Purification, Overproduction, and Partial Characterization of β-RFAP Synthase, a Key Enzyme in the Methanopterin Biosynthesis Pathway†

Abstract

Methanopterin is a folate analog involved in the C 1 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 ␤-ribofuranosylaminobenzene 5-phosphate synthase (␤-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 ␤-RFAP, CO 2 , and inorganic pyrophosphate. The enzyme catalyzing this reaction has not been purified to homogeneity, and the gene encoding ␤-RFAP synthase has not yet been identified. In the present work, we report on the purification to homogeneity of ␤-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 ␤-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 ␤-RFAP synthase gene identified homologs in additional archaea and in a gene cluster required for C 1 metabolism by the bacterium Methylobacterium extorquens. The identification of a gene encoding a potential ␤-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.Methanopterin is a folate analog involved in the C 1 metabolism of methanogenic archaea, sulfate-reducing archaea, and methylotrophic bacteria (5,7,19,31,39). This coenzyme is used during the production of methane by methanogenic archaea and during the oxidation of growth substrates by sulfurmetabolizing archaea and certain methylotrophic bacteria. The recent discovery of methanopterin in the bacterium Methylobacterium extorquens (5) was surprising because methanopterin had been thought to be exclusive to Archaea. This discovery has raised interesting questions about the evolutionary relationships between archaea and bacteria that use methanopterin. While M. extorquens is the only bacterium in which methanopterin itself has been detected, methanopterin-dependent enzyme activity has been observed in a number of other methylotrophic bacteria (34).One approach to investigating the evolutionary relationships among organisms that use methanopterin is to compare the enzymes and genes involved in methanopterin biosynthesis. At present, the pathway of methanopterin biosynthesis in the Bacteria is unknown. However, a methanopterin biosynthetic pathway has been described for the methane-producing archaeon Methanosarcina thermophila (38, 39). To date, the g...

Mechanism of 4-(β-D-Ribofuranosyl)aminobenzene 5′-Phosphate Synthase, a Key Enzyme in the Methanopterin Biosynthetic Pathway

“…1). The specific activity for the enzyme in the cell extract of induced cells was 10.0 nmol of ␤-RFA-P produced per min/mg of protein, in agreement with the enzyme from Archaeoglobus fulgidus (4). Overexpression of the protein was probed by using anti-His tag antibodies and antibodies raised against the pure protein, which stained a band around 37 kDa, indicating that this is the protein of interest.…”

“…Even though H 4 MPT is found in Archaea and one class of Bacterium (e.g. Methylobacterium extorquens), the biosynthetic pathway for these two folates (folate and methanopterin) is different suggesting that they play different functional roles in physiology of the cell (4,5). We are targeting this enzyme to inhibit specifically methanogenesis (6), which has the potential impact of reducing the levels of the greenhouse gas methane, which has doubled over the last 200 years.…”

“…H 4 MPT is involved in multiple steps in methane formation as in one carbon reactions involved in amino acid and nucleotide metabolism. Even though H 4 MPT is found in Archaea and one class of Bacterium (e.g.…”

“…H 4 MPT is involved in multiple steps in methane formation as in one carbon reactions involved in amino acid and nucleotide metabolism. Even though H 4 MPT is found in Archaea and one class of Bacterium (e.g. Methylobacterium extorquens), the biosynthetic pathway for these two folates (folate and methanopterin) is different suggesting that they play different functional roles in physiology of the cell (4,5).…”

Fosmids of novel marine Planctomycetes from the Namibian and Oregon coast upwelling systems and their cross-comparison with planctomycete genomes

“…In all four Planctomycete genomes, ORF19 was found to be linked to mptG. ORF19 is involved in H 4 MPT biosynthesis , while mptG codes for b-RFAP synthase, an enzyme that catalyzes the first reaction distinguishing the methanopterin biosynthesis pathway from that of folate biosynthesis (Scott and Rasche, 2002;Chistoserdova et al, 2004 Genes involved in the H 4 MPT-linked C1 metabolism were also found on two of the four fosmids from the Namibian OMZ (Table 3 and Figure 2). Fosmid 6FN harbors an instance of fmdC plus the gene mobA that encodes the molybdopterin-guanine dinucleotide biosynthesis protein A. Fosmid 8FN contains a larger C1 module comprising ORF20-pabB-pabA-ORF19-mptG.…”