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

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 M for utilizing NADH and NADPH as electron donors, respectively, with corresponding V max values of 6.2 and 16.0 mol 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 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.

Genomic and Physiological Characterization of the Chromate-Reducing, Aquifer-Derived Firmicute Pelosinus sp. Strain HCF1

“…To date, the best-studied chromate reductase is ChrR, a soluble, dimeric, NADH-dependent flavoprotein reported in Pseudomonas putida (38,39). BLASTP searches of the strain HCF1 genome were conducted for the following chromate reductases: ChrR from P. putida (GenBank accession number AF375642); an Old Yellow Enzyme (OYE) homolog from Thermus scotoductis (GenBank accession number AM902709 [40]); YieF, a ChrR homolog from E. coli (GenBank accession number AAK62985.2 [38]); the Fre flavin reductase from E. coli (EcoCyc accession number EG10334 [25]); FerB from Paracoccus denitrificans (putatively GenBank accession number YP_917833.1 [41,42]); and an azoreductase from Bacillus sp. OY1-2 (GenBank accession number BAB13746.1 [43]).…”

Photobiocatalytic chemistry of oxidoreductases using water as the electron donor

“…The model biocatalyst was the old yellow enzyme homologue from Thermus scotoductus SA-01 (TsOYE) 40,41 , catalysing the stereoselective trans-hydrogenation of Ketoisophorone (1) to (R)-Levodione (2) (Fig. 2).…”

Preliminary Characterization of a NADPH Dependent Chromate Reductase from Trichoderma pseudokoningii

“…The optimum temperature of the partially purified enzyme was determined by incubating the reaction mixture of the enzyme at different temperatures ranging from 30 ºC to 90 ºC for 10 min. In order to determine the effects of pH on the stability of the enzyme, the partially purified enzyme protein was exposed to various pHs (6)(7)(8) …”

“…In aerobic conditions, the enzyme chromate reductase is reported to reduce Cr(VI) to Cr(III) inside or outside the plasma membrane and utilize NADH as electron donor, while under anaerobic condition membrane bound chromate reductase reduces Cr(VI) through respiratory chains involving cytochromes [3][4][5]. The most efficient Cr(VI) transforming enzymes that have been described to date are flavo-proteins, but these require expensive NADPH cofactors to power reduction, and stoichiometric addition of these cofactors is not an economically viable proposition for large scale applications [6][7][8][9][10]. Das and Chandra [11] have reported that Cr(VI) was reduced to Cr(III) in the presence of NADPH in the cell extract of Streptomyces species and existence of NADPH-dependent chromium (VI) reductase in Pseudomonas ambigua G-1, E.coli [12,13].…”