A novel chromate reductase from Thermus scotoductus SA-01 related to old yellow enzyme.
Bacteria can reduce toxic and carcinogenic Cr(VI) to insoluble and less toxic Cr(III). Thermus scotoductus SA-01, a South African gold mine isolate, has been shown to be able to reduce a variety of metals, including Cr(VI). Here we report the purification to homogeneity and characterization of a novel chromate reductase. The oxidoreductase is a homodimeric protein, with a monomer molecular mass of approximately 36 kDa, containing a noncovalently bound flavin mononucleotide cofactor. The chromate reductase is optimally active at a pH of 6.3 and at 65 degrees C and requires Ca(2+) or Mg(2+) for activity. Enzyme activity was also dependent on NADH or NADPH, with a preference for NADPH, coupling the oxidation of approximately 2 and 1.5 mol NAD(P)H to the reduction of 1 mol Cr(VI) under aerobic and anaerobic conditions, respectively. The K(m) values for Cr(VI) reduction were 3.5 and 8.4 microM for utilizing NADH and NADPH as electron donors, respectively, with corresponding V(max) values of 6.2 and 16.0 micromol min(-1) mg(-1). The catalytic efficiency (k(cat)/K(m)) of chromate reduction was 1.14 x 10(6) M(-1) s(-1), which was >50-fold more efficient than that of the quinone reductases and >180-fold more efficient than that of the nitroreductases able to reduce Cr(VI). The chromate reductase was identified to be encoded by an open reading frame of 1,050 bp, encoding a single protein of 38 kDa under the regulation of an Escherichia coli sigma(70)-like promoter. Sequence analysis shows the chromate reductase to be related to the old yellow enzyme family, in particular the xenobiotic reductases involved in the oxidative stress response.
Opperman
Diederik Johannes
DJ
Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein 9300, South Africa.
Piater
Lizelle Ann
LA
van Heerden
Esta
E
D000595:Amino Acid Sequence; D001419:Bacteria; D002413:Cations, Divalent / Q000494:pharmacology; D002857:Chromium / Q000378:metabolism*; D003067:Coenzymes / Q000032:analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D004492:Edetic Acid / Q000494:pharmacology; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005486:Flavin Mononucleotide / Q000032:analysis / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000235:genetics*; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013057:Spectrum Analysis; D013696:Temperature; D013824:Thermus / Q000201:enzymology* / Q000235:genetics
D002413:Cations, Divalent; D003067:Coenzymes; D004269:DNA, Bacterial; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D002857:Chromium; D009243:NAD; D009249:NADP; D005486:Flavin Mononucleotide; D004492:Edetic Acid; D010088:Oxidoreductases; D009252:NADPH Dehydrogenase; C063243:chromate reductase