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Hamnevik, Emil
Publications (6 of 6) Show all publications
Hamnevik, E., Maurer, D., Enugala, T. R., Chu, T., Löfgren, R., Dobritzsch, D. & Widersten, M. (2018). Directed Evolution of Alcohol Dehydrogenase for Improved Stereoselective Redox Transformations of 1-Phenylethane-1,2-Diol and Its Corresponding Acyloin. Biochemistry, 57, 1059-1062
Open this publication in new window or tab >>Directed Evolution of Alcohol Dehydrogenase for Improved Stereoselective Redox Transformations of 1-Phenylethane-1,2-Diol and Its Corresponding Acyloin
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2018 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, p. 1059-1062Article in journal (Refereed) Published
Abstract [en]

Laboratory evolution of alcohol dehydrogenase produced enzyme variants with improved turnover numbers with a vicinal 1,2-diol and its corresponding hydroxyketone. Crystal structure and transient kinetics analysis aids in rationalizing the new functions of these variants.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-340574 (URN)10.1021/acs.biochem.8b00055 (DOI)000426013300003 ()29384657 (PubMedID)
Funder
Stiftelsen Olle Engkvist Byggmästare, 183-358
Available from: 2018-01-31 Created: 2018-01-31 Last updated: 2019-10-21Bibliographically approved
Maurer, D., Enugala, T. R., Hamnevik, E., Bauer, P., Lüking, M., Petrovic, D., . . . Widersten, M. (2018). 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. ACS Catalysis, 8(8), 7526-7538
Open this publication in new window or tab >>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
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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

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-355854 (URN)10.1021/acscatal.8b01762 (DOI)000441112400074 ()
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), 2016/34-27
Available from: 2018-07-05 Created: 2018-07-05 Last updated: 2019-10-21Bibliographically approved
Hamnevik, E. (2017). Characterization and Directed Evolution of an Alcohol Dehydrogenase: A Study Towards Understanding of Three Central Aspects of Substrate Selectivity. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
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. p. 95
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1497
Keywords
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
Hamnevik, E., Enugala, T. R., Maurer, D., Ntuku, S., Oliveira, A., Dobritzsch, D. & Widersten, M. (2017). Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer. The FEBS Journal, 284(22), 3895-3914
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
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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: 2019-10-21Bibliographically approved
Hamnevik, E., Blikstad, C., Norrehed, S. & Widersten, M. (2014). Kinetic characterization of Rhodococcus ruber DSM 44541 alcohol dehydrogenase A. Journal of Molecular Catalysis B: Enzymatic, 99, 68-78
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
Eklund, S., Lindås, A.-C., Hamnevik, E., Widersten, M. & Tomkinson, B. (2012). Exploring the active site of tripeptidyl-peptidase II through studies of pH dependence of reaction kinetics. Biochimica et Biophysica Acta - Proteins and Proteomics, 1824(4), 561-570
Open this publication in new window or tab >>Exploring the active site of tripeptidyl-peptidase II through studies of pH dependence of reaction kinetics
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2012 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1824, no 4, p. 561-570Article in journal (Refereed) Published
Abstract [en]

Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine protease which forms a large enzyme complex (> 4 MDa). It is considered a potential drug target due to its involvement in specific physiological processes. However, information is scarce concerning the kinetic characteristics of TPP II and its active site features, which are important for design of efficient inhibitors. To amend this, we probed the active site by determining the pH dependence of TPP II catalysis. Access to pure enzyme is a prerequisite for kinetic investigations and herein we introduce the first efficient purification system for heterologously expressed mammalian TPP II. The pH dependence of kinetic parameters for hydrolysis of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was determined for murine, human and Drosophila melanogaster TPP II as well as mutant variants thereof. The investigation demonstrated that TPP II, in contrast to subtilisin, has a bell-shaped pH dependence of kcatapp/KM probably due to deprotonation of the N-terminal amino group of the substrate at higher pH. Since both the KM and kcatapp are lower for cleavage of AAA-pNA than for AAF-pNA we propose that the former can bind non-productively to the active site of the enzyme, a phenomenon previously observed with some substrates for subtilisin. Two mutant variants, H267A and D387G, showed bell-shaped pH-dependence of kcatapp, possibly due to an impaired protonation of the leaving group. This work reveals previously unknown differences between TPP II orthologues and subtilisin as well as features that might be conserved within the entire family of subtilisin-like serine peptidases.

Keywords
tripeptidyl-peptidase II, AAF-pNA, AAA-pNA, steady-state kinetics, pH-dependence
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-166875 (URN)10.1016/j.bbapap.2012.01.004 (DOI)000302443000004 ()
Available from: 2012-01-16 Created: 2012-01-16 Last updated: 2017-12-08Bibliographically approved
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