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°C and requires Ca²⁺ or Mg²⁺ 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 × 10⁶ 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 σ⁷⁰-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.