Substrate promiscuity in evolved Alcohol Dehydrogenase A (ADH-A)
September 15-19, 2019
Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 tolerates organic solvents, and therefore this became a useful biocatalyst for asymmetric synthesis of organic compounds.1 ADH-A is capable of catalyzing stereoselective oxidation of phenyl-substituted sec-alcohols and reduction of the corresponding ketones. Importantly, these compounds are precursors for the synthesis of a range of biologically active compounds.1,2 Therefore, we have been studying engineering of ADH-A for the purpose of developing new enzymes with pre-designed catalytic properties regarding substrate scope and selectivity. We have been isolated a number of ADH-A variants which have been isolated from CASTing libraries for different purposes and function. Variants isolated from a library originally generated from random mutagenesis of residues Y294 and W295 (called “A”, clones A1, A2, A2C3 and A2C2B1) represent hits from different generation of directed evolution, selected for improved activity for the non-preferred R-enantiomer of 1-phenylethanol.2 Other mutants that were selected (variants B1 and B1F4) for improved activity with a disubstituted sec-alcohol also displayed altered regioselectivity as compared to the wild type.3 In a third evolution effort, enzyme variants C1 and C1B1 were isolated after selection for improved activity with the vicinal diol (R)-1-pheny-1,2-ethanediol.4
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Thilak Reddy Enugala and Mikael Widersten, "Substrate promiscuity in evolved Alcohol Dehydrogenase A (ADH-A)" in "Enzyme Engineering XXV", Huimin Zhao, University of Illinois at Urbana-Champaign, USA John Wong, Pfizer, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/enzyme_xxv/22