Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1

Abstract

Cell extracts of Methylobacterium extorquens AM1 were recently found to catalyze the dehydrogenation of methylene tetrahydromethanopterin (methylene H 4 MPT) with NAD 1 and NADP 1. The purification of a 32-kDa NADP-specific methylene H 4 MPT dehydrogenase (MtdA) was described already. Here we report on the characterization of a second methylene H 4 MPT dehydrogenase (MtdB) from this aerobic a-proteobacterium. Purified MtdB with an apparent molecular mass of 32 kDa was shown to catalyze the oxidation of methylene H 4 MPT to methenyl H 4 MPT with NAD 1 and NADP 1 via a ternary complex catalytic mechanism. The K m for methylene H 4 MPT was 50 mm with NAD 1 (V max 1100 U´mg 21) and 100 mm with NADP 1 (V max 950 U´mg 21 ). The K m value for NAD 1 was 200 mm and for NADP 1 20 mm. In contrast to MtdA, MtdB could not catalyze the dehydrogenation of methylene tetrahydrofolate. Via the N-terminal aminoacid sequence, the MtdB encoding gene was identified to be orfX located in a cluster of genes whose translated products show high sequence identities to enzymes previously found only in methanogenic and sulfate reducing archaea. Despite its location, MtdB did not show sequence similarity to archaeal enzymes. The highest similarity was to MtdA, whose encoding gene is located outside of the archaeal island. Mutants defective in MtdB were unable to grow on methanol and showed a pronounced sensitivity towards formaldehyde. On the basis of the mutant phenotype and of the kinetic properties, possible functions of MtdB and MtdA are discussed. We also report that both MtdB and MtdA can be heterologously overproduced in Escherichia coli making these two enzymes readily available for structural analysis.Keywords: C 1 -metabolism; methylotrophic bacteria; methanogenic archaea; tetrahydromethanopterin; tetrahydrofolate.The methylotrophic a-proteobacterium Methylobacterium extorquens AM1 possesses tetrahydrofolate (H 4 F) and dephospho tetrahydromethanopterin (H 4 MPT), two structural analogue C 1 carriers [1,2]. In cell extracts of this aerobic a-proteobacterium H 4 MPT-and H 4 F-dependent enzymes were found. It was proposed that they participate in two separate C 1 pathways, the one with the H 4 MPT-dependent enzymes being mainly involved in formaldehyde oxidation to CO 2 [1,2].Three [2]. MchA and FfsA were found to have high sequence similarity to the respective enzymes from methanogenic and sulfate reducing archaea and their encoding genes were shown to be located within a cluster of genes whose deduced gene products show, with an exception of OrfX, high sequence similarity to proteins found until now only in methanogenic and sulfate reducing archaea [1]. The gene encoding MtdA is not located within this gene cluster and MtdA does not show significant sequence similarity at the protein level to archaea specific F 420 -dependent or H 2 -forming methylene H 4 MPT dehydrogenases [4]. MtdA shows only minor sequence identity (, 15%) to methylene H 4 F dehydrogenases from bacteria and eucarya [5±9], which is consistent with the fact that ...

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

“…The assay is based on the NAD ϩ -reducing activity of methylene-H 4 MPT dehydrogenase B (MtdB) (16) (Fig. 1).…”

“…The oxidation of methylene-H 4 MPT is coupled to the reduction of NAD ϩ via MtdB from M. extorquens, producing an increase in A 340 . MtdB is highly specific for H 4 MPT and does not react with tetrahydrofolate (16). Thus, the enzyme can be used to distinguish between H 4 MPT and tetrahydrofolate in bacterial cells.…”

“…These organisms may contain previously unidentified forms of H 4 MPT. At least six derivatives of H 4 MPT have been characterized by structural analyses (7,19,20,32), and the MtdB enzyme used in this work reacts with at least three of these analogs (H 4 MPT from Methanothermobacter marburgensis [16], H 4 SPT from Methanosarcina thermophila [ Fig. 2], and dephospho-H 4 MPT from M. extorquens [ Fig.…”

Novel Dephosphotetrahydromethanopterin Biosynthesis Genes Discovered via Mutagenesis in Methylobacterium extorquens AM1

“…While the functions of some genes in the cluster are well established in dH 4 MPT-linked C 1 transfer ( Fig. 1) (5,9,16,17,22), the functions of others remain unknown. We hypothesized that these genes of unknown function homologous to genes of unknown function in archaea might encode the yet-unidentified enzymes in the dH 4 MPT biosynthesis pathway.…”

Characterization of the formyltransferase from Methylobacterium extorquens AM1

“…The following enzymes have been proposed to participate in the metabolic pathway: formaldehyde-activating enzyme (Fae) [1], methyleneH 4 MPT dehydrogenases (MtdA and MtdB) [2,3], and three enzymes with high sequence identity with enzymes involved in methanogenesis: methenylH 4 MPT cyclohydrolase (Mch), formylMFR-H 4 MPT formyltransferase (Ftr), and formylMFR dehydrogenase (Fmd) [4] (Fig. 1).…”