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Characterization and Directed Evolution of an Alcohol Dehydrogenase: A Study Towards Understanding of Three Central Aspects of Substrate Selectivity
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Many different chemicals are used in the everyday life, like detergents and pharmaceuticals. However, their production has a big impact on health and environment as much of the raw materials are not renewable and the standard ways of production in many cases includes toxic and environmentally hazardous components. As the population and as the life standard increases all over the planet, the demand for different important chemicals, like pharmaceuticals, will increase. A way to handle this is to apply the concept of Green chemistry, where biocatalysis, in the form of enzymes, is a very good alternative. Enzymes do not normally function in industrial processes and needs modifications through protein engineering to cope in such conditions. To be able to efficiently improve an enzyme, there is a need to understand the mechanism and characteristics of that enzyme.

Acyloins (α-hydroxy ketones) are important building blocks in the synthesis of pharmaceuticals. In this thesis, the enzyme alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber has been in focus, as it has been shown to display a wide substrate scope, also accepting aryl-substituted alcohols. The aim has been to study the usefulness of ADH-A as a biocatalyst towards production of acyloins and its activity with aryl-substituted vicinal diols and to study substrate-, regio-, and enantioselectivity of this enzyme.

This thesis is based on four different papers where the focus of the first has been to biochemically characterize ADH-A and determine its mechanism, kinetics and its substrate-, regio-, and enantioselectivity. The second and third paper aims towards deeper understanding of some aspects of selectivity of ADH-A. Non-productive binding and its importance for enantioselectivity is studied in the second paper by evolving ADH-A towards increased activity with the least favored enantiomer through protein engineering. In the third paper, regioselectivity is in focus, where an evolved variant displaying reversed regioselectivity is studied. In the fourth and last paper ADH-A is studied towards the possibility to increase its activity towards aryl-substituted vicinal diols, with R-1-phenyl ethane-1,2-diol as the model substrate, and the possibility to link ADH-A with an epoxide hydrolase to produce acyloins from racemic epoxides.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , p. 95
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1497
Keywords [en]
Alcohol dehydrogenase, ADH-A, Biocatalysis, Directed evolution, Enantioselectivity, Enzyme kinetics, Enzyme mechanisms, Protein Engineering, Regioselectivity, Substrate selectivity
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-318984ISBN: 978-91-554-9875-7 (print)OAI: oai:DiVA.org:uu-318984DiVA, id: diva2:1085659
Public defence
2017-05-19, BMC A1:111A, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2017-04-28 Created: 2017-03-30 Last updated: 2017-05-05
List of papers
1. Kinetic characterization of Rhodococcus ruber DSM 44541 alcohol dehydrogenase A
Open this publication in new window or tab >>Kinetic characterization of Rhodococcus ruber DSM 44541 alcohol dehydrogenase A
2014 (English)In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 99, p. 68-78Article in journal (Refereed) Published
Abstract [en]

An increasing interest in biocatalysis and the use of stereoselective alcohol dehydrogenases in synthetic asymmetric catalysis motivates detailed studies of potentially useful enzymes such as alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber. This enzyme is capable of catalyzing enantio-, and regioselective production of phenyl-substituted α-hydroxy ketones (acyloins) which are precursors for the synthesis of a range of biologically active compounds. In this study, we have determined the enzyme activity for a selection of phenyl-substituted vicinal diols and other aryl- or alkyl-substituted alcohols and ketones. In addition, the kinetic mechanism for the oxidation of (R)- and (S)-1-phenylethanol and the reduction of acetophenone has been identified as an Iso Theorell-Chance (hit and run) mechanism with conformational changes of the enzyme-coenzyme binary complexes as rate-determining for the oxidation of (S)-1-phenylethanol and the reduction of acetophenone. The underlying cause of the 270-fold enantiopreference for the (S)-enantiomer of 1-phenylethanol has been attributed to non-productive binding of the R-enantiomer. We have also shown that it is possible to tune the direction of the redox chemistry by adjusting pH with the oxidative reaction being favored at pH values above 7.

Keywords
alcohol dehydrogenase, kinetic mechanism, pre-steady state kinetics, product inhibition
National Category
Other Chemistry Topics Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-207474 (URN)10.1016/j.molcatb.2013.10.023 (DOI)000331340500010 ()
Available from: 2013-09-15 Created: 2013-09-15 Last updated: 2017-12-06Bibliographically approved
2. Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer
Open this publication in new window or tab >>Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer
Show others...
2017 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, no 22, p. 3895-3914Article in journal (Refereed) Published
Abstract [en]

Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 is a promising biocatalyst for redox transformations of arylsubstituted sec-alcohols and ketones. The enzyme is stereoselective in the oxidation of 1-phenylethanol with a 300-fold preference for the (S)-enantiomer. The low catalytic efficiency with (R)-1-phenylethanol has been attributed to nonproductive binding of this substrate at the active site. Aiming to modify the enantioselectivity, to rather favor the (R)-alcohol, and also test the possible involvement of nonproductive substrate binding as a mechanism in substrate discrimination, we performed directed laboratory evolution of ADH-A. Three targeted sites that contribute to the active-site cavity were exposed to saturation mutagenesis in a stepwise manner and the generated variants were selected for improved catalytic activity with (R)-1-phenylethanol. After three subsequent rounds of mutagenesis, selection and structure-function analysis of isolated ADH-A variants, we conclude: (1) W295 has a key role as a structural determinant in the discrimination between (R)- and (S)-1-phenylethanol and a W295A substitution fundamentally changes the stereoselectivity of the protein. One observable effect is a faster rate of NADH release, which changes the rate-limiting step of the catalytic cycle from coenzyme release to hydride transfer. (2) The obtained change in enantiopreference, from the (S)- to the (R)-alcohol, can be partly explained by a shift in the nonproductive substrate binding modes.

Keywords
alcohol dehydrogenase, biocatalysis, stereoselectivity, directed evolution, crystal structures, enzyme kinetics
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-318981 (URN)10.1111/febs.14279 (DOI)000415877100011 ()
Funder
Swedish Research Council, 621-2011-6055
Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2018-03-09Bibliographically approved
3. Stereoselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases
Open this publication in new window or tab >>Stereoselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases
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(English)In: Article in journal (Other academic) Submitted
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-318982 (URN)
Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2018-02-18
4. Laboratory Evolution of Alcohol Dehydrogenase ADH-A for Efficient Transformation of Vicinal Diols and Acyloins. Synthesis of 2-Hydroxy Acetophenone from Racemic Styrene Oxide
Open this publication in new window or tab >>Laboratory Evolution of Alcohol Dehydrogenase ADH-A for Efficient Transformation of Vicinal Diols and Acyloins. Synthesis of 2-Hydroxy Acetophenone from Racemic Styrene Oxide
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-318983 (URN)
Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2017-03-30

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