THE ANGLE OF A SIDE-CHAIN DECIDES REGIO- AND ENANTIOSELECTIVITY IN ALCOHOL DEHYDROGENASE A

摘要

Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 is a promising biocatalyst for asymmetric synthesis of organic compounds. This enzyme is capable of catalyzing enantio- and regioselectivity of phenyl-substitute a-hydroxy ketones (acyloins), which are precursors for the synthesis of a range of biologically active compounds. ADH-A catalyzes the oxidation of (S)-1-phenylethanol 3000-fold more efficiently as compared to the 2-hydroxylated derivatives (R)-phenyl-1,2-ethanediol. ADH-A is highly selective towards secondary-alcohols and displays very low activities with corresponding primary-alcohol derivatives. Apparently, when this selectivity was tested with substrate contained two secondary-alcohols, we analyzed the catalytic efficiency and the regioselectivity towards (1R,2S)-2. The conclusions were yielded that ADH-A is a comparably inefficient catalyst for oxidation of vicinal diols, but displays regioselectivity, oxidizing primarily the benzylic carbon of this substrate. To further investigate the structural requirements for more efficient oxidation of vicinal diols, we conducted iterative CASTing on this enzyme. One isolated variant, B1 was displayed improved activity by a three-fold higher k_(cat) value with (1R,2S)-2. Moreover, B1 variant displays a shift in the regioselectivity in the oxidation of (1R,2S)-2, and also enantioselectivity towards (S)-phenyl-1,2-ethanediol by a 70-fold increase in the turnover number. However, the crystal structure of the B1 describes the structural alterations to the active site; the positioning of a Tyr side-chain located distal to the coenzyme and the catalytic zinc ion has been changed. The effects on function by this subtle structural change was tested kinetically substrate binding. Therefore, we conclude that the increases in the k_(cat) values with (1R,2S)-2 and (S)-phenyl-1,2-ethanediol are mainly due to decreased relative levels of nonproductive substrate binding.