Cloning and Sequencing of the Genes Encoding the Periplasmic-Cytochrome<i>B</i>-Containing Selenate Reductase of<i>Thauera selenatis</i>

Krafft, Torsten; Bowen, A. R.; Theis, Fabian J.; Macy, Joan M.

doi:10.3109/10425170009015604

Cited by 93 publications

(74 citation statements)

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“…This proposal takes into account our finding that the selenate reductase in E. coli requires a molybdopterin cofactor and is located in the cytoplasm. This proposal is also in line with the demonstration that the selenate reductase of T. selenatis is composed of several polypeptides encoded by the serABDC loci (Krafft et al, 2000). Several attempts were made to provide biochemical evidence that YgfKMN is a selenate-reducing complex.…”

Section: Identification Of a Putative Selenate Reductase In E Colimentioning

confidence: 50%

Involvement of a putative molybdenum enzyme in the reduction of selenate by Escherichia coli

et al. 2002

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Selenium oxyanions, particularly selenite, can be highly toxic to living organisms. Few bacteria reduce both selenate and selenite into the less toxic elemental selenium. Insights into the mechanisms of the transport and the reduction of selenium oxyanions in Escherichia coli were provided by a genetic analysis based on transposon mutagenesis. Ten mutants impaired in selenate reduction were analysed. Three of them were altered in genes encoding transport proteins including a porin, an inner-membrane protein and a sulfate carrier. Two mutants were altered in genes required for molybdopterin biosynthesis, strongly suggesting that the selenate reductase of E. coli is a molybdoenzyme. However, mutants deleted in various oxomolybdenum enzymes described so far in this species still reduced selenate. Finally, a mutant in the gene ygfK encoding a putative oxidoreductase was obtained. This gene is located upstream of ygfN and ygfM in the ygfKLMN putative operon. YgfN and YgfM code for a molybdopterin-containing enzyme and a polypeptide carrying a FAD domain, respectively. It is therefore proposed that the selenate reductase of E. coli is a structural complex including the proteins YgfK, YgfM and YgfN. In addition, all the various mutants were still able to reduce selenite into elemental selenium. This implies that the transport and reduction of this compound are clearly distinct from those of selenate.Keywords : molybdenum iron-sulfur protein, oxyanion reduction, selenite INTRODUCTIONSelenium is an essential trace nutrient for most living organisms (McKeehan et al., 1976 ;Shamberger, 1983). Small amounts of selenium are required to synthesize the amino acid selenocysteine present in a few proteins such as formate dehydrogenases and glycine reductase in prokaryotes (reviewed by Stadtman, 1996). In aerated environments, selenium occurs predominantly and naturally as the high-valence soluble forms selenate (SeO# − % , jVI) and selenite (SeO# − $ , jIV). These inorganic oxidized forms are abundant in some habitats, particularly through contamination of soil and drainage waters as a result of widespread use in industrial and agricultural processes (Losi & Frankenberger, 1997 toxic and mutagenic for bacteria and mammals (Noda et al., 1979 ;Stadtman, 1974). Consequently, selenium accumulation can cause important ecological problems such as in the Kesterson reservoir, in the San Joaquin Valley (California, USA), where selenium concentration resulted in extensive deformities and deaths in waterfowl and other wildlife (Ohlendorf & Santolo, 1994 ;Saiki & Lowe, 1987).In the biogeochemical cycle of selenium, various redox reactions are carried out by microorganisms. Several bacteria, including Escherichia coli (Turner et al., 1998) are able to reduce both selenate and selenite into elemental selenium (Se!), while certain species like Rhodobacter sphaeroides (Bebien et al., 2001 ;Van Fleet-Stalder et al., 2000) or Ralstonia metallidurans (Roux et al., 2001) reduce only selenite. The reduction of the bioavailable selenium oxyanions int...

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Section: Identification Of a Putative Selenate Reductase In E Colimentioning

confidence: 50%

Involvement of a putative molybdenum enzyme in the reduction of selenate by Escherichia coli

et al. 2002

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“…In analogy to other known three-subunit molybdoenzymes, it may be assumed that the ␣ subunit contains the molybdenum cofactor; the ␤ subunit carries the Fe/S clusters; and the ␥ subunit, the heme cofactor. The enzyme, which is most similar to ethylbenzene dehydrogenase with respect to subunit composition, cofactor content and location, is presumably the recently characterized selenate reductase from a closely related species, T. selenatis (19,20). However, ethylbenzene dehydrogenase did not catalyze reduction of selenate or nitrate.…”

Section: Discussionmentioning

confidence: 99%

“…(19,20). Other metals detected by ICP-OES in significant amounts were magnesium and calcium (2.5 mol/mol each), but no further transition metals or selenium were present in purified enzyme.…”

Section: ) Lanes 1-3 Strain Pbn1 Grown On Ethylbenzene (Lane 1) mentioning

confidence: 99%

Ethylbenzene Dehydrogenase, a Novel Hydrocarbon-oxidizing Molybdenum/Iron-Sulfur/Heme Enzyme

Kniemeyer

Heider

2001

Journal of Biological Chemistry

176

128

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The initial enzyme of ethylbenzene metabolism in denitrifying Azoarcus strain EbN1, ethylbenzene dehydrogenase, was purified and characterized. The soluble periplasmic enzyme is the first known enzyme oxidizing a nonactivated hydrocarbon without molecular oxygen as cosubstrate. It is a novel molybdenum/iron-sulfur/ heme protein of 155 kDa, which consists of three subunits (96, 43, and 23 kDa) in an ␣␤␥ structure. The Nterminal amino acid sequence of the ␣ subunit is similar to that of other molybdenum proteins such as selenate reductase from the related species Thauera selenatis. Ethylbenzene dehydrogenase is unique in that it oxidizes the hydrocarbon ethylbenzene, a compound without functional groups, to (S)-1-phenylethanol. Formation of the product was evident by coupling to an enantiomer-specific (S)-1-phenylethanol dehydrogenase from the same organism. The apparent K m of the enzyme for ethylbenzene is very low at <2 M. Oxygen does not affect ethylbenzene dehydrogenase activity in extracts but inactivates the purified enzyme, if the heme b cofactor is in the reduced state. A variant of ethylbenzene dehydrogenase exhibiting significant activity also with the homolog n-propylbenzene was detected in a related Azoarcus strain (PbN1).

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“…Thauera selenatis (a β-proteobacterium) is by far the best-studied selenate respiring bacterium (5)(6)(7)(8). The selenate reductase (SerABC) isolated from T. selenatis (6) is a soluble periplasmic enzyme.…”

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A bacterial process for selenium nanosphere assembly

Debieux

Dridge

Mueller

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

177

143

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During selenate respiration by Thauera selenatis, the reduction of selenate results in the formation of intracellular selenium (Se) deposits that are ultimately secreted as Se nanospheres of approximately 150 nm in diameter. We report that the Se nanospheres are associated with a protein of approximately 95 kDa. Subsequent experiments to investigate the expression and secretion profile of this protein have demonstrated that it is up-regulated and secreted in response to increasing selenite concentrations. The protein was purified from Se nanospheres, and peptide fragments from a tryptic digest were used to identify the gene in the draft T. selenatis genome. A matched open reading frame was located, encoding a protein with a calculated mass of 94.5 kDa. N-terminal sequence analysis of the mature protein revealed no cleavable signal peptide, suggesting that the protein is exported directly from the cytoplasm. The protein has been called Se factor A (SefA), and homologues of known function have not been reported previously. The sefA gene was cloned and expressed in Escherichia coli, and the recombinant His-tagged SefA purified. In vivo experiments demonstrate that SefA forms larger (approximately 300 nm) Se nanospheres in E. coli when treated with selenite, and these are retained within the cell. In vitro assays demonstrate that the formation of Se nanospheres upon the reduction of selenite by glutathione are stabilized by the presence of SefA. The role of SefA in selenium nanosphere assembly has potential for exploitation in bionanomaterial fabrication.nanoparticles | biomineralization | anaerobic respiration

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Cloning and Sequencing of the Genes Encoding the Periplasmic-CytochromeB-Containing Selenate Reductase ofThauera selenatis

Cited by 93 publications

References 49 publications

Involvement of a putative molybdenum enzyme in the reduction of selenate by Escherichia coli

Involvement of a putative molybdenum enzyme in the reduction of selenate by Escherichia coli

Ethylbenzene Dehydrogenase, a Novel Hydrocarbon-oxidizing Molybdenum/Iron-Sulfur/Heme Enzyme

A bacterial process for selenium nanosphere assembly

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