Publications

• Biosynthesis of the 5-(aminomethyl)-3-furanmethanol moiety of methanofuran.

  Miller D., Wang Y., Xu H., Harich K., White R.H.

  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 conde ... >> More

  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 [(15)N]alanine to produce [(15)N] 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 extracts and the measurement of the incorporation of stable isotopically labeled precursors into F1. After incubation of cell extracts with [1,2,3-(13)C3]pyruvate and DHAP, 4-([(2)H2]hydroxymethyl)-2-furancarboxylic acid phosphate (4-HFCA-P) or 4-([(2)H2]hydroxymethyl)-2-furancarboxaldehyde phosphate (4-HFC-P) was found to be incorporated into F1-P. 4-HFCA-P and 4-HFC-P were confirmed in cell extracts after removal of the phosphate. The low level of incorporation of [1,2,3-(13)C3]pyruvate into F1-P in these experiments is explained by the fact that the labeled pyruvate must first be converted into Ga-3-P through gluconeogenesis before being incorporated into 4-HFC-P. Cell extracts incubated with 4-HFC-P and a mixture of [(15)N]aspartate, [(15)N]glutamate, and [(15)N]alanine produced [(15)N]F1-P. We also demonstrated that aqueous solutions of methylglyoxal or pyruvate heated with dihydroxyacetone led to the formation of 4-HFC and 4-HFCA, suggesting a possible prebiotic route to this moiety of methanofuran. << Less

  Biochemistry 53:4635-4647(2014) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structure of the methanofuran/methanopterin-biosynthetic enzyme MJ1099 from Methanocaldococcus jannaschii.

  Bobik T.A., Morales E.J., Shin A., Cascio D., Sawaya M.R., Arbing M., Yeates T.O., Rasche M.E.

  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 sol ... >> More

  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 Å 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. << Less

  Acta Crystallogr. F 70:1472-1479(2014) [PubMed] [EuropePMC]

• Identification and characterization of a tyramine-glutamate ligase (MfnD) involved in methanofuran biosynthesis.

  Wang Y., Xu H., Harich K.C., White R.H.

  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 attach ... >> More

  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. jannaschii. Our steady-state kinetic analysis of the recombinant enzyme showed that MfnD exhibits a catalytic ability comparable to other ATP-grasp enzymes such as the Escherichia coli glutathione synthetase (GS), with a similar apparent kcat and KM. In addition, its activity is divalent metal-dependent, with the highest activity observed with Mn(2+). The previously solved crystal structure of MfnD from Archaeoglobus fulgidus exhibits a classical ATP-grasp fold with three structural domains; the ATP-binding and metal-binding motifs are conserved in MfnD as seen in other ATP-grasp enzymes. We used site-directed mutagenesis and kinetic analysis to demonstrate that Arg251 is an important residue for both catalysis and glutamate binding. By comparing the active site of MfnD with GS and by molecular docking substrates to the MfnD active site, we predicted the possible glutamate- and tyramine-binding pocket. This is the first report describing the enzymology of the incorporation of the initial l-glutamate molecule into the methanofuran structure. It also provides the first example of an ATP-grasp enzyme activating the γ-carboxylate of glutamate as substrate. << Less

  Biochemistry 53:6220-6230(2014) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Mechanism of the enzymatic synthesis of 4-(hydroxymethyl)-2-furancarboxaldehyde-phosphate (4-HFC-P) from glyceraldehyde-3-phosphate catalyzed by 4-HFC-P synthase.

  Wang Y., Jones M.K., Xu H., Ray W.K., White R.H.

  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), ... >> More

  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. << Less

  Biochemistry 54:2997-3008(2015) [PubMed] [EuropePMC]