Publications

• Mechanism of 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate synthase, a key enzyme in the methanopterin biosynthetic pathway.

  Dumitru R.V., Ragsdale S.W.

  The first committed step in methanopterin biosynthesis is catalyzed by 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate (RFA-P) synthase. Unlike all known phosphoribosyltransferases, beta-RFA-P synthase catalyzes the unique formation of a C-riboside instead of an N-riboside in the condensation of ... >> More

  The first committed step in methanopterin biosynthesis is catalyzed by 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate (RFA-P) synthase. Unlike all known phosphoribosyltransferases, beta-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-alpha-D-ribosyl-1-pyrophosphate (PRPP) to produce 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate (beta-RFA-P), CO(2), and inorganic pyrophosphate (PP(i)). 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 CO(2), then beta-RFA-P, and finally PP. The Michaelis parameters are as follows: K(m)pABA, 0.15 mm; K(m)PRPP, 1.50 mm; V(max), 375 nmol/min/mg; k(cat), 0.23 s(-1). CO(2) showed uncompetitive inhibition, K(i) = 0.990 mm, under varied PRPP and saturated pABA, and a mixed type of inhibition, K(1) = 1.40 mm and K = 3.800 mm, under varied pABA and saturated PRPP. RFA-P showed uncompetitive inhibition, K(i) = 0.210 mm, under varied PRPP and saturated pABA, and again uncompetitive, K(i) = 0.300 mm, under saturated PRPP and varied pABA. PP(i) exhibits competitive inhibition, K(i) = 0.320 mm, under varied PRPP and saturated pABA, and a mixed type of inhibition, K(1) = 0.60 mm and K(2) = 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. << Less

  J. Biol. Chem. 279:39389-39395(2004) [PubMed] [EuropePMC]

• 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.

  White R.H.

  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-hydroxyben ... >> More

  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. << Less

  Biochemistry 50:6041-6052(2011) [PubMed] [EuropePMC]

• Mechanism for the enzymatic formation of 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate during the biosynthesis of methanopterin.

  Rasche M.E., White R.H.

  A central step in the biosynthesis of the modified folate methanopterin is the condensation of p-aminobenzoic acid (pAB) and 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRPP) which produce 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate (beta-RFA-P) [White, R. H. (1996) Biochemistry 35, 3447-3456 ... >> More

  A central step in the biosynthesis of the modified folate methanopterin is the condensation of p-aminobenzoic acid (pAB) and 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRPP) which produce 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate (beta-RFA-P) [White, R. H. (1996) Biochemistry 35, 3447-3456]. This reaction, catalyzed by the enzyme beta-RFA-P synthase, is unique among known phosphoribosyltransferases in that a decarboxylation of one of the substrates (pAB) occurs during the reaction and a C-riboside rather than an N-riboside is the product. In this work, the reaction catalyzed by the enzyme from Methanosarcina thermophila is shown to be analogous to other phosphoribosyltransferase reactions in that pyrophosphate is released as a product of the reaction, which is dependent upon magnesium ions. The molecular weight of the enzyme was estimated to be 65 000 using gel filtration chromatography, and the pH optimum was 4.8. Kinetic analysis indicated that the reaction involved a sequential pattern of substrate binding. Benzoic acid and several para-substituted benzoic acids inhibited beta-RFA-P synthase activity, while aniline, 4-aminobenzamide, and the methyl ester of pAB did not, indicating that an ionized carboxylic group plays a role in the binding of pAB. The observation that the enzyme was not inhibited by carbonyl reagents and that 4-hydroxybenzoic acid served as an alternate substrate, producing 4-(beta-D-ribofuranosyl)hydroxybenzene 5'-phosphate as the product, indicated that pyridoxal phosphate was not directly involved in the reaction mechanism. Incubation of the enzyme with PRPP and either pAB or 4-aminothiobenzoic acid in the presence of sodium cyanoborohydride led to the decreased production of beta-RFA-P and the accumulation of a reduced form of the proposed cyclohexadienimine reaction intermediates. These compounds were characterized by their acid-catalyzed decomposition which produces beta-D-ribofuranosylbenzene 5'-phosphate. On the basis of these results, a concerted mechanism is proposed for beta-RFA-P synthase in which an SN1-like reaction produces oxonium ion character at C-1 of PRPP which undergoes an ipso electrophilic aromatic substitution reaction at the carboxylic acid-bound carbon of pAB. Decarboxylation of the resulting cyclohexadienimine intermediate leads to the formation of beta-RFA-P. << Less

  Biochemistry 37:11343-11351(1998) [PubMed] [EuropePMC]

• Purification, overproduction, and partial characterization of beta-RFAP synthase, a key enzyme in the methanopterin biosynthesis pathway.

  Scott J.W., Rasche M.E.

  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 biosynthet ... >> More

  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 beta-ribofuranosylaminobenzene 5'-phosphate synthase (beta-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 beta-RFAP, CO2, and inorganic pyrophosphate. The enzyme catalyzing this reaction has not been purified to homogeneity, and the gene encoding beta-RFAP synthase has not yet been identified. In the present work, we report on the purification to homogeneity of beta-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 beta-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 beta-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 beta-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. << Less

  J. Bacteriol. 184:4442-4448(2002) [PubMed] [EuropePMC]