A new member of the short-chain dehydrogenases/reductases supe...

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 35kDa on SDS-PAGE and only required NADPH as an electron donor. The K(m) value of PsCR for ethyl 4-chloro-3-oxobutanoate was 4.9mg/mL and the corresponding V(max) was 337mumol/mgprotein/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.