A new member of the short-chain dehydrogenases/reductases superfamily: purification, characterization and substrate specificity of a recombinant carbonyl reductase from Pichia stipitis.
A novel short-chain dehydrogenases/reductases superfamily (SDRs) reductase (PsCR) from Pichia stipitis that produced ethyl (S)-4-chloro-3-hydroxybutanoate with greater than 99% enantiomeric excess, was purified to homogeneity using fractional ammonium sulfate precipitation followed by DEAE-Sepharose chromatography. The enzyme purified from recombinant Escherichia coli had a molecular mass of about 35 kDa on SDS-PAGE and only required NADPH as an electron donor. The K(m) value of PsCR for ethyl 4-chloro-3-oxobutanoate was 4.9 mg/mL and the corresponding V(max) was 337 micromol/mg protein/min. The catalytic efficiency value was the highest ever reported for reductases from yeasts. Moreover, PsCR exhibited a medium-range substrate spectrum toward various keto and aldehyde compounds, i.e., ethyl-3-oxobutanoate with a chlorine substitution at the 2 or 4-position, or alpha,beta-diketones. In addition, the activity of the enzyme was strongly inhibited by SDS and beta-mercaptoethanol, but not by ethylene diamine tetra acetic acid.
Ye
Qi
Q
State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing 210009, PR China.
Yan
Ming
M
Yao
Zhong
Z
Xu
Lin
L
Cao
Hou
H
Li
Zhengjiang
Z
Chen
Yong
Y
Li
Shuya
S
Bai
Jianxin
J
Xiong
Jian
J
Ying
Hanjie
H
Ouyang
Pingkai
P
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics*; D000449:Aldehyde Reductase; D000447:Aldehydes / Q000737:chemistry; D000074408:Aldo-Keto Reductases; D000645:Ammonium Sulfate / Q000494:pharmacology; D001709:Biotechnology / Q000379:methods; D004492:Edetic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008623:Mercaptoethanol / Q000737:chemistry; D010088:Oxidoreductases / Q000737:chemistry*; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry*; D013237:Stereoisomerism; D013379:Substrate Specificity
D000447:Aldehydes; D011994:Recombinant Proteins; D008623:Mercaptoethanol; D004492:Edetic Acid; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; D000645:Ammonium Sulfate