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Characterization of an O-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 (?70%) 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.

SUBMITTER: Studenik S

PROVIDER: S-EPMC3434752 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Characterization of an O-demethylase of Desulfitobacterium hafniense DCB-2.

Studenik Sandra S Vogel Michaela M Diekert Gabriele G

Journal of bacteriology 20120420 13

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 med ...[more]

PMID: 22522902

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Project description:IntroductionDesulfitobacterium hafniense was isolated for its ability to use organohalogens as terminal electron acceptors via organohalide respiration (OHR). In contrast to obligate OHR bacteria, Desulfitobacterium spp. show a highly versatile energy metabolism with the capacity to use different electron donors and acceptors and to grow fermentatively. Desulfitobacterium genomes display numerous and apparently redundant members of redox enzyme families which confirm their metabolic potential. Nonetheless, the enzymes responsible for many metabolic traits are not yet identified.MethodsIn the present work, we conducted an extended proteomic study by comparing the proteomes of Desulfitobacterium hafniense strain DCB-2 cultivated in combinations of electron donors and acceptors, triggering five alternative respiratory metabolisms that include OHR, as well as fermentation. Tandem Mass Tag labelling proteomics allowed us to identify and quantify almost 60% of the predicted proteome of strain DCB-2 (2,796 proteins) in all six growth conditions. Raw data are available via ProteomeXchange with identifier PXD030393.Results and discussionThis dataset was analyzed in order to highlight the proteins that were significantly up-regulated in one or a subset of growth conditions and to identify possible key players in the different energy metabolisms. The addition of sodium sulfide as reducing agent in the medium - a very widespread practice in the cultivation of strictly anaerobic bacteria - triggered the expression of the dissimilatory sulfite reduction pathway in relatively less favorable conditions such as fermentative growth on pyruvate, respiration with H2 as electron donor and OHR conditions. The presence of H2, CO2 and acetate in the medium induced several metabolic pathways involved in carbon metabolism including the Wood-Ljungdahl pathway and two pathways related to the fermentation of butyrate that rely on electron-bifurcating enzymes. While the predicted fumarate reductase appears to be constitutively expressed, a new lactate dehydrogenase and lactate transporters were identified. Finally, the OHR metabolism with 3-chloro-4-hydroxyphenylacetate as electron acceptor strongly induced proteins encoded in several reductive dehalogenase gene clusters, as well as four new proteins related to corrinoid metabolism. We believe that this extended proteomic database represents a new landmark in understanding the metabolic versatility of Desulfitobacterium spp. and provides a solid basis for addressing future research questions.

Dataset Information

Characterization of an O-demethylase of Desulfitobacterium hafniense DCB-2.

Publications

Characterization of an O-demethylase of Desulfitobacterium hafniense DCB-2.

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