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

By Diederik Johannes Opperman, Lizelle Ann Piater and Esta van Heerden

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 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 Km values for Cr(VI) reduction were 3.5 and 8.4 μM for utilizing NADH and NADPH as electron donors, respectively, with corresponding Vmax values of 6.2 and 16.0 μmol min−1 mg−1. The catalytic efficiency (kcat/Km) 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

Topics: Enzymes and Proteins
Publisher: American Society for Microbiology (ASM)
OAI identifier: oai:pubmedcentral.nih.gov:2293266
Provided by: PubMed Central
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