A Novel Chromate Reductase from Thermus scotoductus SA-01 Related to Old Yellow Enzyme

Abstract

ABSTRACT 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°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 μM for utilizing NADH and NADPH as electron donors, respectively, with corresponding V max values of 6.2 and 16.0 μmol min −1 mg −1 . The catalytic efficiency ( k cat / K m ) of chromate reduction was 1.14 × 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 σ 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.