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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
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC. (Dobritzsch)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. (Widersten)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC. (Widersten)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Molecular Cell Biology. (Kamerlin)
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2018 (English)In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 8, no 8, p. 7526-7538Article in journal (Refereed) Published
Abstract [en]

ADH-A from Rhodococcus ruber DSM 44541 catalyzes the oxidation of (S)-1-phenylethanol 3000-fold more efficiently as compared with 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 2-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 repositioning 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.

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

Place, publisher, year, edition, pages
2018. Vol. 8, no 8, p. 7526-7538
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-355854DOI: 10.1021/acscatal.8b01762ISI: 000441112400074OAI: oai:DiVA.org:uu-355854DiVA, id: diva2:1231232
Funder
Stiftelsen Olle Engkvist ByggmästareSwedish Research Council, 2015-04928Knut and Alice Wallenberg Foundation, KAW 2013.0124EU, FP7, Seventh Framework Programme, 283570Swedish National Infrastructure for Computing (SNIC), 2015/16-12Swedish National Infrastructure for Computing (SNIC), 2016/34-27Available from: 2018-07-05 Created: 2018-07-05 Last updated: 2018-11-29Bibliographically approved

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Maurer, DirkEnugala, Thilak ReddyHamnevik, EmilBauer, PaulPetrovic, DusanWidersten, Mikael

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Maurer, DirkEnugala, Thilak ReddyHamnevik, EmilBauer, PaulLüking, MalinPetrovic, DusanKamerlin, Shina C. L.Widersten, Mikael
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Department of Chemistry - BMCBiochemistryMolecular Cell BiologyDepartment of Cell and Molecular BiologyDepartment of Chemistry - Ångström
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