Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
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 Univ, BMC, Dept Chem, Box 576, SE-751236 Uppsala, Sweden..
Uppsala Univ, BMC, Dept Chem, Box 576, SE-751236 Uppsala, Sweden..
Uppsala Univ, BMC, Dept Chem, Box 576, SE-751236 Uppsala, Sweden..
KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. Uppsala Univ, Dept Cell & Mol Biol, Box 596, SE-75124 Uppsala, Sweden.;.
Show others and affiliations
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 k(cat) value with 1-phenylpropane-(1R,25)-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.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018. Vol. 8, no 8, p. 7526-7538
Keywords [en]
alcohol dehydrogenase, alcohol oxidation, directed evolution, enzyme engineering, biocatalysis, stereoselectivity, crystal structure, molecular dynamics simulations
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-235137DOI: 10.1021/acscatal.8b01762ISI: 000441112400074Scopus ID: 2-s2.0-85049666919OAI: oai:DiVA.org:kth-235137DiVA, id: diva2:1248582
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), 2015/16-12 2016/34-27
Note

QC 20180917

Available from: 2018-09-17 Created: 2018-09-17 Last updated: 2018-09-17Bibliographically approved

Open Access in DiVA

No full text in DiVA

Other links

Publisher's full textScopus
By organisation
Biophysics
In the same journal
ACS Catalysis
Chemical Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 89 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf