Abstract: Based on homology modeling analysis, the sites, P124 and W125, in cofactor-binding domain of the carbonyl reductase SCR1 were found to have a steric effect on the binding of NADPH. The site-directed mutagenesis of these two sites using the amino acid residues with small side group showed that the substrate sepcificity and stereoselectivity of the enzyme were both changed in some level, indicating that these sites have a critical role in binding the cofactor, and the configuration of catalytic active center formed from enzyme/cofactor complex determines the recognition of the enzyme to substrate of different type and also the product of different stereo-configuration. Regarding the substrate specificity, the wild-type enzyme showed activity toward 2-hydroxyacetophenone, 2-bromoacetophenone and its derivatives, while the catalytic efficiency of mutants toward acetophenone and its derivatives and 2-octanone was enhanced. On the other hand, the stereospecificity of some mutants was even found to be inverted. The mutants P124A/W125A and P124G/W125G exhibited a shift of enantioselectivity toward 2-hydroxyacetophenone and ethyl 4-chloro-3-oxobutanoate to give the product in (R)-configuration.

Key words: carbonyl reductase, site-directed mutagenesis, catalytic activity, substrate specificity, stereoselectivity