JB Accepts, published online ahead of print on 8 February 2008

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J. Bacteriol. doi:10.1128/JB.01766-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A novel chromate reductase from Thermus scotoductus SA-01 related to Old Yellow Enzyme

Diederik Johannes Opperman, Lizelle Ann Piater, and Esta van Heerden^*

Department of Microbial, Biochemical and Food Biotechnology; University of the Free State; Bloemfontein; 9300; South Africa

^* To whom correspondence should be addressed. Email: vheerde.sci{at}mail.uovs.ac.za.

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 non-covalently bound FMN co-factor. 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 was 3.5 and 8.4 µM when utilizing NADH or NADPH as electron donor 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 x 10⁶ M^-1 s^-1, more than 50 fold more efficient than the quinone reductases and more than 180 fold more efficient than the nitroreductases able to reduce Cr(VI). The chromate reductase was identified to be encoded for by an ORF of 1050 bp, encoding a single protein of 38 kDa under the regulation of an E. coli 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.