Mutation of cysteine-295 to alanine in secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus affects the enantioselectivity and substrate specificity of ketone reductions

The mutation of Cys-295 to alanine in Thermoanaerobacter ethanolicus second ary alcohol dehydrogenase (SADH) was performed to give C295A SADH, on the b asis of molecular modeling studies utilizing the X-ray crystal structure co ordinates of the highly homologous T. brockii secondary alcohol dehydrogena se (IYKF.PDB). This mutant SADH has activity for 2-propanol comparable to w ild-type SADH. However, the C295A mutation was found to cause a significant shift of enantioselectiviry toward the (S)-configuration in the reduction of some ethynylketones to the corresponding chiral propargyl alcohols. This result confirms our prediction that Cys-295 is part of a small alkyl group binding pocket whose size determines the binding orientation of ketone sub strates, and, hence, the stereochemical configuration of the product alcoho l. Furthermore, C295A SADH has much higher activity towards t-butyl and som e alpha -branched ketones than does wild-type SADH. The C295A mutation does not affect the thioester reductase activity of SADH. The broader substrate specificity and altered stereoselectivity for C295A SADH make it a potenti ally useful tool for asymmetric reductions. (C) 2001 Elsevier Science Ltd. All rights reserved.