chlamydomonas reinhardtii has been studied extensively and belongs to the A1 subclass of [FeFe] hydrogenases that have the highest turnover frequencies amongst hydrogenases (21,000 ± 12,000 H 2 s −1 for Ca HydA from Clostridium acetobutyliticum ). Yet to date, limitations in C. reinhardtii H 2 production pathways have h ered commercial scale implementation, in part due to O 2 sensitivity of hydrogenases and competing metabolic pathways, resulting in low H 2 production efficiency. Here, we describe key processes in the biogenesis of HydA1 and H 2 production pathways in C. reinhardtii . We also summarize recent advancements of algal H 2 production using synthetic biology and describe valuable tools such as high‐throughput screening (HTS) assays to accelerate the process of engineering algae for commercial biological H 2 production.
Biosynthesis of chlorophyll and bilins in algae
Publisher: Springer International Publishing
Date: 2020
2,7-Dimethylocta-2,4-dienedioic acid is not a by-product of abscisic acid biosynthesis
Publisher: Elsevier BV
Date: 1988
Heterologous expression of the Rhodobacter capsulatus BchI, -D, and -H genes that encode magnesium chelatase subunits and characterization of the reconstituted enzyme
Publisher: Elsevier BV
Date: 12-1998
Detection of rare prostate cancer cells in human urine offers prospect of non-invasive diagnosis
Publisher: Springer Science and Business Media LLC
Date: 02-11-2022
DOI: 10.1038/S41598-022-21656-9
Abstract: Two molecular cytology approaches, (i) time-gated immunoluminescence assay (TGiA) and (ii) Raman-active immunolabeling assay (RiA), have been developed to detect prostate cancer (PCa) cells in urine from five prostate cancer patients. For TGiA, PCa cells stained by a biocompatible europium chelate antibody-conjugated probe were quantitated by automated time-gated microscopy (OSAM). For RiA, PCa cells labeled by antibody-conjugated Raman probe were detected by Raman spectrometer. TGiA and RiA were first optimized by the detection of PCa cultured cells (DU145) spiked into control urine, with TGiA-OSAM showing single-cell PCa detection sensitivity, while RiA had a limit of detection of 4–10 cells/mL. Blinded analysis of each patient urine s le, using MIL-38 antibody specific for PCa cells, was performed using both assays in parallel with control urine. Both assays detected very low abundance PCa cells in patient urine (3–20 PCa cells per mL by TGiA, 4–13 cells/mL by RiA). The normalized mean of the detected PCa cells per 1 ml of urine was plotted against the clinical data including prostate specific antigen (PSA) level and Clinical Risk Assessment for each patient. Both cell detection assays showed correlation with PSA in the high risk patients but aligned with the Clinical Assessment rather than with PSA levels of the low/intermediate risk patients. Despite the limited available urine s les of PCa patients, the data presented in this proof-of-principle work is promising for the development of highly sensitive diagnostic urine tests for PCa.
GUN4-Protoporphyrin IX Is a singlet oxygen generator with consequences for plastid retrograde signaling
Publisher: Elsevier BV
Date: 04-2016
Characterization of red-shifted phycobilisomes isolated from the chlorophyll f-containing cyanobacterium Halomicronema hongdechloris
Publisher: Elsevier BV
Date: 2016
DOI: 10.1016/J.BBABIO.2015.10.009
Abstract: Phycobilisomes are the main light-harvesting protein complexes in cyanobacteria and some algae. It is commonly accepted that these complexes only absorb green and orange light, complementing chlorophyll absorbance. Here, we present a new phycobilisome derived complex that consists only of allophycocyanin core subunits, having red-shifted absorption peaks of 653 and 712 nm. These red-shifted phycobiliprotein complexes were isolated from the chlorophyll f-containing cyanobacterium, Halomicronema hongdechloris, grown under monochromatic 730 nm-wavelength (far-red) light. The 3D model obtained from single particle analysis reveals a double disk assembly of 120-145 Å with two α/β allophycocyanin trimers fitting into the two separated disks. They are significantly smaller than typical phycobilisomes formed from allophycocyanin subunits and core-membrane linker proteins, which fit well with a reduced distance between thylakoid membranes observed from cells grown under far-red light. Spectral analysis of the dissociated and denatured phycobiliprotein complexes grown under both these light conditions shows that the same bilin chromophore, phycocyanobilin, is exclusively used. Our findings show that red-shifted phycobilisomes are required for assisting efficient far-red light harvesting. Their discovery provides new insights into the molecular mechanisms of light harvesting under extreme conditions for photosynthesis, as well as the strategies involved in flexible chromatic acclimation to erse light conditions.
A Red-Shifted Chlorophyll: Fig. 1
Publisher: American Association for the Advancement of Science (AAAS)
Date: 10-09-2010
Abstract: Among the first facts students learn about the natural world is that plants owe their green color to the pigment chlorophyll. There have actually been a handful of slightly different chlorophyll variants uncovered over the years, and Chen et al. (p. 1318 , published online 19 August) have found another in bacteria from Shark Bay, Australia. The chlorophyll variant displayed a red-shifted absorption spectrum, which extended into the near-infrared region due to the insertion of a formyl group on the molecule's periphery. The precise cellular function of the pigment awaits further study.
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View Funded ActivityLinkage Projects - Grant ID: LP130100517
Start Date: 09-2013
End Date: 09-2016
Amount: $427,510.00
Funder: Australian Research Council
View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE120100038
Start Date: 01-2012
End Date: 06-2014
Amount: $654,000.00
Funder: Australian Research Council
View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE150100177
Start Date: 2015
End Date: 12-2015
Amount: $440,000.00
Funder: Australian Research Council
View Funded Activity