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Stereo- and regioselectivity in catalyzed transformation of a 1,2-disubstituted vicinal diol and the corresponding diketone by wild type and laboratory evolved alcohol dehydrogenases

DOI: 10.1021/acscatal.8b01762 DOI Help

Authors: Dirk Maurer (Uppsala University) , Thilak Reddy Enugala (Uppsala University) , Emil Hamnevik (Uppsala University) , Paul Bauer (Uppsala University) , Malin Lüking (Uppsala University) , Dušan Petrović (Uppsala University) , Heidi Hillier (Uppsala University) , Shina C. L. Kamerlin (Uppsala University) , Doreen Dobritzsch (Uppsala University) , Mikael Widersten (Uppsala University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Catalysis

State: Published (Approved)
Published: July 2018
Diamond Proposal Number(s): 11171 , 15868

Abstract: ADH-A from Rhodococcus ruber DSM 44541 catalyzes the oxidation of (S)-1-phenylethanol 3,000-fold more efficiently as compared to the 2-hydroxylated derivative (R)-phenylethane-1,2-diol. The enzyme is also highly selective for sec-alcohols with comparably low activities with the corresponding primary alcohols. When challenged with a substrate containing two secondary alcohols, such as 1-phenylpropane-(1R,2S)-diol, ADH-A favors the oxidation of the benzylic carbon of this alcohol. The catalytic efficiency, however, is modest in comparison to the activity with (S)-1-phenylethanol. To investigate the structural requirements for improved oxidation of vicinal diols we conducted iterative saturation mutagenesis combined with activity screening. A first-generation variant, B1 (Y54G, L119Y) displays a two-fold higher kcat value with 1-phenylpropane-(1R,2S)-diol and a shift in the cooperative behavior in alcohol binding, from negative in the wild type, to positive in B1, suggesting a shift from a less active enzyme form (T) in the wild type to a more active form (R) in the B1 variant. Also, the regiopreference changed to favor oxidation of C-2. A second-generation variant, B1F4 (F43T, Y54G, L119Y, F282W), shows further improvement in the turnover and regioselectivity in oxidation of 1-phenylpropane-(1R,2S)-diol. The crystal structures of the B1 and B1F4 variants describe the structural alterations to the active site the most significant of which is a re-positioning of a Tyr side-chain located distal to the coenzyme and the catalytic zinc ion. The links between the changes in structures and stereoselectivities are rationalized by molecular dynamics simulations of substrate binding at the respective active sites.

Journal Keywords: Alcohol dehydrogenase; alcohol oxidation; directed evolution; enzyme engineering; biocatalysis; stereoselectivity; crystal structure; molecular dynamics simulations

Subject Areas: Chemistry

Instruments: I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography