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Structure of the cobalamin-binding protein of a putative O-demethylase from Desulfitobacterium hafniense DCB-2.

ABSTRACT: This study describes the identification and the structural and spectroscopic analysis of a cobalamin-binding protein (termed CobDH) implicated in O-demethylation by the organohalide-respiring bacterium Desulfitobacterium hafniense DCB-2. The 1.5?Å resolution crystal structure of CobDH is presented in the cobalamin-bound state and reveals that the protein is composed of an N-terminal helix-bundle domain and a C-terminal Rossmann-fold domain, with the cobalamin coordinated in the base-off/His-on conformation similar to other cobalamin-binding domains that catalyse methyl-transfer reactions. EPR spectroscopy of CobDH confirms cobalamin binding and reveals the presence of a cob(III)alamin superoxide, indicating binding of oxygen to the fully oxidized cofactor. These data provide the first structural insights into the methyltransferase reactions that occur during O-demethylation by D. hafniense.

SUBMITTER: Sjuts H

PROVIDER: S-EPMC3727330 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Structure of the cobalamin-binding protein of a putative O-demethylase from Desulfitobacterium hafniense DCB-2.

Sjuts Hanno H Dunstan Mark S MS Fisher Karl K Leys David D

Acta crystallographica. Section D, Biological crystallography 20130720 Pt 8

This study describes the identification and the structural and spectroscopic analysis of a cobalamin-binding protein (termed CobDH) implicated in O-demethylation by the organohalide-respiring bacterium Desulfitobacterium hafniense DCB-2. The 1.5 Å resolution crystal structure of CobDH is presented in the cobalamin-bound state and reveals that the protein is composed of an N-terminal helix-bundle domain and a C-terminal Rossmann-fold domain, with the cobalamin coordinated in the base-off/His-on c ...[more]

PMID: 23897483

Similar Datasets

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

Project description: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.

| S-EPMC3434752 | biostudies-literature

Desulfitobacterium hafniense DCB-2

Project description:Degrades chlorinated pollutants

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Desulfitobacterium hafniense DCB-2

Project description:Desulfitobacterium hafniense DCB-2 Gene Expression Differences from Pyruvate Fermentation when Grown with Ferric Citrate, Selenate, Nitrate, or Uranyl Acetate

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Diversity of cobalamin riboswitches in the corrinoid-producing organohalide respirer Desulfitobacterium hafniense.

Project description:The strategic adaptation of prokaryotes in polluted niches involves the efficient regulation of their metabolism. The obligate anaerobe and metabolically versatile Desulfitobacterium hafniense reductively dechlorinates halogenated organic compounds (so-called organohalides). Some D. hafniense strains carry out organohalide respiration (OHR), a process which requires the use of corrinoid as a cofactor in reductive dehalogenases, the key enzymes in OHR. We report here the diversity of the cobalamin riboswitches that possibly regulate the corrinoid metabolism for D. hafniense. The analysis of available D. hafniense genomes indicates the presence of 18 cobalamin riboswitches located upstream of genes whose products are mainly involved in corrinoid biosynthesis and transport. To obtain insight into their function, the secondary structures of three of these RNA elements were predicted by Mfold, as well as analyzed by in-line probing. These RNA elements both display diversity in their structural elements and exhibit various affinities toward adenosylcobalamin that possibly relates to their role in the regulation of corrinoid metabolism. Furthermore, adenosylcobalamin-induced in vivo repression of RNA synthesis of the downstream located genes indicates that the corrinoid transporters and biosynthetic enzymes in D. hafniense strain TCE1 are regulated at the transcriptional level. Taken together, the riboswitch-mediated regulation of the complex corrinoid metabolism in D. hafniense could be of crucial significance in environments polluted with organohalides both to monitor their intracellular corrinoid level and to coexist with corrinoid-auxotroph OHR bacteria.

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Genome sequence of Desulfitobacterium hafniense DCB-2, a Gram-positive anaerobe capable of dehalogenation and metal reduction.

Project description:BACKGROUND: The genome of the Gram-positive, metal-reducing, dehalorespiring Desulfitobacterium hafniense DCB-2 was sequenced in order to gain insights into its metabolic capacities, adaptive physiology, and regulatory machineries, and to compare with that of Desulfitobacterium hafniense Y51, the phylogenetically closest strain among the species with a sequenced genome. RESULTS: The genome of Desulfitobacterium hafniense DCB-2 is composed of a 5,279,134-bp circular chromosome with 5,042 predicted genes. Genome content and parallel physiological studies support the cell's ability to fix N2 and CO2, form spores and biofilms, reduce metals, and use a variety of electron acceptors in respiration, including halogenated organic compounds. The genome contained seven reductive dehalogenase genes and four nitrogenase gene homologs but lacked the Nar respiratory nitrate reductase system. The D. hafniense DCB-2 genome contained genes for 43 RNA polymerase sigma factors including 27 sigma-24 subunits, 59 two-component signal transduction systems, and about 730 transporter proteins. In addition, it contained genes for 53 molybdopterin-binding oxidoreductases, 19 flavoprotein paralogs of the fumarate reductase, and many other FAD/FMN-binding oxidoreductases, proving the cell's versatility in both adaptive and reductive capacities. Together with the ability to form spores, the presence of the CO2-fixing Wood-Ljungdahl pathway and the genes associated with oxygen tolerance add flexibility to the cell's options for survival under stress. CONCLUSIONS: D. hafniense DCB-2's genome contains genes consistent with its abilities for dehalogenation, metal reduction, N2 and CO2 fixation, anaerobic respiration, oxygen tolerance, spore formation, and biofilm formation which make this organism a potential candidate for bioremediation at contaminated sites.

| S-EPMC3306737 | biostudies-literature

Structure of Desulfitobacterium hafniense PylSc, a pyrrolysyl-tRNA synthetase.

Project description:Pyrrolysine, the 22nd genetically-encoded amino acid, is charged onto its specific tRNA by PylS, a pyrrolysyl-tRNA synthetase. While PylS is found as a single protein in certain archaeal methanogens, in the gram-positive bacterium Desulfitobacterium hafniense, PylS is divided into two separate proteins, PylSn and PylSc, corresponding to the N-terminal and C-terminal domains of the single PylS protein found in methanogens. Previous crystallographic studies have provided the structure of a truncated C-terminal portion of the archaeal Methanosarcina mazei PylS associated with catalysis. Here, we report the apo 2.1A resolution structure of the intact D. hafniense PylSc protein and compare it to structures of the C-terminal truncated PylS from methanogenic species. In PylSc, the hydrophobic pocket binding the ring of pyrrolysine is more constrained than in the archaeal enzyme; other structural differences are also apparent.

| S-EPMC2572580 | biostudies-literature

Noncanonical vancomycin resistance cluster from Desulfitobacterium hafniense Y51.

Project description:The glycopeptide vancomycin is a drug of last resort for infection with gram-positive organisms, and three genes are vital to resistance: vanH, vanA, and vanX. These genes are found in a vanHAX cluster, which is conserved across pathogenic bacteria, glycopeptide antibiotic producers, and other environmental bacteria. The genome sequence of the anaerobic, gram-positive, dehalogenating bacterium Desulfitobacterium hafniense Y51 revealed a predicted vanA homolog; however, it exists in a vanAWK-murFX cluster, unlike those of other vancomycin-resistant organisms. Using purified recombinant VanA from D. hafniense Y51, we determined its substrate specificity and found it to have a 42-fold preference for D-lactate over D-alanine, confirming its activity as a D-Ala-D-Lac ligase and its annotation as VanA. Furthermore, we showed that D. hafniense Y51 is highly resistant to vancomycin, with a MIC for growth of 64 microg/ml. Finally, vanA(Dh) is expressed during growth in vancomycin, as demonstrated by reverse transcription-PCR. This finding represents a new glycopeptide antibiotic resistance gene cluster and expands the genetic diversity of resistance to this important class of antibiotic.

| S-EPMC2704641 | biostudies-other

Desulfitobacterium hafniense DCB-2 Gene Expression Differences from Pyruvate Fermentation when Grown with Ferric Citrate, Selenate, Nitrate, or Uranyl Acetate

Project description:Desulfitobacterium hafniense DCB-2, a halo-respirer and versatile inorganic ion reducer, grows in hazardous waste contaminated environments. Gene expression changes were determined when the strain was grown in the presence of high concentrations of Fe(+3), Se(+6), U(+6), or NO3(-) as alternative electron acceptors. (Thesis by Christina L. Harzman at Michigan State University, 2009)

2012-11-29 | GSE33988 | GEO

Desulfitobacterium hafniense

Project description:Hydrocarbon dehalogenator

| PRJNA16640 | ENA

Desulfitobacterium hafniense DCB-2 Gene Expression Differences from Pyruvate Fermentation when Grown with Ferric Citrate, Selenate, Nitrate, or Uranyl Acetate

Project description:Desulfitobacterium hafniense DCB-2, a halo-respirer and versatile inorganic ion reducer, grows in hazardous waste contaminated environments. M-BM- Gene expression changes were determined when the strain was grown in the presence of high concentrations of Fe(+3), Se(+6), U(+6), or NO3(-) as alternative electron acceptors. (Thesis by Christina L. Harzman at Michigan State University, 2009) RNA was used to directly compare gene expression in control samples of pyruvate fermentation (20 mM) to fermentation in the presence inorganic electron acceptors or respiration-inducing conditions. M-BM- The inorganic electron acceptors added to media for fermentation were 1 mM selenate or 0.5 mM uranyl acetate. The respiration-inducing conditions were 50 mM ferric citrate (20 mM lactate carbon source) or 10 mM nitrate (20 mM lactate carbon source). Microarray hybridizations were performed with a dye swap and 3 biological replicates for a total of 6 slides per condition for comparison.

2012-11-29 | E-GEOD-33988 | biostudies-arrayexpress

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