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Robert H. White - One of the best experts on this subject based on the ideXlab platform.
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Identification of the Radical SAM Enzymes Involved in the Biosynthesis of Methanopterin and Coenzyme F420 in Methanogens.
Methods in enzymology, 2018Co-Authors: Kylie D Allen, Robert H. WhiteAbstract:Methanogenic archaea represent a source of unique and fascinating anaerobic biochemistry that includes the involvement of many radical S-adenosyl-l-methionine (SAM) enzymes, some of which have well-established functions, while the majority have currently unknown or only partially understood functions. Here, we describe our strategy for the identification of the radical SAM enzyme that catalyzes the two methylation reactions in Methanopterin biosynthesis in Methanocaldococcus jannaschii. Additionally, we describe the similar strategy carried out for the identification of the two radical SAM enzymes required for the biosynthesis of the 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F0) moiety of coenzyme F420 in M. jannaschii. This approach can be employed for future functional identification of radical SAM enzymes with currently unknown functions.
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identification of a unique radical s adenosylmethionine methylase likely involved in Methanopterin biosynthesis in methanocaldococcus jannaschii
Journal of Bacteriology, 2014Co-Authors: Kylie D Allen, Robert H. WhiteAbstract:Methanopterin (MPT) and its analogs are coenzymes required for methanogenesis and methylotrophy in specialized microorganisms. The methyl groups at C-7 and C-9 of the pterin ring distinguish MPT from all other pterin-containing natural products. However, the enzyme(s) responsible for the addition of these methyl groups has yet to be identified. Here we demonstrate that a putative radical S-adenosyl-l-methionine (SAM) enzyme superfamily member encoded by the MJ0619 gene in the methanogen Methanocaldococcus jannaschii is likely this missing methylase. When MJ0619 was heterologously expressed in Escherichia coli, various methylated pterins were detected, consistent with MJ0619 catalyzing methylation at C-7 and C-9 of 7,8-dihydro-6-hydroxymethylpterin, a common intermediate in both folate and MPT biosynthesis. Site-directed mutagenesis of Cys77 present in the first of two canonical radical SAM CX3CX2C motifs present in MJ0619 did not inhibit C-7 methylation, while mutation of Cys102, found in the other radical SAM amino acid motif, resulted in the loss of C-7 methylation, suggesting that the first motif could be involved in C-9 methylation, while the second motif is required for C-7 methylation. Further experiments demonstrated that the C-7 methyl group is not derived from methionine and that methylation does not require cobalamin. When E. coli cells expressing MJ0619 were grown with deuterium-labeled acetate as the sole carbon source, the resulting methyl group on the pterin was predominantly labeled with three deuteriums. Based on these results, we propose that this archaeal radical SAM methylase employs a previously uncharacterized mechanism for methylation, using methylenetetrahydrofolate as a methyl group donor.
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Biochemical Characterization of a Dihydroneopterin Aldolase Used for Methanopterin Biosynthesis in Methanogens
Journal of bacteriology, 2014Co-Authors: Yu Wang, Laura L. Grochowski, Robert H. WhiteAbstract:The gene encoding 7,8-dihydroneopterin aldolase (DHNA) was recently identified in archaea through comparative genomics as being involved in Methanopterin biosynthesis (V. Crecy-Lagard, G. Phillips, L. L. Grochowski, B. El Yacoubi, F. Jenney, M. W. Adams, A. G. Murzin, and R. H. White, ACS Chem. Biol. 7:1807–1816, 2012, doi:10.1021/cb300342u). Archaeal DHNA shows a unique secondary and quaternary structure compared with bacterial and plant DHNAs. Here, we report a detailed biochemical examination of DHNA from the methanogen Methanocaldococcus jannaschii. Kinetic studies show that M. jannaschii DHNA possesses a catalytic capability with a kcat/Km above 105 M−1 s−1 at 70°C, and at room temperature it exhibits a turnover number (0.07 s−1) comparable to bacterial DHNAs. We also found that this enzyme follows an acid-base catalytic mechanism similar to the bacterial DHNAs, except when using alternative catalytic residues. We propose that in the absence of lysine, which is considered to be the general base in bacterial DHNAs, an invariant water molecule likely functions as the catalytic base, and the strictly conserved His35 and Gln61 residues serve as the hydrogen bond partners to adjust the basicity of the water molecule. Indeed, substitution of either His35 or Gln61 causes a 20-fold decrease in kcat. An invariant Tyr78 is also shown to be important for catalysis, likely functioning as a general acid. Glu25 plays an important role in substrate binding, since replacing Glu25 by Gln caused a ≥25-fold increase in Km. These results provide important insights into the catalytic mechanism of archaeal DHNAs.
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The conversion of a phenol to an aniline occurs in the biochemical formation of the 1-(4-aminophenyl)-1-deoxy-D-ribitol moiety in Methanopterin.
Biochemistry, 2011Co-Authors: Robert H. WhiteAbstract:Recent work has demonstrated that 4-hydroxybenzoic acid is the in vivo precursor to the 1-(4-aminophenyl)-1-deoxy-D-ribitol (APDR) moiety present in the C(1) carrier coenzyme Methanopterin present in the methanogenic archaea. For this transformation to occur, the hydroxyl group of the 4-hydroxybenzoic acid must be replaced with an amino group at some point in the biosynthetic pathway. Using stable isotopically labeled precursors and liquid chromatography with electrospray-ionization mass spectroscopy, the first step of this transformation in Methanocaldococcus jannaschii occurs by the reaction of 4-hydroxybenzoic acid with phosphoribosyl pyrophosphate (PRPP) to form 4-(β-d-ribofuranosyl)hydroxybenzene 5'-phosphate (β-RAH-P). The β-RAH-P then condenses with l-aspartate in the presence of ATP to form 4-(β-d-ribofuranosyl)-N-succinylaminobenzene 5'-phosphate (β-RFSA-P). Elimination of fumarate from β-RFSA-P produces 4-(β-D-ribofuranosyl)aminobenzene 5'-phosphate (β-RFA-P), the known precursor to the APDR moiety of Methanopterin [White, R. H. (1996) Biochemistry 35, 3447-3456]. This work represents the first biochemical example of the conversion of a phenol to an aniline.
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Biosynthesis of the Methanogenic Coenzymes
Comprehensive Natural Products II, 2010Co-Authors: Laura L. Grochowski, Robert H. WhiteAbstract:Our current knowledge of the genes, enzymes, and pathways involved in the biosynthesis of the methanogenic coenzymes methanofuran, Methanopterin, coenzyme F420, FMN, FAD, coenzyme B, coenzyme M, coenzyme F430, and corrinoid factor III is updated. Proposed reaction mechanisms for several of the novel reactions involved in their biosynthesis are presented and discussed.
Ludmila Chistoserdova - One of the best experts on this subject based on the ideXlab platform.
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Analysis of Gene Islands Involved in Methanopterin-Linked C1 Transfer Reactions Reveals New Functions and Provides Evolutionary Insights
Journal of bacteriology, 2005Co-Authors: Marina G. Kalyuzhnaya, Christopher J. Marx, Natalia Korotkova, Gregory J. Crowther, Mary E. Lidstrom, Ludmila ChistoserdovaAbstract:In this study, the occurrence and chromosomal clustering of genes encoding C1 transfer reactions linked to tetrahydroMethanopterin (H4MPT) were analyzed in a variety of proteobacteria and in representatives of the Planctomycetes via genomic analysis or via partial sequencing by cosmid walking. Although a tendency for clustering was found common for the genes of interest, significant variations in gene order and the degree of clustering were uncovered both between and within different groups of Proteobacteria and between Proteobacteria and Planctomycetes. Phylogenetic analyses suggested that the evolution of genes encoding H4MPT-linked reactions in Proteobacteria involved lateral transfers within Proteobacteria and possibly between Proteobacteria and other phyla. Gene cluster comparisons revealed a number of novel genes potentially involved in the C1 transfer reactions, and these were analyzed by mutation and expression analyses. Four genes, a homolog of pabB, and three genes conserved between methanogenic Archaea and Bacteria possessing H4MPT-linked functions, orfY, orf1, and afpA were shown to be involved in formaldehyde oxidation/detoxification, as judged by specific mutant phenotypes. In particular, pabB contributes to the biosynthesis of para-aminobenzoic acid, a precursor of both tetrahydrofolate and H4MPT, and afpA apparently encodes a novel dihydroMethanopterin reductase, based on mutant complementation experiments.
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Community-level analysis: genes encoding Methanopterin-dependent enzymes.
Methods in enzymology, 2005Co-Authors: Marina G. Kalyuzhnaya, Ludmila ChistoserdovaAbstract:Abstract This chapter describes a set of novel tools for the environmental detection of C 1 transfer functions linked to the cofactor Methanopterin. These tools include degenerate environmental primers targeting four of the most conserved genes in the Methanopterin‐linked C 1 transfer pathway in bacteria , fae, mtdB, mch, and fhcD , and extensive databases of the respective genes. The tools described are suitable for detecting Methanopterin‐linked formaldehyde‐oxidizing capacity in natural microbial communities and for determining the phylogenetic affiliations of major phyla involved in single‐carbon cycling in the environment. The range of detection includes a variety of methano‐ and methylotrophic groups, other proteobacterial species capable of Methanopterin‐mediated reactions, and a variety of planctomycetes, as well as groups of microbes with currently unknown phylogenetic affiliations.
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Highly divergent genes for Methanopterin-linked C1 transfer reactions in Lake Washington, assessed via metagenomic analysis and mRNA detection.
Applied and environmental microbiology, 2005Co-Authors: Marina G. Kalyuzhnaya, Olivier Nercessian, Mary E. Lidstrom, Sarah Bowerman, Ludmila ChistoserdovaAbstract:The origins and the evolutionary history of tetrahydroMethanopterin-linked C1 transfer reactions that are part of two environmentally important biotransformations, methylotrophy and methanogenesis, are still not well understood. In previous studies, we have expanded the known phylogenetic diversity of these reactions by identifying genes highly diverging from the ones associated with cultivated Proteobacteria, Planctomycetes, or Archaea (M. G. Kalyuzhnaya, M. E. Lidstrom, and L. Chistoserdova, Microb. Ecol. 48:463-472, 2004; M. G. Kalyuzhnaya, O. Nercessian, M. E. Lidstrom, and L. Chistoserdova, Environ. Microbiol. 7:1269-1274, 2005). Here we used a metagenomic approach to demonstrate that these divergent genes are present with high abundance in the microbial community inhabiting Lake Washington sediment. We also gained preliminary insights into the genomic composition of the organisms possessing these genes by sequencing genomic fragments from three uncultured microbes possessing the genes of interest. Phylogenetic analyses suggested that, although distantly related to each other, these organisms deeply diverge from known Bacteria and Archaea, with more relation to the former, suggesting their affiliation with a new bacterial phylum. We also demonstrate, via specific mRNA detection, that these divergent genes are expressed in the environment, pointing toward their potential role in local carbon cycling.
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Development and application of polymerase chain reaction primers based on fhcD for environmental detection of Methanopterin-linked C1-metabolism in bacteria.
Environmental microbiology, 2005Co-Authors: Marina G. Kalyuzhnaya, Olivier Nercessian, Mary E. Lidstrom, Ludmila ChistoserdovaAbstract:In this work we describe development and testing of a novel pair of environmental primers targeting fhcD, a conserved gene in the H4MTP-linked C1-transfer pathway, and demonstrate that these primers enable confident detection of a broad variety of fhcD genes originating from phylogenetically diverse bacteria. The new primer pair was employed to analyse fhcD diversity in Lake Washington sediment, uncovering the presence of 40 fhcD phylotypes. Based on phylogenetic analyses, the phylotypes identified were affiliated with alpha-, beta- and gamma-proteobacteria, and Planctomycetes, while a number of sequences formed deep branches suggesting the presence of unknown groups of microorganisms. To assess the physiological potential and the possible substrate repertoire of the fhcD-containing species in Lake Washington, we conducted enrichments of natural populations on a variety of C1 substrates, and observed specific shifts in community structure in response to different C1 substrates. A specific shift in community structure was also observed in the presence of humic acids suggesting that C1 transfer metabolism linked to H4MPT may be part of the degradation pathway for this natural polymer, possibly involving formaldehyde production. Overall, our data suggest that C1 oxidation reactions linked to H4MPT are much more widespread in natural environments than previously thought.
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Fishing for biodiversity: Novel Methanopterin-linked C1 transfer genes deduced from the Sargasso Sea metagenome
Lawrence Berkeley National Laboratory, 2004Co-Authors: Marina G. Kalyuzhnaya, Olivier Nercessian, Alla Lapidus, Ludmila ChistoserdovaAbstract:The recently generated database of microbial genes from an oligotrophic environment populated by a calculated 1,800 of major phylotypes (the Sargasso Sea metagenome) presents a great source for expanding local databases of genes indicative of a specific function. In this paper we analyze the Sargasso Sea metagenome in terms of the presence of Methanopterin-linked C1 transfer genes that are signature for methylotrophy. We conclude that more than 10 phylotypes possessing genes of interest are present in this environment, and a few of these are relatively abundant species. The sequences representative of the major phylotypes do not appear to belong to any known microbial group capable of Methanopterin-linked C1 transfer. Instead, they separate from all known sequences on phylogenetic trees, pointing towards their affiliation with a novel microbial phylum. These data imply a broader distribution of Methanopterin-linked functions in the microbial world than previously known.
Marina G. Kalyuzhnaya - One of the best experts on this subject based on the ideXlab platform.
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Analysis of Gene Islands Involved in Methanopterin-Linked C1 Transfer Reactions Reveals New Functions and Provides Evolutionary Insights
Journal of bacteriology, 2005Co-Authors: Marina G. Kalyuzhnaya, Christopher J. Marx, Natalia Korotkova, Gregory J. Crowther, Mary E. Lidstrom, Ludmila ChistoserdovaAbstract:In this study, the occurrence and chromosomal clustering of genes encoding C1 transfer reactions linked to tetrahydroMethanopterin (H4MPT) were analyzed in a variety of proteobacteria and in representatives of the Planctomycetes via genomic analysis or via partial sequencing by cosmid walking. Although a tendency for clustering was found common for the genes of interest, significant variations in gene order and the degree of clustering were uncovered both between and within different groups of Proteobacteria and between Proteobacteria and Planctomycetes. Phylogenetic analyses suggested that the evolution of genes encoding H4MPT-linked reactions in Proteobacteria involved lateral transfers within Proteobacteria and possibly between Proteobacteria and other phyla. Gene cluster comparisons revealed a number of novel genes potentially involved in the C1 transfer reactions, and these were analyzed by mutation and expression analyses. Four genes, a homolog of pabB, and three genes conserved between methanogenic Archaea and Bacteria possessing H4MPT-linked functions, orfY, orf1, and afpA were shown to be involved in formaldehyde oxidation/detoxification, as judged by specific mutant phenotypes. In particular, pabB contributes to the biosynthesis of para-aminobenzoic acid, a precursor of both tetrahydrofolate and H4MPT, and afpA apparently encodes a novel dihydroMethanopterin reductase, based on mutant complementation experiments.
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Community-level analysis: genes encoding Methanopterin-dependent enzymes.
Methods in enzymology, 2005Co-Authors: Marina G. Kalyuzhnaya, Ludmila ChistoserdovaAbstract:Abstract This chapter describes a set of novel tools for the environmental detection of C 1 transfer functions linked to the cofactor Methanopterin. These tools include degenerate environmental primers targeting four of the most conserved genes in the Methanopterin‐linked C 1 transfer pathway in bacteria , fae, mtdB, mch, and fhcD , and extensive databases of the respective genes. The tools described are suitable for detecting Methanopterin‐linked formaldehyde‐oxidizing capacity in natural microbial communities and for determining the phylogenetic affiliations of major phyla involved in single‐carbon cycling in the environment. The range of detection includes a variety of methano‐ and methylotrophic groups, other proteobacterial species capable of Methanopterin‐mediated reactions, and a variety of planctomycetes, as well as groups of microbes with currently unknown phylogenetic affiliations.
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Highly divergent genes for Methanopterin-linked C1 transfer reactions in Lake Washington, assessed via metagenomic analysis and mRNA detection.
Applied and environmental microbiology, 2005Co-Authors: Marina G. Kalyuzhnaya, Olivier Nercessian, Mary E. Lidstrom, Sarah Bowerman, Ludmila ChistoserdovaAbstract:The origins and the evolutionary history of tetrahydroMethanopterin-linked C1 transfer reactions that are part of two environmentally important biotransformations, methylotrophy and methanogenesis, are still not well understood. In previous studies, we have expanded the known phylogenetic diversity of these reactions by identifying genes highly diverging from the ones associated with cultivated Proteobacteria, Planctomycetes, or Archaea (M. G. Kalyuzhnaya, M. E. Lidstrom, and L. Chistoserdova, Microb. Ecol. 48:463-472, 2004; M. G. Kalyuzhnaya, O. Nercessian, M. E. Lidstrom, and L. Chistoserdova, Environ. Microbiol. 7:1269-1274, 2005). Here we used a metagenomic approach to demonstrate that these divergent genes are present with high abundance in the microbial community inhabiting Lake Washington sediment. We also gained preliminary insights into the genomic composition of the organisms possessing these genes by sequencing genomic fragments from three uncultured microbes possessing the genes of interest. Phylogenetic analyses suggested that, although distantly related to each other, these organisms deeply diverge from known Bacteria and Archaea, with more relation to the former, suggesting their affiliation with a new bacterial phylum. We also demonstrate, via specific mRNA detection, that these divergent genes are expressed in the environment, pointing toward their potential role in local carbon cycling.
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Development and application of polymerase chain reaction primers based on fhcD for environmental detection of Methanopterin-linked C1-metabolism in bacteria.
Environmental microbiology, 2005Co-Authors: Marina G. Kalyuzhnaya, Olivier Nercessian, Mary E. Lidstrom, Ludmila ChistoserdovaAbstract:In this work we describe development and testing of a novel pair of environmental primers targeting fhcD, a conserved gene in the H4MTP-linked C1-transfer pathway, and demonstrate that these primers enable confident detection of a broad variety of fhcD genes originating from phylogenetically diverse bacteria. The new primer pair was employed to analyse fhcD diversity in Lake Washington sediment, uncovering the presence of 40 fhcD phylotypes. Based on phylogenetic analyses, the phylotypes identified were affiliated with alpha-, beta- and gamma-proteobacteria, and Planctomycetes, while a number of sequences formed deep branches suggesting the presence of unknown groups of microorganisms. To assess the physiological potential and the possible substrate repertoire of the fhcD-containing species in Lake Washington, we conducted enrichments of natural populations on a variety of C1 substrates, and observed specific shifts in community structure in response to different C1 substrates. A specific shift in community structure was also observed in the presence of humic acids suggesting that C1 transfer metabolism linked to H4MPT may be part of the degradation pathway for this natural polymer, possibly involving formaldehyde production. Overall, our data suggest that C1 oxidation reactions linked to H4MPT are much more widespread in natural environments than previously thought.
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Fishing for biodiversity: Novel Methanopterin-linked C1 transfer genes deduced from the Sargasso Sea metagenome
Lawrence Berkeley National Laboratory, 2004Co-Authors: Marina G. Kalyuzhnaya, Olivier Nercessian, Alla Lapidus, Ludmila ChistoserdovaAbstract:The recently generated database of microbial genes from an oligotrophic environment populated by a calculated 1,800 of major phylotypes (the Sargasso Sea metagenome) presents a great source for expanding local databases of genes indicative of a specific function. In this paper we analyze the Sargasso Sea metagenome in terms of the presence of Methanopterin-linked C1 transfer genes that are signature for methylotrophy. We conclude that more than 10 phylotypes possessing genes of interest are present in this environment, and a few of these are relatively abundant species. The sequences representative of the major phylotypes do not appear to belong to any known microbial group capable of Methanopterin-linked C1 transfer. Instead, they separate from all known sequences on phylogenetic trees, pointing towards their affiliation with a novel microbial phylum. These data imply a broader distribution of Methanopterin-linked functions in the microbial world than previously known.
Stephen W. Ragsdale - One of the best experts on this subject based on the ideXlab platform.
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mechanism of 4 β d ribofuranosyl aminobenzene 5 phosphate synthase a key enzyme in the Methanopterin biosynthetic pathway
Journal of Biological Chemistry, 2004Co-Authors: Razvan Dumitru, Stephen W. RagsdaleAbstract:Abstract The first committed step in Methanopterin biosynthesis is catalyzed by 4-(β-d-ribofuranosyl)aminobenzene 5′-phosphate (RFA-P) synthase. Unlike all known phosphoribosyltransferases, β-RFA-P synthase catalyzes the unique formation of a C-riboside instead of an N-riboside in the condensation of p-aminobenzoic acid (pABA) and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to produce 4-(β-d-ribofuranosyl)aminobenzene 5′-phosphate (β-RFA-P), CO2, and inorganic pyrophosphate (PPi). Here we report the successful cloning, active overexpression in Escherichia coli, and purification of this homodimeric enzyme containing two 36.2-kDa subunits from the methanogen Methanococcus jannaschii. Steady-state initial velocity and product inhibition kinetic studies indicate an ordered Bi-Ter mechanism involving binding of PRPP, then pABA, followed by release of the products CO2, then β-RFA-P, and finally PP. The Michaelis parameters are as follows: KmpABA, 0.15 mm; KmPRPP, 1.50 mm; Vmax, 375 nmol/min/mg; kcat, 0.23 s–1. CO2 showed uncompetitive inhibition, Ki = 0.990 mm, under varied PRPP and saturated pABA, and a mixed type of inhibition, K1 = 1.40 mm and K = 3.800 mm, under varied pABA and saturated PRPP. RFA-P showed uncompetitive inhibition, Ki = 0.210 mm, under varied PRPP and saturated pABA, and again uncompetitive, Ki = 0.300 mm, under saturated PRPP and varied pABA. PPi exhibits competitive inhibition, Ki = 0.320 mm, under varied PRPP and saturated pABA, and a mixed type of inhibition, K1 = 0.60 mm and K2 = 1.900 mm, under saturated PRPP and varied pABA. Synthase lacks any chromogenic cofactor, and the presence of pyridoxal phosphate and the mechanistically related pyruvoyl cofactors has been strictly excluded.
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Mechanism of 4-(β-D-Ribofuranosyl)aminobenzene 5′-Phosphate Synthase, a Key Enzyme in the Methanopterin Biosynthetic Pathway
The Journal of biological chemistry, 2004Co-Authors: Razvan Dumitru, Stephen W. RagsdaleAbstract:Abstract The first committed step in Methanopterin biosynthesis is catalyzed by 4-(β-d-ribofuranosyl)aminobenzene 5′-phosphate (RFA-P) synthase. Unlike all known phosphoribosyltransferases, β-RFA-P synthase catalyzes the unique formation of a C-riboside instead of an N-riboside in the condensation of p-aminobenzoic acid (pABA) and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to produce 4-(β-d-ribofuranosyl)aminobenzene 5′-phosphate (β-RFA-P), CO2, and inorganic pyrophosphate (PPi). Here we report the successful cloning, active overexpression in Escherichia coli, and purification of this homodimeric enzyme containing two 36.2-kDa subunits from the methanogen Methanococcus jannaschii. Steady-state initial velocity and product inhibition kinetic studies indicate an ordered Bi-Ter mechanism involving binding of PRPP, then pABA, followed by release of the products CO2, then β-RFA-P, and finally PP. The Michaelis parameters are as follows: KmpABA, 0.15 mm; KmPRPP, 1.50 mm; Vmax, 375 nmol/min/mg; kcat, 0.23 s–1. CO2 showed uncompetitive inhibition, Ki = 0.990 mm, under varied PRPP and saturated pABA, and a mixed type of inhibition, K1 = 1.40 mm and K = 3.800 mm, under varied pABA and saturated PRPP. RFA-P showed uncompetitive inhibition, Ki = 0.210 mm, under varied PRPP and saturated pABA, and again uncompetitive, Ki = 0.300 mm, under saturated PRPP and varied pABA. PPi exhibits competitive inhibition, Ki = 0.320 mm, under varied PRPP and saturated pABA, and a mixed type of inhibition, K1 = 0.60 mm and K2 = 1.900 mm, under saturated PRPP and varied pABA. Synthase lacks any chromogenic cofactor, and the presence of pyridoxal phosphate and the mechanistically related pyruvoyl cofactors has been strictly excluded.
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Targeting Methanopterin Biosynthesis To Inhibit Methanogenesis
Applied and environmental microbiology, 2003Co-Authors: Razvan Dumitru, Madeline E. Rasche, Hector Palencia, Scott D. Schroeder, Bree A. Demontigny, James M. Takacs, Jess L. Miner, Stephen W. RagsdaleAbstract:This paper describes the design, synthesis, and successful employment of inhibitors of 4-(beta-D-ribofuranosyl)aminobenzene-5'-phosphate (RFA-P) synthase, which catalyzes the first committed step in the biosynthesis of Methanopterin, to specifically halt the growth of methane-producing microbes. RFA-P synthase catalyzes the first step in the synthesis of tetrahydroMethanopterin, a key cofactor required for methane formation and for one-carbon transformations in methanogens. A number of inhibitors, which are N-substituted derivatives of p-aminobenzoic acid (pABA), have been synthesized and their inhibition constants with RFA-P synthase have been determined. Based on comparisons of the inhibition constants among various inhibitors, we propose that the pABA binding site in RFA-P synthase has a relatively large hydrophobic pocket near the amino group. These enzyme-targeted inhibitors arrest the methanogenesis and growth of pure cultures of methanogens. Supplying pABA to the culture relieves the inhibition, indicating a competitive interaction between pABA and the inhibitor at the cellular target, which is most likely RFAP synthase. The inhibitors do not adversely affect the growth of pure cultures of the bacteria (acetogens) that play a beneficial role in the rumen. Inhibitors added to dense ruminal fluid cultures (artificial rumena) halt methanogenesis; however, they do not inhibit volatile fatty acid (VFA) production and, in some cases, VFA levels are slightly elevated in the methanogenesis-inhibited cultures. We suggest that inhibiting Methanopterin biosynthesis could be considered in strategies to decrease anthropogenic methane emissions, which could have an environmental benefit since methane is a potent greenhouse gas.
Madeline E. Rasche - One of the best experts on this subject based on the ideXlab platform.
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structure of the methanofuran Methanopterin biosynthetic enzyme mj1099 from methanocaldococcus jannaschii
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2014Co-Authors: Thomas A. Bobik, Erick J. Morales, Annie Shin, Duilio Cascio, Michael R. Sawaya, Mark A. Arbing, Todd O. Yeates, Madeline E. RascheAbstract:Prior studies have indicated that MJ1099 from Methanocaldococcus jannaschii has roles in the biosynthesis of tetrahydroMethanopterin and methanofuran, two key cofactors of one-carbon (C1) metabolism in diverse organisms including the methanogenic archaea. Here, the structure of MJ1099 has been solved to 1.7 A resolution using anomalous scattering methods. The results indicate that MJ1099 is a member of the TIM-barrel superfamily and that it is a homohexamer. Bioinformatic analyses identified a potential active site that is highly conserved among MJ1099 homologs and the key amino acids involved were identified. The results presented here should guide further studies of MJ1099 including mechanistic studies and possibly the development of inhibitors that target the methanogenic archaea in the digestive tracts of humans and that are a source of the greenhouse gas methane.
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Structure of the methanofuran/Methanopterin-biosynthetic enzyme MJ1099 from Methanocaldococcus jannaschii
Acta Crystallographica Section F Structural Biology Communications, 2014Co-Authors: Thomas A. Bobik, Erick J. Morales, Annie Shin, Duilio Cascio, Michael R. Sawaya, Mark A. Arbing, Todd O. Yeates, Madeline E. RascheAbstract:Prior studies have indicated that MJ1099 from Methanocaldococcus jannaschii has roles in the biosynthesis of tetrahydroMethanopterin and methanofuran, two key cofactors of one-carbon (C1) metabolism in diverse organisms including the methanogenic archaea. Here, the structure of MJ1099 has been solved to 1.7 A resolution using anomalous scattering methods. The results indicate that MJ1099 is a member of the TIM-barrel superfamily and that it is a homohexamer. Bioinformatic analyses identified a potential active site that is highly conserved among MJ1099 homologs and the key amino acids involved were identified. The results presented here should guide further studies of MJ1099 including mechanistic studies and possibly the development of inhibitors that target the methanogenic archaea in the digestive tracts of humans and that are a source of the greenhouse gas methane.
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Characterization of Two Methanopterin Biosynthesis Mutants of Methylobacterium extorquens AM1 by Use of a TetrahydroMethanopterin Bioassay
Journal of bacteriology, 2004Co-Authors: Madeline E. Rasche, Stephanie A. Havemann, Mariana RosenzvaigAbstract:An enzymatic assay was developed to measure tetrahydroMethanopterin (H 4 MPT) levels in wild-type and mutant cells of Methylobacterium extorquens AM1. H 4 MPT was detectable in wild-type cells but not in strains with a mutation of either the orf4 or the dmrA gene, suggesting a role for these two genes in H 4 MPT biosynthesis. The protein encoded by orf4 catalyzed the reaction of ribofuranosylaminobenzene 5′-phosphate synthase, the first committed step of H 4 MPT biosynthesis. These results provide the first biochemical evidence for H 4 MPT biosynthesis genes in bacteria.
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Targeting Methanopterin Biosynthesis To Inhibit Methanogenesis
Applied and environmental microbiology, 2003Co-Authors: Razvan Dumitru, Madeline E. Rasche, Hector Palencia, Scott D. Schroeder, Bree A. Demontigny, James M. Takacs, Jess L. Miner, Stephen W. RagsdaleAbstract:This paper describes the design, synthesis, and successful employment of inhibitors of 4-(beta-D-ribofuranosyl)aminobenzene-5'-phosphate (RFA-P) synthase, which catalyzes the first committed step in the biosynthesis of Methanopterin, to specifically halt the growth of methane-producing microbes. RFA-P synthase catalyzes the first step in the synthesis of tetrahydroMethanopterin, a key cofactor required for methane formation and for one-carbon transformations in methanogens. A number of inhibitors, which are N-substituted derivatives of p-aminobenzoic acid (pABA), have been synthesized and their inhibition constants with RFA-P synthase have been determined. Based on comparisons of the inhibition constants among various inhibitors, we propose that the pABA binding site in RFA-P synthase has a relatively large hydrophobic pocket near the amino group. These enzyme-targeted inhibitors arrest the methanogenesis and growth of pure cultures of methanogens. Supplying pABA to the culture relieves the inhibition, indicating a competitive interaction between pABA and the inhibitor at the cellular target, which is most likely RFAP synthase. The inhibitors do not adversely affect the growth of pure cultures of the bacteria (acetogens) that play a beneficial role in the rumen. Inhibitors added to dense ruminal fluid cultures (artificial rumena) halt methanogenesis; however, they do not inhibit volatile fatty acid (VFA) production and, in some cases, VFA levels are slightly elevated in the methanogenesis-inhibited cultures. We suggest that inhibiting Methanopterin biosynthesis could be considered in strategies to decrease anthropogenic methane emissions, which could have an environmental benefit since methane is a potent greenhouse gas.
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Purification, Overproduction, and Partial Characterization of β-RFAP Synthase, a Key Enzyme in the Methanopterin Biosynthesis Pathway†
Journal of bacteriology, 2002Co-Authors: Joseph W. Scott, Madeline E. RascheAbstract:Methanopterin is a folate analog involved in the C1 metabolism of methanogenic archaea, sulfate-reducing archaea, and methylotrophic bacteria. Although a pathway for Methanopterin biosynthesis has been described in methanogens, little is known about the enzymes and genes involved in the biosynthetic pathway. The enzyme β-ribofuranosylaminobenzene 5′-phosphate synthase (β-RFAP synthase) catalyzes the first unique step to be identified in the pathway of Methanopterin biosynthesis, namely, the condensation of p-aminobenzoic acid with phosphoribosylpyrophosphate to form β-RFAP, CO2, and inorganic pyrophosphate. The enzyme catalyzing this reaction has not been purified to homogeneity, and the gene encoding β-RFAP synthase has not yet been identified. In the present work, we report on the purification to homogeneity of β-RFAP synthase. The enzyme was purified from the methane-producing archaeon Methanosarcina thermophila, and the N-terminal sequence of the protein was used to identify corresponding genes from several archaea, including the methanogen Methanococcus jannaschii and the sulfate-reducing archaeon Archaeoglobus fulgidus. The putative β-RFAP synthase gene from A. fulgidus was expressed in Escherichia coli, and the enzymatic activity of the recombinant gene product was verified. A BLAST search using the deduced amino acid sequence of the β-RFAP synthase gene identified homologs in additional archaea and in a gene cluster required for C1 metabolism by the bacterium Methylobacterium extorquens. The identification of a gene encoding a potential β-RFAP synthase in M. extorquens is the first report of a putative Methanopterin biosynthetic gene found in the Bacteria and provides evidence that the pathways of Methanopterin biosynthesis in Bacteria and Archaea are similar.