Characterization of Two Methanopterin Biosynthesis Mutants of Methylobacterium extorquens AM1 by Use of a Tetrahydromethanopterin Bioassay

Abstract

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.Methylobacterium extorquens AM1 is a facultative methylotrophic bacterium capable of growth on succinate and onecarbon (C 1 ) compounds. Growth on C 1 compounds requires several clusters of genes found on the chromosomal DNA (5, 6), and a number of these genes code for enzymes which have archaeal homologs that depend on tetrahydromethanopterin (H 4 MPT) or structurally related coenzymes (6,7,24,25). Previously, these coenzymes had been found only in methanogenic or hyperthermophilic sulfur-dependent archaea (9,19,22,29,32).M. extorquens cells contain a form of H 4 MPT called dephospho-H 4 MPT (7). Although it has been assumed that this bacterium produces dephospho-H 4 MPT biosynthetic enzymes, these proteins have not yet been identified, and their evolutionary relationship to archaeal enzymes is unknown. In archaea, the genes encoding only 4 of the 18 putative H 4 MPT biosynthesis enzymes have been identified (14,15,28,33,34). One of these enzymes, ribofuranosylaminobenzene 5Ј-phosphate (RFAP) synthase, catalyzes the first committed step of H 4 MPT biosynthesis (26, 28). In M. extorquens, a gene encoding an RFAP synthase homolog (orf4, also called mptG) has been found clustered among several genes encoding H 4 MPTdependent enzymes (6, 7). The orf4 gene product is 29% identical to RFAP synthase from Archaeoglobus fulgidus (28). The protein encoded by a second putative H 4 MPT biosynthesis gene (dmrA) shows homology to bacterial dihydrofolate reductases and has been proposed by Marx et al. (21) to encode dihydromethanopterin reductase, which would catalyze the final step of H 4 MPT biosynthesis. The dmrA mutant cannot grow on C 1 compounds and exhibits a methanol-and formaldehyde-sensitive phenotype characteristic of mutants deficient in H 4 MPT-dependent metabolism.To test the hypotheses that orf4 and dmrA encode H 4 MPT biosynthesis enzymes, we have developed an enzymatic assay to measure H 4 MPT levels in M. extorquens mutants. The assay is based on the NAD ϩ -reducing activity of methylene-H 4 MPT dehydrogenase B (MtdB) (16) (Fig. 1). Here, we provide the initial biochemical evidence for two H 4 MPT biosynthetic genes in M. extorquens and demonstrate that the protein encoded by orf4 has RFAP synthase activity.Methods. Methanosarcina thermophila cells were grown anaerobically on acetate as previously described (28). M. extorquens AM1 wild-type and mutant strains were generously provided by the laboratory of Mary Lidstrom. It has previously been ...

Analysis of Gene Islands Involved in Methanopterin-Linked C 1 Transfer Reactions Reveals New Functions and Provides Evolutionary Insights

“…All of the mutants investigated showed increased formaldehyde sensitivity similar to the previously described mutant in mch; however, the new mutants showed slightly lower formaldehyde sensitivity (between 2.5 and 3 mM versus 2 mM for the mch mutant) ( Table 1). The functions of mtdB and mptG have been determined (12,22), and a function has been proposed for orf5 in H 4 MPT biosynthesis (6). Based on the phenotypes of the new mutants and also on gene location within the formaldehyde oxidation islands, orfY, orf1, and afpA are likely to be involved in the H 4 MPT-linked pathway as well.…”

Methenyl-Dephosphotetrahydromethanopterin Is a Regulatory Signal for Acclimation to Changes in Substrate Availability in Methylobacterium extorquens AM1

“…The mtdB gene was amplified and cloned into pET28b as described by Rasche et al (34). The construct was transformed into Escherichia coli BL21-AI.…”

Novel Dephosphotetrahydromethanopterin Biosynthesis Genes Discovered via Mutagenesis in Methylobacterium extorquens AM1

“…Some of these archaeal-type genes have been shown to catalyze reactions of dH 4 MPTlinked C 1 transfers, similar to reactions of methanogenesis (9,16,17,22). One was shown to catalyze the first reaction in dH 4 MPT biosynthesis (18), while the functions of others remained unknown. We sequenced a DNA region adjacent to this cluster and discovered four more genes whose translated polypeptides revealed high similarity to archaeal polypeptides of unknown function.…”

“…The facultative methylotrophic bacterium Methylobacterium extorquens AM1 has been shown to contain a modified form of H 4 MPT, dephosphoH 4 MPT (dH 4 MPT) (5). Two genes have been shown to be involved so far in dH 4 MPT biosynthesis via gene overexpression, activity detection, and mutant analysis (1,15,18), demonstrating that M. extorquens AM1 presents an excellent model for identifying other genes in the dH 4 MPT biosynthetic pathway. This ␣-proteobacterium contains a cluster of genes whose translated polypeptides have homologs in archaea (3)(4)(5).…”

“…To determine whether the mutants were capable of synthesizing dH 4 MPT, we used an enzymatic assay developed previously to detect H 4 MPT analogs in archaea and M. extorquens AM1 (18). Of the eight mutants tested, only ORF7 and ORF17 mutants produced dH 4 MPT, while ORF5, ORF9, ORF19, ORF20, ORF21, and ORF22 mutants did not produce detectable levels of dH 4 MPT (Table 1).…”