Characterization of anO-Demethylase of Desulfitobacterium hafniense DCB-2

Abstract

Besides acetogenic bacteria, only Desulfitobacterium has been described to utilize and cleave phenyl methyl ethers under anoxic conditions; however, no ether-cleaving O-demethylases from the latter organisms have been identified and investigated so far. In this study, genes of an operon encoding O-demethylase components of Desulfitobacterium hafniense strain DCB-2 were cloned and heterologously expressed in Escherichia coli. Methyltransferases I and II were characterized. Methyltransferase I mediated the ether cleavage and the transfer of the methyl group to the superreduced corrinoid of a corrinoid protein. Desulfitobacterium methyltransferase I had 66% identity (80% similarity) to that of the vanillate-demethylating methyltransferase I (OdmB) of Acetobacterium dehalogenans. The substrate spectrum was also similar to that of the latter enzyme; however, Desulfitobacterium methyltransferase I showed a higher level of activity for guaiacol and used methyl chloride as a substrate. Methyltransferase II catalyzed the transfer of the methyl group from the methylated corrinoid protein to tetrahydrofolate. It also showed a high identity (ϳ70%) to methyltransferases II of A. dehalogenans. The corrinoid protein was produced in E. coli as cofactor-free apoprotein that could be reconstituted with hydroxocobalamin or methylcobalamin to function in the methyltransferase I and II assays. Six COG3894 proteins, which were assumed to function as activating enzymes mediating the reduction of the corrinoid protein after an inadvertent oxidation of the corrinoid cofactor, were studied with respect to their abilities to reduce the recombinant reconstituted corrinoid protein. Of these six proteins, only one was found to catalyze the reduction of the corrinoid protein.A cetogenic bacteria were the first anaerobes described to utilize phenyl methyl ethers as energy substrates (2). These organisms mediate the cleavage of the substrate ether bond and utilize the methyl group, which is oxidized to CO 2 in the oxidative part of catabolism. The reducing equivalents derived from methyl group oxidation are transferred to CO 2 upon the formation of an enzyme-bound carbon monoxide, which is then combined with further methyl groups to finally yield acetate (6). The key enzymes in the methylotrophic phenyl methyl ether metabolism of acetogens such as Acetobacterium dehalogenans (23) and Moorella thermoacetica (13) are the O-demethylases. These inducible enzyme systems mediate the ether cleavage and transfer of the methyl group to tetrahydrofolate (FH 4 ). Until now, three of these acetogenic O-demethylase systems were purified and characterized (7,14,25). In general, they consist of four protein components: two methyltransferases (MTs) (MT I and MT II), a corrinoid protein (CP), and an activating enzyme (AE). Both MTs and CP are involved in the catalytic cycle (Fig. 1) (Fig. 1). In A. dehalogenans, the genes encoding MT I, MT II, and CP are usually organized into an operon (31). Only one AE gene has been detected so far; this gene is not part of a...

A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase

“…Each nonPyl MttB is encoded in gene clusters that are potential transcriptional units and has, within the unit or nearby, a gene encoding a member of the BCCT family of proteins (2). Additionally, each gene cluster encodes homologs of a methylotrophic corrinoid protein (30) and a methylcorrinoid:THF methyltransferase (31,32). Such proteins are part of the systems that initiate catabolism by Gram-positive bacteria such as D. hafniense during growth on methoxylated aromatic compounds (31,32).…”

Desulfitobacterium contributes to the microbial transformation of 2,4,5‐T by methanogenic enrichment cultures from a Vietnamese active landfill

“…The methyl group is finally oxidized to CO 2 by acetogenesis. The resulting reducing equivalents allow growth using terminal electron acceptors such as fumarate (Kreher et al ., 2010; Studenik et al ., 2012). Despite the presence of several O‐demethylases encoded in the genomes of Desulfitobacterium (Nonaka et al ., 2006; Kim et al ., 2012), none of these proteins was detected in the metaproteome (Tables S3 and S6), strongly suggesting that they do not play a role in 2,4,5‐T ether cleavage.…”

Cloning, expression, and characterization of a four-component O-demethylase from human intestinal bacterium Eubacterium limosum ZL-II

“…1 a). According to the currently identified O-demethylase (Schilhabel et al 2009;Studenik et al 2012), MT-I, CP, and MT-II were organized in one operon. Therefore, the genes in gene clusters 4 and 6 were predicted as the putative components of O-demethylase.…”

“…In the four-component O-demethylase system, genes encoding methyltransferase-I (MT-I), corrinoid protein (CP), and methyltransferase-II (MT-II) are usually organized in an operon, while the activating enzyme (AE) gene is separated from the O-demethylase gene transcription unit (Schilhabel et al 2009;Studenik et al 2012). Therefore, putative genetic loci encoding MT-I, CP, and MT-II in the genome of E. limosum KIST612 were screened, with the amino acid sequences of OdmB (MT-I) (GenBank accession no.…”

“…The ultraviolet-visible (UV) absorption spectroscopy was applied to determine the activities of putative O-demethylases based on the protocols described by Studenik et al (2012). The reaction volume was 100 μl in quartz cuvettes.…”