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Kinetic characterization of Rhodococcus ruber DSM 44541 alcohol dehydrogenase A
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
2014 (English)In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 99, 68-78 p.Article 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.

Place, publisher, year, edition, pages
2014. Vol. 99, 68-78 p.
Keyword [en]
alcohol dehydrogenase, kinetic mechanism, pre-steady state kinetics, product inhibition
National Category
Other Chemistry Topics Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-207474DOI: 10.1016/j.molcatb.2013.10.023ISI: 000331340500010OAI: oai:DiVA.org:uu-207474DiVA: diva2:648326
Available from: 2013-09-15 Created: 2013-09-15 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Oxidation of 1,2-Diols Using Alcohol Dehydrogenases: From Kinetic Characterization to Directed Evolution
Open this publication in new window or tab >>Oxidation of 1,2-Diols Using Alcohol Dehydrogenases: From Kinetic Characterization to Directed Evolution
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The use of enzymes as catalysts for chemical transformations has emerged as a “greener” alternative to traditional organic synthesis. An issue to solve though, is that enzymes are designed by nature to catalyze reactions in a living cell and therefore, in many cases, do not meet the requirements of a suitable biocatalyst. By mimicking Darwinian evolution these problems can be addressed in vitro by different types of directed evolution strategies.

α-Hydroxy aldehydes and α-hydroxy ketones are important building blocks in the synthesis of natural products, fine chemicals and pharmaceuticals. In this thesis, two alcohol dehydrogenases, FucO and ADH-A, have been studied. Their potentials to serve as useful biocatalysts for the production of these classes of molecules have been investigated, and shown to be good. FucO for its strict regiospecificity towards primary alcohols and that it strongly prefers the S-enantiomer of diol substrates. ADH-A for its regiospecificity towards secondary alcohols, its enantioselectivity and that is has the ability to use a wide variety of bulky substrates. The kinetic mechanisms of these enzymes were investigated using pre-steady state kinetics, product inhibition, kinetic isotope effects and solvent viscosity effects, and in both cases, the rate limiting steps were pin-pointed to conformational changes occurring at the enzyme-nucleotide complex state. These characterizations provide an important foundation for further studies on these two enzymes.  

FucO is specialized for activity with small aliphatic substrates but is virtually inactive with aryl-substituted compounds. By the use of iterative saturation mutagenesis, FucO was re-engineered and several enzyme variants active with S-3-phenylpropane-1,2-diol and phenylacetaldehyde were obtained. It was shown that these variants capability to act on larger substrates are mainly due to an enlargement of the active site cavity. Furthermore, several amino acids which are important for catalysis and specificity were identified. Phe254 interacts with aryl-substituted substrates through π-π stacking and may be essential for activity with these larger substrates. One mutation caused a loss in the interactions made between the enzyme and the nucleotide and thereby enhanced the turnover number for the preferred substrate

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 59 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1081
Keyword
enzyme kinetics, alcohol dehydrogenase, directed evolution, enzyme engineering, diol, α-hydroxy aldehyde
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-208139 (URN)978-91-554-8763-8 (ISBN)
Public defence
2013-11-08, B42, Husargatan 3, BMC, Uppsala universitet, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2013-10-18 Created: 2013-09-24 Last updated: 2014-01-23Bibliographically approved
2. Characterization and Directed Evolution of an Alcohol Dehydrogenase: A Study Towards Understanding of Three Central Aspects of Substrate Selectivity
Open this publication in new window or tab >>Characterization and Directed Evolution of an Alcohol Dehydrogenase: A Study Towards Understanding of Three Central Aspects of Substrate Selectivity
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. 95 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1497
Keyword
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:nbn:se:uu:diva-318984 (URN)978-91-554-9875-7 (ISBN)
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

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Hamnevik, EmilBlikstad, CeciliaNorrehed, SaraWidersten, Mikael

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