Characterization of a novel NADPH-dependent oxidoreductase from Gluconobacter oxydans.
A novel protein from Gluconobacter oxydans DSM2003 which shows 60-70% similarity with members of aldo-keto reductase (AKR) superfamily was overexpressed in Escherichia coli BL21 (DE3) and purified by one step affinity chromatography with a Ni-NTA agarose column. The recombinant protein (named GOX0644) consists of 279 amino acids with an apparent molecular mass of 32 kDa in the soluble fraction, and the gene sequence encoding the protein GOX0644 is 100% identical to the ORF of gox0644 in G. oxydans 621H (DSM2343). For a detailed analysis of its enzymatic activity, the substrate specificity of the recombinant protein GOX0644 was determined. With NADPH as a cofactor, GOX0644 exhibited better activity to aromatic aldehydes, especially o-chlorobenzaldehyde, compared to aliphatic aldehydes. It showed almost no activity toward glyceraldehyde, xylose, glucose, and ketones. The protein was unable to oxidize primary- or secondary alcohols. Based on these results, GOX0644 was defined as a novel NADPH-dependent aldehyde reductase. Kinetic parameters of the protein and the dependence of its activity on temperature and pH were also determined.
Chen
Minmin
M
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China. minmin855@163.com
Lin
Jinping
J
Ma
Yushu
Y
Wei
Dongzhi
D
D000429:Alcohol Oxidoreductases; D000449:Aldehyde Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000074408:Aldo-Keto Reductases; D001547:Benzaldehydes / Q000378:metabolism; D004926:Escherichia coli; D020600:Gluconobacter oxydans / Q000201:enzymology* / Q000235:genetics; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019255:NADPH Oxidases; D010084:Oxidation-Reduction; D013379:Substrate Specificity
D001547:Benzaldehydes; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases
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