Mutagenesis of Met-151 and Thr-153 to alanine in Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase changes substrate specificity for acetophenones

作者：Nealon Christopher M; Kim Chang Sup; Dwamena Amos K; Phillips Robert S^*

来源：Enzyme and Microbial Technology, 2017, 105: 59-63.

DOI：10.1016/j.enzmictec.2017.06.010

• Kinetic resolution
• Mutagenesis
• Substrate specificity
• Alcohol dehydrogenase
• Stereoselectivity
• Dehydrogenase
• NADP
• Ketone

Secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus reduces ketones to chiral alcohols, and generally obeys Prelog's Rule, with binding pockets for large and small alkyl substituents, giving (S) alcohols. We have previously shown that mutations in both the large and small pockets can alter both substrate specificity and stereoselectivity. In the present work, Met-151 and Thr-153, residues located in the small pocket, were mutated to alanine. The M151A mutant SADH shows significantly lower activity and lower stereo selectivity for reduction of aliphatic ketones than wild-type SADH. Furthermore, M151A showed non-linear kinetics for reduction of acetone. T153A SADH shows lower activity but similar stereoselectivity for ketone reduction compared to wild-type SADH. The I86A/M151A/C295A and I86A/T153A/C295A triple mutant SADH show altered specificity for reduction of substituted acetophenones. These results confirm that these mutations are useful to combine with I86A/C295A SADH to expand the small pocket of SADH and broaden the substrate specificity.

• 出版日期2017-10
• 单位中国人民解放军海军大连舰艇学院