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Robert H. White - One of the best experts on this subject based on the ideXlab platform.
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-Alanine Biosynthesis in Methanocaldococcus jannaschii
2016Co-Authors: Yu Wang, Robert H. WhiteAbstract:One efficient approach to assigning function to unannotated genes is to establish the enzymes that are missing in known biosyn-thetic pathways. One group of such pathways is those involved in coenzyme biosynthesis. In the case of the methanogenic ar-chaeonMethanocaldococcus jannaschii as well as most methanogens, none of the expected enzymes for the biosynthesis of the -alanine and pantoic acid moieties required for coenzyme A are annotated. To identify the gene(s) for -alanine biosynthesis, we have established the pathway for the formation of -alanine in this organism after experimentally eliminating other known and proposed pathways to-alanine frommalonate semialdehyde, L-alanine, spermine, dihydrouracil, and acryloyl-coenzyme A (CoA). Our data showed that the decarboxylation of aspartate was the only source of -alanine in cell extracts ofM. jannaschii. Unlike other prokaryotes where the enzyme producing -alanine from L-aspartate is a pyruvoyl-containing L-aspartate decar-boxylase (PanD), the enzyme inM. jannaschii is a pyridoxal phosphate (PLP)-dependent L-aspartate decarboxylase encoded by MJ0050, the same enzyme that was found to decarboxylate tyrosine for Methanofuran biosynthesis. AKm of0.80 mM for L-as-partate with a specific activity of 0.09mol min1 mg1 at 70°C for the decarboxylation of L-aspartate was measured for the re-combinant enzyme. The MJ0050 gene was also demonstrated to complement the Escherichia coli panD deletion mutant cells, in which panD encoding aspartate decarboxylase in E. coli had been knocked out, thus confirming the function of this gene in vivo. Coenzyme A (CoA) is an important coenzyme in all knownliving organisms where it functions as an acyl carrier for amide-, ester-, and thioester-forming reactions as well as activat
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identification of the final two genes functioning in Methanofuran biosynthesis in methanocaldococcus jannaschii
Journal of Bacteriology, 2015Co-Authors: Yu Wang, Michael K Jones, Robert H. WhiteAbstract:All Methanofuran structural variants contain a basic core structure of 4-[N-(-L-glutamyl)-p-(-aminoethyl)phenoxymethyl](aminomethyl)furan (APMF-Glu) but have different side chains depending on the source organism. Recently, we identified four genes (MfnA, MfnB, MfnC, and MfnD) that are responsible for the biosynthesis of the Methanofuran precursor -glutamyltyramine and 5-(aminomethyl)-3-furanmethanol-phosphate (F1-P) from tyrosine, glutamate, glyceraldehyde-3-P, and alanine in Methanocaldococcus jannaschii. How -glutamyltyramine and F1-P couple together to form the core structure of Methanofuran was previously unknown. Here, we report the identification of two enzymes encoded by the genes mj0458andmj0840that catalyze the formation of F1-PP from ATP and F1-P and the condensation of F1-PP with -glutamyltyramine, respectively, to form APMF-Glu. We have annotated these enzymes as MfnE and MfnF, respectively, representing thefifth and sixth enzymes in the Methanofuran biosynthetic pathway to be identified. Although MfnE was previously reported as an archaeal adenylate kinase, our present results show that MfnE is a promiscuous enzyme and that its possible physiological role is to produce F1-PP. Unlike other enzymes catalyzing coupling reactions involving pyrophosphate as the leaving group, MfnF exhibits a distinctive /twolayer sandwich structure. By comparing MfnF with thiamine synthase and dihydropteroate synthase, a substitution nucleophilic unimolecular (S N-1) reaction mechanism is proposed for MfnF. With the identification of MfnE and MfnF, the biosynthetic pathway for the Methanofuran core structure APMF-Glu is complete. IMPORTANCE This work describes the identification of thefinal two enzymes responsible for catalyzing the biosynthesis of the core structure of Methanofuran. The gene products of mj0458andmj0840catalyze the formation of F1-PP and the coupling of F1-PP with -glutamyltyramine, respectively, to form APMF-Glu. Although the chemistry of such a coupling reaction is widespread in biochemistry, we provide here thefirst evidence that such a mechanism is used in Methanofuran biosynthesis. MfnF belongs to the hydantoinase A family (PF01968) and exhibits a unique /two-layer sandwich structure that is different from the enzymes catalyzing similar reactions. Our results show that MfnF catalyzes the formation of an ether bond during Methanofuran biosynthesis. Therefore, this work further expands the functionality of this enzyme family.
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Mechanism of the Enzymatic Synthesis of 4‑(Hydroxymethyl)-2-furancarboxaldehyde-phosphate (4-HFC-P) from Glyceraldehyde-3-phosphate Catalyzed by 4‑HFC‑P Synthase
2015Co-Authors: Yu Wang, Michael K Jones, Keith W. Ray, Robert H. WhiteAbstract:A single enzyme, 4-(hydroxymethyl)-2-furancarboxaldehyde-phosphate synthase (MfnB), from the methanogen Methanocaldococcus jannaschii catalyzed at least 10 separate chemical reactions in converting two molecules of glyceraldehyde-3-P (GA-3-P) to 4-(hydroxymethyl)-2-furancarboxaldehyde-P (4-HFC-P), the first discrete intermediate in the biosynthetic pathway to the furan moiety of the coenzyme Methanofuran. Here we describe the biochemical characterization of the recombinantly expressed MfnB to understand its catalytic mechanism. Site-directed mutagenesis showed that the strictly conserved residues (Asp25, Lys27, Lys85, and Asp151) around the active site are all essential for enzyme catalysis. Matrix-assisted laser desorption/ionization analysis of peptide fragments of MfnB incubated with GA-3-P followed by NaBH4 reduction and trypsin digestion identified a peptide with a mass/charge ratio of 1668.8 m/z present only in the D25N, D151N, and K155R mutants, which is consistent with Lys27 having increased by a mass of 58 m/z, indicating that Lys27 forms a Schiff base with a methylglyoxal-like intermediate. In addition, incubation of MfnB with GA-3-P in the presence of deuterated water or incubation of MfnB with C-2 deuterated GA-3-P showed essentially no deuterium incorporated into the 4-HFC-P. Combined with structural analysis and molecular docking, we predict the potential binding sites for two GA-3P molecules in the active site. On the basis of our observations, a possible catalytic mechanism of MfnB is proposed in this study. A phosphate elimination reaction and a triose phosphate isomerase-like reaction occur at the GA-3-P binding site I and II, respectively, prior to the aldol condensation between the enzyme-bound enol form of methylglyoxal and dihydroxyacetone phosphate (DHAP), after which the catalytic cycle is completed by a cyclization and two dehydration reactions assisted by several general acids/bases at the same active site
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identification and characterization of a tyramine glutamate ligase mfnd involved in Methanofuran biosynthesis
Biochemistry, 2014Co-Authors: Yu Wang, Kim Harich, Robert H. WhiteAbstract:Methanofuran is the first in a series of coenzymes involved in the reduction of carbon dioxide to methane. All Methanofuran structural variants contain a basic core structure of 4-[N-(γ-l-glutamyl-γ-l-glutamyl)-p-(β-aminoethyl)phenoxymethyl]-2-(aminomethyl)furan (APMF-(Glu)2) with different attached side chains depending on the source organism. Recently, we discovered the biosynthetic route for the production of 5-(aminomethyl)-3-furanmethanol-phosphate (F1-P), a precursor to the furan moiety of Methanofuran. However, how the γ-linked glutamates are incorporated into Methanofuran’s structure remains unknown. Here, we report the identification of an ATP-grasp enzyme encoded by the gene Mefer_1180 in Methanocaldococcus fervens (the homologue of MJ0815 in Methanocaldococcus jannaschii, annotated as MfnD) that catalyzes the ATP-dependent addition of one glutamate to tyramine via a γ-linked amide bond. The occurrence of this reaction is consistent with the presence of γ-glutamyltyramine in cell extracts of M. ...
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identification of structurally diverse Methanofuran coenzymes in methanococcales that are both n formylated and n acetylated
Biochemistry, 2014Co-Authors: Kylie D Allen, Robert H. WhiteAbstract:Methanofuran (MF) is a coenzyme necessary for the first step of methanogenesis from CO2. The well-characterized MF core structure is 4-[N-(γ-l-glutamyl-γ-l-glutamyl)-p-(β-aminoethyl)phenoxymethyl]-2-(aminomethyl)furan (APMF-γ-Glu2). Three different MF structures that differ on the basis of the composition of their side chains have been determined previously. Here, we use liquid chromatography coupled with high-resolution mass spectrometry and a variety of biochemical methods to deduce the unique structures of MFs present in four different methanogens in the order Methanococcales. This is the first detailed characterization of the MF occurring in methanogens of this order. MF in each of these organisms contains the expected APMF-γ-Glu2; however, the composition of the side chain is different from that of the previously described MF structures. In Methanocaldococcus jannaschii, additional γ-linked glutamates that range from 7 to 12 residues are present. The MF coenzymes in Methanococcus maripaludis, Methano...
Julia A. Vorholt - One of the best experts on this subject based on the ideXlab platform.
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methylofuran is a prosthetic group of the formyltransferase hydrolase complex and shuttles one carbon units between two active sites
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Jethro L Hemmann, Tristan Wagner, Seigo Shima, Julia A. VorholtAbstract:Methylotrophy, the ability of microorganisms to grow on reduced one-carbon substrates such as methane or methanol, is a feature of various bacterial species. The prevailing oxidation pathway depends on tetrahydromethanopterin (H4MPT) and methylofuran (MYFR), an analog of Methanofuran from methanogenic archaea. Formyltransferase/hydrolase complex (Fhc) generates formate from formyl-H4MPT in two consecutive reactions where MYFR acts as a carrier of one-carbon units. Recently, we chemically characterized MYFR from the model methylotroph Methylorubrum extorquens and identified an unusually long polyglutamate side chain of up to 24 glutamates. Here, we report on the crystal structure of Fhc to investigate the function of the polyglutamate side chain in MYFR and the relatedness of the enzyme complex with the orthologous enzymes in archaea. We identified MYFR as a prosthetic group that is tightly, but noncovalently, bound to Fhc. Surprisingly, the structure of Fhc together with MYFR revealed that the polyglutamate side chain of MYFR is branched and contains glutamates with amide bonds at both their α- and γ-carboxyl groups. This negatively charged and branched polyglutamate side chain interacts with a cluster of conserved positively charged residues of Fhc, allowing for strong interactions. The MYFR binding site is located equidistantly from the active site of the formyltransferase (FhcD) and metallo-hydrolase (FhcA). The polyglutamate serves therefore an additional function as a swinging linker to shuttle the one-carbon carrying amine between the two active sites, thereby likely increasing overall catalysis while decreasing the need for high intracellular MYFR concentrations.
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the one carbon carrier methylofuran from methylobacterium extorquens am1 contains a large number of α and γ linked glutamic acid residues
Journal of Biological Chemistry, 2016Co-Authors: Jethro L Hemmann, Olivier Saurel, Alain Milon, Andrea M Ochsner, Barbara K Stodden, Patrick Kiefer, Julia A. VorholtAbstract:Methylobacterium extorquens AM1 uses dedicated cofactors for one-carbon unit conversion. Based on the sequence identities of enzymes and activity determinations, a Methanofuran analog was proposed to be involved in formaldehyde oxidation in Alphaproteobacteria. Here, we report the structure of the cofactor, which we termed methylofuran. Using an in vitro enzyme assay and LC-MS, methylofuran was identified in cell extracts and further purified. From the exact mass and MS-MS fragmentation pattern, the structure of the cofactor was determined to consist of a polyglutamic acid side chain linked to a core structure similar to the one present in archaeal Methanofuran variants. NMR analyses showed that the core structure contains a furan ring. However, instead of the tyramine moiety that is present in Methanofuran cofactors, a tyrosine residue is present in methylofuran, which was further confirmed by MS through the incorporation of a (13)C-labeled precursor. Methylofuran was present as a mixture of different species with varying numbers of glutamic acid residues in the side chain ranging from 12 to 24. Notably, the glutamic acid residues were not solely γ-linked, as is the case for all known Methanofurans, but were identified by NMR as a mixture of α- and γ-linked amino acids. Considering the unusual peptide chain, the elucidation of the structure presented here sets the basis for further research on this cofactor, which is probably the largest cofactor known so far.
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The one-carbon carrier methylofuran from Methylobacterium extorquens AM1 contains a large number of alpha- and gamma-linked glutamic acid residues
Journal of Biological Chemistry, 2016Co-Authors: Jethro L Hemmann, Olivier Saurel, Alain Milon, Andrea M Ochsner, Barbara K Stodden, Patrick Kiefer, Julia A. VorholtAbstract:Methylobacterium extorquens AM1 uses dedicated cofactors for one-carbon unit conversion. Based on the sequence identities of enzymes and activity determinations, a Methanofuran analog was proposed to be involved in formaldehyde oxidation in Alphaproteobacteria. Here, we report the structure of the cofactor, which we termed methylofuran. Using an in vitro enzyme assay and LC-MS, methylofuran was identified in cell extracts and further purified. From the exact mass and MS-MS fragmentation pattern, the structure of the cofactor was determined to consist of a polyglutamic acid side chain linked to a core structure similar to the one present in archaeal Methanofuran variants. NMR analyses showed that the core structure contains a furan ring. However, instead of the tyramine moiety that is present in Methanofuran cofactors, a tyrosine residue is present in methylofuran, which was further confirmed by MS through the incorporation of a C-13-labeled precursor. Methylofuran was present as a mixture of different species with varying numbers of glutamic acid residues in the side chain ranging from 12 to 24. Notably, the glutamic acid residues were not solely gamma-linked, as is the case for all known Methanofurans, but were identified by NMR as a mixture of alpha- and gamma-linked amino acids. Considering the unusual peptide chain, the elucidation of the structure presented here sets the basis for further research on this cofactor, which is probably the largest cofactor known so far.
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generation of formate by the formyltransferase hydrolase complex fhc from methylobacterium extorquens am1
FEBS Letters, 2002Co-Authors: Olivier Saurel, Barbara K Pomper, Alain Milon, Julia A. VorholtAbstract:Methylobacterium extorquens AM1 possesses a formyltransferase (Ftr) complex that is essential for growth in the presence of methanol and involved in formaldehyde oxidation to CO2. One of the subunits of the complex carries the catalytic site for transfer of the formyl group from tetrahydromethanopterin to Methanofuran (MFR). We now found via nuclear magnetic resonance-based studies that the Ftr complex also catalyzes the hydrolysis of formyl-MFR and generates formate. The enzyme was therefore renamed Ftr/hydrolase complex (Fhc). FhcA shares a sequence pattern with amidohydrolases and is assumed to be the catalytic site where the hydrolysis takes place.
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Characterization of the formyltransferase from Methylobacterium extorquens AM1
European journal of biochemistry, 2001Co-Authors: Barbara K Pomper, Julia A. VorholtAbstract:1 Methylobacterium extorquens AM1 possesses a formaldehyde-oxidation pathway that involves enzymes with high sequence identity with enzymes from methanogenic and sulfate-reducing archaea. Here we describe the purification and characterization of formylMethanofuran–tetrahydromethanopterin formyltransferase (Ftr), which catalyzes the reversible formation of formylMethanofuran (formylMFR) and tetrahydromethanopterin (H4MPT) from N5-formylH4MPT and Methanofuran (MFR). Formyltransferase from M. extorquens AM1 showed activity with MFR and H4MPT isolated from the methanogenic archaeon Methanothermobacter marburgensis (apparent Km for formylMFR = 50 µm; apparent Km for H4MPT = 30 µm). The enzyme is encoded by the ffsA gene and exhibits a sequence identity of ≈ 40% with Ftr from methanogenic and sulfate-reducing archaea. The 32-kDa Ftr protein from M. extorquens AM1 copurified in a complex with three other polypeptides of 60 kDa, 37 kDa and 29 kDa. Interestingly, these are encoded by the genes orf1, orf2 and orf3 which show sequence identity with the formylMFR dehydrogenase subunits FmdA, FmdB and FmdC, respectively. The clustering of the genes orf2, orf1, ffsA, and orf3 in the chromosome of M. extorquens AM1 indicates that, in the bacterium, the respective polypeptides form a functional unit. Expression studies in Escherichia coli indicate that Ftr requires the other subunits of the complex for stability. Despite the fact that three of the polypeptides of the complex showed sequence similarity to subunits of Fmd from methanogens, the complex was not found to catalyze the oxidation of formylMFR. Detailed comparison of the primary structure revealed that Orf2, the homolog of the active site harboring subunit FmdB, lacks the binding motifs for the active-site cofactors molybdenum, molybdopterin and a [4Fe−4S] cluster. Cytochrome c was found to be spontaneously reduced by H4MPT. On the basis of this property, a novel assay for Ftr activity and MFR is described.
Yu Wang - One of the best experts on this subject based on the ideXlab platform.
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-Alanine Biosynthesis in Methanocaldococcus jannaschii
2016Co-Authors: Yu Wang, Robert H. WhiteAbstract:One efficient approach to assigning function to unannotated genes is to establish the enzymes that are missing in known biosyn-thetic pathways. One group of such pathways is those involved in coenzyme biosynthesis. In the case of the methanogenic ar-chaeonMethanocaldococcus jannaschii as well as most methanogens, none of the expected enzymes for the biosynthesis of the -alanine and pantoic acid moieties required for coenzyme A are annotated. To identify the gene(s) for -alanine biosynthesis, we have established the pathway for the formation of -alanine in this organism after experimentally eliminating other known and proposed pathways to-alanine frommalonate semialdehyde, L-alanine, spermine, dihydrouracil, and acryloyl-coenzyme A (CoA). Our data showed that the decarboxylation of aspartate was the only source of -alanine in cell extracts ofM. jannaschii. Unlike other prokaryotes where the enzyme producing -alanine from L-aspartate is a pyruvoyl-containing L-aspartate decar-boxylase (PanD), the enzyme inM. jannaschii is a pyridoxal phosphate (PLP)-dependent L-aspartate decarboxylase encoded by MJ0050, the same enzyme that was found to decarboxylate tyrosine for Methanofuran biosynthesis. AKm of0.80 mM for L-as-partate with a specific activity of 0.09mol min1 mg1 at 70°C for the decarboxylation of L-aspartate was measured for the re-combinant enzyme. The MJ0050 gene was also demonstrated to complement the Escherichia coli panD deletion mutant cells, in which panD encoding aspartate decarboxylase in E. coli had been knocked out, thus confirming the function of this gene in vivo. Coenzyme A (CoA) is an important coenzyme in all knownliving organisms where it functions as an acyl carrier for amide-, ester-, and thioester-forming reactions as well as activat
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identification of the final two genes functioning in Methanofuran biosynthesis in methanocaldococcus jannaschii
Journal of Bacteriology, 2015Co-Authors: Yu Wang, Michael K Jones, Robert H. WhiteAbstract:All Methanofuran structural variants contain a basic core structure of 4-[N-(-L-glutamyl)-p-(-aminoethyl)phenoxymethyl](aminomethyl)furan (APMF-Glu) but have different side chains depending on the source organism. Recently, we identified four genes (MfnA, MfnB, MfnC, and MfnD) that are responsible for the biosynthesis of the Methanofuran precursor -glutamyltyramine and 5-(aminomethyl)-3-furanmethanol-phosphate (F1-P) from tyrosine, glutamate, glyceraldehyde-3-P, and alanine in Methanocaldococcus jannaschii. How -glutamyltyramine and F1-P couple together to form the core structure of Methanofuran was previously unknown. Here, we report the identification of two enzymes encoded by the genes mj0458andmj0840that catalyze the formation of F1-PP from ATP and F1-P and the condensation of F1-PP with -glutamyltyramine, respectively, to form APMF-Glu. We have annotated these enzymes as MfnE and MfnF, respectively, representing thefifth and sixth enzymes in the Methanofuran biosynthetic pathway to be identified. Although MfnE was previously reported as an archaeal adenylate kinase, our present results show that MfnE is a promiscuous enzyme and that its possible physiological role is to produce F1-PP. Unlike other enzymes catalyzing coupling reactions involving pyrophosphate as the leaving group, MfnF exhibits a distinctive /twolayer sandwich structure. By comparing MfnF with thiamine synthase and dihydropteroate synthase, a substitution nucleophilic unimolecular (S N-1) reaction mechanism is proposed for MfnF. With the identification of MfnE and MfnF, the biosynthetic pathway for the Methanofuran core structure APMF-Glu is complete. IMPORTANCE This work describes the identification of thefinal two enzymes responsible for catalyzing the biosynthesis of the core structure of Methanofuran. The gene products of mj0458andmj0840catalyze the formation of F1-PP and the coupling of F1-PP with -glutamyltyramine, respectively, to form APMF-Glu. Although the chemistry of such a coupling reaction is widespread in biochemistry, we provide here thefirst evidence that such a mechanism is used in Methanofuran biosynthesis. MfnF belongs to the hydantoinase A family (PF01968) and exhibits a unique /two-layer sandwich structure that is different from the enzymes catalyzing similar reactions. Our results show that MfnF catalyzes the formation of an ether bond during Methanofuran biosynthesis. Therefore, this work further expands the functionality of this enzyme family.
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Mechanism of the Enzymatic Synthesis of 4‑(Hydroxymethyl)-2-furancarboxaldehyde-phosphate (4-HFC-P) from Glyceraldehyde-3-phosphate Catalyzed by 4‑HFC‑P Synthase
2015Co-Authors: Yu Wang, Michael K Jones, Keith W. Ray, Robert H. WhiteAbstract:A single enzyme, 4-(hydroxymethyl)-2-furancarboxaldehyde-phosphate synthase (MfnB), from the methanogen Methanocaldococcus jannaschii catalyzed at least 10 separate chemical reactions in converting two molecules of glyceraldehyde-3-P (GA-3-P) to 4-(hydroxymethyl)-2-furancarboxaldehyde-P (4-HFC-P), the first discrete intermediate in the biosynthetic pathway to the furan moiety of the coenzyme Methanofuran. Here we describe the biochemical characterization of the recombinantly expressed MfnB to understand its catalytic mechanism. Site-directed mutagenesis showed that the strictly conserved residues (Asp25, Lys27, Lys85, and Asp151) around the active site are all essential for enzyme catalysis. Matrix-assisted laser desorption/ionization analysis of peptide fragments of MfnB incubated with GA-3-P followed by NaBH4 reduction and trypsin digestion identified a peptide with a mass/charge ratio of 1668.8 m/z present only in the D25N, D151N, and K155R mutants, which is consistent with Lys27 having increased by a mass of 58 m/z, indicating that Lys27 forms a Schiff base with a methylglyoxal-like intermediate. In addition, incubation of MfnB with GA-3-P in the presence of deuterated water or incubation of MfnB with C-2 deuterated GA-3-P showed essentially no deuterium incorporated into the 4-HFC-P. Combined with structural analysis and molecular docking, we predict the potential binding sites for two GA-3P molecules in the active site. On the basis of our observations, a possible catalytic mechanism of MfnB is proposed in this study. A phosphate elimination reaction and a triose phosphate isomerase-like reaction occur at the GA-3-P binding site I and II, respectively, prior to the aldol condensation between the enzyme-bound enol form of methylglyoxal and dihydroxyacetone phosphate (DHAP), after which the catalytic cycle is completed by a cyclization and two dehydration reactions assisted by several general acids/bases at the same active site
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identification and characterization of a tyramine glutamate ligase mfnd involved in Methanofuran biosynthesis
Biochemistry, 2014Co-Authors: Yu Wang, Kim Harich, Robert H. WhiteAbstract:Methanofuran is the first in a series of coenzymes involved in the reduction of carbon dioxide to methane. All Methanofuran structural variants contain a basic core structure of 4-[N-(γ-l-glutamyl-γ-l-glutamyl)-p-(β-aminoethyl)phenoxymethyl]-2-(aminomethyl)furan (APMF-(Glu)2) with different attached side chains depending on the source organism. Recently, we discovered the biosynthetic route for the production of 5-(aminomethyl)-3-furanmethanol-phosphate (F1-P), a precursor to the furan moiety of Methanofuran. However, how the γ-linked glutamates are incorporated into Methanofuran’s structure remains unknown. Here, we report the identification of an ATP-grasp enzyme encoded by the gene Mefer_1180 in Methanocaldococcus fervens (the homologue of MJ0815 in Methanocaldococcus jannaschii, annotated as MfnD) that catalyzes the ATP-dependent addition of one glutamate to tyramine via a γ-linked amide bond. The occurrence of this reaction is consistent with the presence of γ-glutamyltyramine in cell extracts of M. ...
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biosynthesis of the 5 aminomethyl 3 furanmethanol moiety of Methanofuran
Biochemistry, 2014Co-Authors: Danielle V Miller, Yu Wang, Kim Harich, Robert H. WhiteAbstract:We have established the biosynthetic pathway and the associated genes for the biosynthesis of the 5-(aminomethyl)-3-furanmethanol (F1) moiety of Methanofuran in the methanogenic archaeon Methanocaldococcus jannaschii. The recombinant enzyme, derived from the MJ1099 gene, was shown to readily condense glyceraldehyde 3-phosphate (Ga-3P) and dihydroxyacetone-P (DHAP) to form 4-(hydroxymethyl)-2-furancarboxaldehyde phosphate (4-HFC-P). The recombinant purified pyridoxal 5′-phosphate-dependent aminotransferase, derived from the MJ0684 gene, was found to be specific for catalyzing the transamination reaction between 4-HFC-P and [15N]alanine to produce [15N] 5-(aminomethyl)-3-furanmethanol-P (F1-P) and pyruvate. To confirm these results in cell extracts, we developed sensitive analytical methods for the liquid chromatography–ultraviolet–electrospray ionization mass spectrometry analysis of F1 as a 7-nitrobenzofurazan derivative. This method has allowed for the quantitation of trace amounts of F1 and F1-P in cell...
Reiner Hedderich - One of the best experts on this subject based on the ideXlab platform.
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co2 reduction to the level of formylMethanofuran in methanosarcina barkeri is non energy driven when co is the electron donor
Fems Microbiology Letters, 2004Co-Authors: Alexander Stojanowic, Reiner HedderichAbstract:A Methanosarcina barkeri mutant lacking Ech hydrogenase does not catalyze CH4 formation from H2/CO2 since, as was shown previously, the energy-driven reduction of CO2 to formylMethanofuran by H2 is blocked. CH4 formation by this mutant could be restored in the presence of CO or pyruvate. Furthermore, CH4 formation from H2/CO2 plus CO by the Δech mutant was not inhibited by the protonophore TCS. These data show that in vivo the reduction of CO2 to formylMethanofuran can be coupled to the oxidation of CO or pyruvate via a common electron carrier and that the reduction of this electron carrier by H2, catalyzed by Ech hydrogenase, is the energy-driven step in formylMethanofuran-synthesis from CO2, H2 and Methanofuran.
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methanobacterium thermoautotrophicum encodes two multisubunit membrane bound nife hydrogenases transcription of the operons and sequence analysis of the deduced proteins
FEBS Journal, 1999Co-Authors: Adrian Tersteegen, Reiner HedderichAbstract:Two gene groups, designated energy converting hydrogenase A (eha) and energy converting hydrogenase B (ehb), each encoding a putative multisubunit membrane-bound [NiFe] hydrogenase, were identified in the genome of Methanobacterium thermoautotrophicum. The length of the transcription units was determined using reverse transcription (RT)-PCR. The eha operon (12.5 kb) and the ehb operon (9.6 kb) were found to be composed of 20 and 17 open reading frames, respectively. Competitive RT-PCR was used to compare the amounts of eha and ehb transcripts with the amounts of transcripts of genes encoding the M. thermoautotrophicum catabolic enzymes cyclohydrolase (mch) and a subunit of heterodisulfide reductase (hdrC). In cells grown under conditions in which H2 was nonlimiting, the eha transcripts were 250-fold and 125-fold less abundant and the ehb transcripts were approximately sixfold and threefold less abundant than the hdrC and mch transcripts, respectively. In cells grown under H2 limitation, the amounts of eha and ehb transcripts were about threefold higher than in cells grown with sufficient H2 when compared to the amounts of hdrC and mch transcripts. Sequence analysis of the deduced proteins indicated that the eha and ehb operons each encode a [NiFe] hydrogenase large subunit, a [NiFe] hydrogenase small subunit, and two conserved integral membrane proteins. These proteins show high sequence similarity to subunits of the Ech hydrogenase from Methanosarcina barkeri, Escherichia coli hydrogenases 3 and 4, and CO-induced hydrogenase from Rhodospirillum rubrum, all of which form a distinct group of multisubunit membrane-bound [NiFe] hydrogenases and show high sequence similarity to the energy-conserving NADH:quinone oxidoreductase (complex I) from various organisms. In addition to these four subunits, the eha operon encodes a 6[4Fe–4S] polyferredoxin, a 10[4F–4S] polyferredoxin, four nonconserved hydrophilic subunits, and 10 nonconserved integral membrane proteins; the ehb operon encodes a 2[4Fe–4S] ferredoxin, a 14[4Fe–4S] polyferredoxin, two nonconserved hydrophilic subunits, and nine nonconserved integral membrane proteins. A function of these putative membrane-bound [NiFe] hydrogenases as proton pumps involved in endergonic reactions, such as the synthesis of formylMethanofuran from CO2, H2 and Methanofuran, is discussed.
Barbara K Pomper - One of the best experts on this subject based on the ideXlab platform.
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generation of formate by the formyltransferase hydrolase complex fhc from methylobacterium extorquens am1
FEBS Letters, 2002Co-Authors: Olivier Saurel, Barbara K Pomper, Alain Milon, Julia A. VorholtAbstract:Methylobacterium extorquens AM1 possesses a formyltransferase (Ftr) complex that is essential for growth in the presence of methanol and involved in formaldehyde oxidation to CO2. One of the subunits of the complex carries the catalytic site for transfer of the formyl group from tetrahydromethanopterin to Methanofuran (MFR). We now found via nuclear magnetic resonance-based studies that the Ftr complex also catalyzes the hydrolysis of formyl-MFR and generates formate. The enzyme was therefore renamed Ftr/hydrolase complex (Fhc). FhcA shares a sequence pattern with amidohydrolases and is assumed to be the catalytic site where the hydrolysis takes place.
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Characterization of the formyltransferase from Methylobacterium extorquens AM1
European journal of biochemistry, 2001Co-Authors: Barbara K Pomper, Julia A. VorholtAbstract:1 Methylobacterium extorquens AM1 possesses a formaldehyde-oxidation pathway that involves enzymes with high sequence identity with enzymes from methanogenic and sulfate-reducing archaea. Here we describe the purification and characterization of formylMethanofuran–tetrahydromethanopterin formyltransferase (Ftr), which catalyzes the reversible formation of formylMethanofuran (formylMFR) and tetrahydromethanopterin (H4MPT) from N5-formylH4MPT and Methanofuran (MFR). Formyltransferase from M. extorquens AM1 showed activity with MFR and H4MPT isolated from the methanogenic archaeon Methanothermobacter marburgensis (apparent Km for formylMFR = 50 µm; apparent Km for H4MPT = 30 µm). The enzyme is encoded by the ffsA gene and exhibits a sequence identity of ≈ 40% with Ftr from methanogenic and sulfate-reducing archaea. The 32-kDa Ftr protein from M. extorquens AM1 copurified in a complex with three other polypeptides of 60 kDa, 37 kDa and 29 kDa. Interestingly, these are encoded by the genes orf1, orf2 and orf3 which show sequence identity with the formylMFR dehydrogenase subunits FmdA, FmdB and FmdC, respectively. The clustering of the genes orf2, orf1, ffsA, and orf3 in the chromosome of M. extorquens AM1 indicates that, in the bacterium, the respective polypeptides form a functional unit. Expression studies in Escherichia coli indicate that Ftr requires the other subunits of the complex for stability. Despite the fact that three of the polypeptides of the complex showed sequence similarity to subunits of Fmd from methanogens, the complex was not found to catalyze the oxidation of formylMFR. Detailed comparison of the primary structure revealed that Orf2, the homolog of the active site harboring subunit FmdB, lacks the binding motifs for the active-site cofactors molybdenum, molybdopterin and a [4Fe−4S] cluster. Cytochrome c was found to be spontaneously reduced by H4MPT. On the basis of this property, a novel assay for Ftr activity and MFR is described.