Thauera selenatis is one of two isolated bacterial species that can obtain energy by respiring anaerobically with selenate as the terminal electron acceptor. The reduction of selenate to selenite is catalyzed by a selenate reductase, previously shown to be located in the periplasmic space of the cell. This study describes the purification of the enzyme from T. selenatis grown anaerobically with selenate. The enzyme is a trimeric ␣␥ complex with an apparent M r of 180,000. The ␣, , and ␥ subunits are 96 kDa, 40 kDa, and 23 kDa, respectively, in size. The selenate reductase contains molybdenum, iron, and acid-labile sulfur as prosthetic group constituents. UV-visible absorption spectroscopy also revealed the presence of one cytochrome b per ␣␥ complex. The K m for selenate was determined to be 16 M, and the V max was 40 mol/min/mg of protein. The enzyme is specific for the reduction of selenate; nitrate, nitrite, chlorate, and sulfate were not reduced at detectable rates. These studies constitute the first description of a selenate reductase, which represents a new class of enzymes. The significance of this enzyme in relation to cell growth and energy generation is discussed.
Chrysiogenes arsenatis is the only bacterium known that respires anaerobically using arsenate as the terminal electron acceptor and the respiratory substrate acetate as the electron donor. During growth, the arsenate is reduced to arsenite; the reduction is catalyzed by an arsenate reductase. This study describes the purification and characterization of a respiratory arsenate reductase (Arr). The enzyme consists of two subunits with molecular masses of 87 kDa (ArrA) and 29 kDa (ArrB), and is a heterodimer A 1 β 1 with a native molecular mass of 123 kDa. The arsenate reductase contains molybdenum, iron, acid-labile sulfur and zinc as cofactor constituents. The K m of the enzyme for arsenate is 0.3 mM and the Vmax is 7013 µmol arsenate reduced · min Ϫ1 · mg protein Ϫ1 . Nitrate, sulfate, selenate and fumarate cannot serve as alternative electron acceptors for the arsenate reductase. Synthesis of the protein is regulated, as arsenate must be present during growth for the enzyme to be fully induced. The N-terminus of ArrA is similar to a number of procaryotic molybdenum-containing polypeptides (e.g. the formate dehydrogenases H and N of Escherichia coli). The N-terminus of ArrB is similar to iron-sulfur proteins. The respiratory arsenate reductase of C. arsenatis is different from the non-respiratory arsenate reductases of E. coli and Staphylococcus aureus.Keywords : respiratory arsenate reductase; arsenate respiration ; Chrysiogenes arsenatis.Arsenic is naturally present in soil, water and air and can occur in the oxidation states ϩ5 (arsenate), ϩ3 (arsenite), 0 (elemental arsenic) and Ϫ3 (arsine) [1]. The two soluble forms, arsenate and arsenite, are commonly found in water and soil. Both forms are toxic, although arsenite more so than arsenate [1].Arsenic resistance appears to be widespread among bacteria [2]. The mechanism for arsenate and arsenite resistance has, however, been investigated in depth only in organisms where resistance is conferred by proteins encoded by similar ars operons. These operons are located on the chromosome of Escherichia coli [2], on plasmid R773 of E. coli [3], on the IncN plasmid R46 found originally in Salmonella typhimurium [4], on plasmid pI258 of Staphylococcus aureus [5] and on plasmid pSX267 of Staphylococcus xylosus [6]. With each of these systems, arsenate, that has entered the cell via the phosphate-transport system, is first reduced to arsenite by a soluble cytoplasmic arsenate reductase, and the arsenite is then transported out of the cell via an energy-dependent arsenite transporter [7,8]. The arsenate reductase of these arsenic-resistance systems does not appear to be involved in energy conservation when catalyzing the reduction of arsenate to arsenite [7,8].Arsenate is also reduced to arsenite by a group of bacteria that grow anaerobically using the non-respiratory substrate lactate as the electron donor ; the lactate is oxidised to acetate. Energy is conserved during this reduction (i.e. arsenate respiration),
The hydrogenase (Hyd) isolated from the cytoplasmic membrane of Wolinellu succinogenes consists of three polypeptides (HydA, HydB and HydC) and contains cytochrome h (6.4 pmol/g protein), which was reduced upon the addition of H2. The enzyme catalyzed the reduction of 2,3-dimethyl-1, 4-naphthoquinone with H2, in contrast to an earlier preparation which was made up of HydA and HydB only and did not contain cytochrome b (Unden, G., Bocher, R., Knecht, J. & Kroger, A. (1982) FEBS Lett. 145, 230-234). This suggests that HydC is a cytochrome b which serves as a mediator in the electron transfer from H2 to the quinone.The hydrogenase genes were cloned, sequenced and identified by sequence comparison with the N-termini of the three subunits. The three genes were arranged in the order hydA, hydB, hydC, with the transcription start site in front of hydA, and were present only once on the genome. Separated by an intergene region of 69 nucleotides, hydC was followed by at least two more open reading frames of unknown function. The amino acid sequences derived from hydA, hydB and hydC were similar to those of the membrane Ni-hydrogenases of seven other bacteria. HydA and HydB also showed similarity to the small and the large subunits of periplasmic Ni-hydrogenases. HydC was predicted to contain four hydrophobic segments which might span the bacterial membrane. Two histidine residues located in hydrophobic segments are conserved in the corresponding sequences of the other membrane hydrogenases and might ligate the haem B.
The periplasmic selenate reductase (Ser) of Thauera selennatis is a component of the electron transport chain catalyzing selenate reduction with acetate as the electron donor (i.e., selenate respiration). The purified enzyme consists of three subunits (SerA, SerB and SerC). Using transposon (i.e., Tn5) mutagenesis selenate reductase mutants were isolated. Junction fragments of DNA adjacent to the integrated Tn5 were used, together with oligonucleotides derived from the N-termini of SerA and SerB, to clone from a gene bank a DNA fragment that contained the corresponding genes. After sequencing, serA, serB and serC were identified by sequence comparison with the N-termini of the three subunits. The genes are arranged in the order serA, serB, serC; a fourth open reading frame (serD) in between, but overlapping serB and serC, is also present. The serA gene product contains an apparent leader peptide with a twin-arginine motif. The remainder of the translated amino acid sequence is similar to that of a number of prokaryotic molybdenum-containing enzymes (e.g., nitrate reductases and formate dehydrogenases of Escherichia coli). The serB gene product contains four cysteine clusters and is similar to various iron-sulfur protein subunits. The serC gene product contains a putative Sec-dependent leader peptide, but there are no similarities between the remainder of the translated protein and other protein subunits. The SerC contains two histidine and four methionine residues, and these may noncovalently bind heme b--which is a component of the active selenate reductase. The serD gene product encodes a putative protein that shows no significant sequence similarities to other proteins. However, the location of the serD within the other ser genes is similar to that of narJ within the E. coli narGHJI operon (nitrate reductase A); thus suggesting that the role of SerD may be similar to that of NarJ, which is a system-specific chaperone protein.
The polysulphide reductase (formerly sulphur reductase) of Wolinella succinogenes is a component of the phosphorylative electron transport system with polysulphid as the terminal acceptor. Using an antiserum raised against the major subunit (PsrA, 85 kDa) of the enzyme, the corresponding gene (psr A) was cloned from a λ‐gene bank. The N‐terminal amino acid sequence of PsrA mapped within the psr A gene product, which also contained an apparent signal peptide. Downstream of the Psr A gene two more open reading frames (psrB and psrC) were found. the three genes may form a transcriptional unit with the transcription start site in front of psr A. The three genes were present only once on the genome. PsrA is a hydrophilic protein homologous to the largest subunits of six prokaryotic molybdoenzymes. PsrB is predicted to be hydrophilic, to contain ferredoxin‐like cysteine clusters and to be homologous to the smaller hydrophilic subunits of four molybdoenzymes. PsrC is predicted to be a hydrophobic protein that could possibly serve as the membrane anchor of the enzyme.
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