Directed Evolution of Alcohol Dehydrogenase for Improved Stereoselective Redox Transformations of 1-Phenylethane-1,2-diol and Its Corresponding Acyloin (original) (raw)

Abstract

Laboratory evolution of alcohol dehydrogenase produced enzyme variants with improved turnover numbers with a vicinal 1,2-diol and its corresponding hydroxyketone. Crystal structure and transient kinetics analysis aids in rationalizing the new functions of these variants. E nzymes can provide effective alternative routes as catalysts in synthetic chemistry, 1−4 and the "green-ness" of biocatalysis can contribute to a more sustainable manufacturing of chemicals. An important chemical transformation in synthetic chemistry is the oxidation of secondary alcohols and the reduction of the corresponding ketones. Partial oxidation of 1,2-substituted (vicinal) diols can produce the corresponding αhydroxy ketones (acyloins) that are attractive building blocks for chiral auxiliaries, natural products, and pharmaceuticals. 5−7 Enzyme-catalyzed production of acyloins has been demonstrated to be feasible using monooxygenases or alcohol dehydrogenases. 8−10 Alcohol dehydrogenase A (ADH-A) from the bacterium Rhodococcus ruber DSM 44541 is an interesting candidate biocatalyst of redox reactions. ADH-A is unusually tolerant toward organic solvents, is highly regio-and enantioselective, and displays activity with a wide range of alcohols and ketones, including aryl-substituted vicinal diols. 11−14 The catalyzed oxidation of 1,2-diols by the wild-type enzyme is, however, relatively inefficient; the k cat /K M for oxidation of (R)-2 (Chart 1) into the corresponding hydroxy ketone 4 is approximately 3000-fold lower than that for the oxidation of (S)-1 into acetophenone (3) (Table 1). ADH-A follows an ordered sequential bi-bi mechanism with a low degree of accumulation of the ternary complex at the steady state. 14 The rate-limiting step for the turnover of preferred substrates such as (S)-1 is NADH release (k 5 in Scheme 1, Table 2). It is noteworthy that in a strictly ordered mechanism the NADH off rate is expected to be independent of the alcohol substrate (or ketone product) because this step involves only the binary enzyme−nucleotide complex. ADH-A displays a k cat with (R)-2 of 0.73 s −1 , which is 70-fold lower than the NADH release rate (Table 2). Assuming that oxidation of (R)-2 still obeys an ordered mechanism, the lower turnover rate is presumably due to nonproductive substrate binding, 15 where the alcohol is bound in the ternary

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