title: Detoxification of hexavalent chromium by Leucobacter sp. uses a reductase with specificity for dihydrolipoamide.
abstract: Leucobacter sp. belongs to the metal stressed community and possesses higher tolerance to metals including chromium and can detoxify toxic hexavalent chromium by reduction to less toxic trivalent chromium. But, the mechanism of reduction of hexavalent chromium by Leucobacter sp. has not been studied. Understanding the enzyme catalyzing reduction of chromium is important to improve the species for application in bioremediation. Hence, a soluble reductase catalyzing the reduction of hexavalent chromium was purified from a Leucobacter sp. and characterized. The pure chromate reductase was obtained from the cell-free extract through hydrophobic interaction and gel filtration column chromatographic methods. It was a monomeric enzyme and showed similar molecular weights in both gel filtration (∼68 KDa) and SDS-PAGE (64 KDa). It reduced Cr(VI) using both NADH and NADPH as the electron donor, but exhibited higher activity with NADH. The optimal activity was found at pH 5.5 and 30 °C. The K(m) and V(max) for Cr(VI) reduction with NADH were 46.57 μM and 0.37 μmol min(-1) (mg protein) (-1), respectively. The activity was inhibited by p-hydroxy mercury benzoate, Ag(2+) and Hg(2+) indicating the role of thiol groups in the catalysis. The spectrophotometric analysis of the purified enzyme showed the absence of bound flavin in the enzyme. The N-terminal amino acid sequence and LC/MS analysis of trypsin digested purified enzyme showed similarity to dihydrolipoyl dehydrogenase. The purified enzyme had dihydrolipoyl dehydrogenase activity with dihydrolipoamide as the substrate, which suggested that Leucobacter sp. uses reductase with multiple substrate specificity for reduction of Cr(VI) detoxification.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras, Chennai, India.
authors.forename: Abhipsa
authors.initials: A
authors.lastname: Sarangi
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras, Chennai, India.
authors.forename: Chandraraj
authors.initials: C
authors.lastname: Krishnan
mesh_terms: D039903:Actinobacteria / Q000201:enzymology* / Q000378:metabolism*; D001711:Biotransformation; D002853:Chromatography, Liquid; D002857:Chromium / Q000378:metabolism* / Q000633:toxicity*; D003067:Coenzymes / Q000378:metabolism; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013053:Spectrophotometry; D013696:Temperature; D008063:Thioctic Acid / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D003067:Coenzymes; D004791:Enzyme Inhibitors; D002857:Chromium; D009243:NAD; C074702:chromium hexavalent ion; C007409:dihydrolipoamide; D009249:NADP; D008063:Thioctic Acid; D010088:Oxidoreductases; D008058:Dihydrolipoamide Dehydrogenase; C063243:chromate reductase
keywords: Bioremediation; Chromate reductase; Hexavalent chromium; Leucobacter sp