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Characterization and Directed Evolution of an Alcohol Dehydrogenase Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1497 Characterization and Directed Evolution of an Alcohol Dehydrogenase A Study Towards Understanding of Three Central Aspects of Substrate Selectivity EMIL HAMNEVIK ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-554-9875-7 UPPSALA urn:nbn:se:uu:diva-318984 2017 Dissertation presented at Uppsala University to be publicly examined in BMC A1:111A, Husargatan 3, Uppsala, Friday, 19 May 2017 at 09:15 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Gerrit Poelarends (University of Groningen). Abstract Hamnevik, E. 2017. Characterization and Directed Evolution of an Alcohol Dehydrogenase. A Study Towards Understanding of Three Central Aspects of Substrate Selectivity. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1497. 95 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9875-7. 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. Keywords: Alcohol dehydrogenase, ADH-A, Biocatalysis, Directed evolution, Enantioselectivity, Enzyme kinetics, Enzyme mechanisms, Protein Engineering, Regioselectivity, Substrate selectivity Emil Hamnevik, Department of Chemistry - BMC, Biochemistry, Box 576, Uppsala University, SE-75123 Uppsala, Sweden. © Emil Hamnevik 2017 ISSN 1651-6214 ISBN 978-91-554-9875-7 urn:nbn:se:uu:diva-318984 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-318984) Till min kära familj When you talk, you are only repeat- ing what you know; But when you listen, you may learn something new – Dalai Lama List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Hamnevik, E., Blikstad, C., Norrehed, S., Widersten, M. (2014) Kinetic characterization of Rhodococcus ruber DSM 44541 al- cohol dehydrogenase A. J. Mol. Catal. B-Enzyme 99: 68-78 II Hamnevik, E., Enugala, T. R., Maurer, D., Ntuku, S., Do- britzsch, 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. Submitted III Maurer, D., Hamnevik, E., Enugala, T. R., Hillier, H., Do- britzsch, D., Widersten, M., (2017) The angle of a phenol side- chain decides regio- and enantioselectivity in Rhodococcus ruber alcohol dehydrogenase A. Submitted IV Hamnevik, E., Maurer, D., Chu, T., Löfgren, R., Dobritzsch, D., Widersten, M., (2017) Laboratory evolution of alcohol dehy- drogenase ADH-A for efficient transformation of vicinal diols and acyloins. Synthesis of 2-hydroxy acetophenone from race- mic styrene oxide. Manuscript Reprints were made with permission from the respective publishers. The following publications are not included in this thesis V Eklund, S, Lindås, A-C., Hamnevik, E., Widersten, M., Tom- kinson, B. (2012) Exploring the active site of tripeptidyl- peptidase II through studies of pH dependence of reaction kinet- ics. Biochim. Biophys. Acta 1824: 561-570 Contribution report My contribution to the included papers is as followed: I Performed all experimental work, except stopped-flow meas- urements and NMR studies. Contributed to the writing of the manuscript. II Designed enzyme libraries and isolated hits, performed most of the characterization work, not including the stopped-flow meas- urements or the enzyme crystallization. Contributed in the writ- ing of the manuscript. III Designed libraries and isolated hits. Contributed in designing characterization experiments and contributed to writing the manuscript. IV Designed libraries and isolated hits. Contributed in designing characterization experiments and contributed to writing the manuscript Contents Introduction ................................................................................................... 11 Green Chemistry ...................................................................................... 11 Biocatalysis .............................................................................................. 12 Benefits of enzymes in biocatalysis ..................................................... 13 Limitations of enzymes in biocatalysis ................................................ 13 Ways of finding new and better enzymes ................................................ 14 Finding novel enzymes ........................................................................ 16 Protein engineering .............................................................................. 17 Screening techniques ........................................................................... 21 Challenges and limitations with improving enzymes .......................... 22 Enzyme concepts for catalysis and biocatalysis ....................................... 22 Enzyme kinetics ................................................................................... 23 Deuterium kinetic isotope effect .......................................................... 25 Inhibition ............................................................................................. 26 Enzyme stability .................................................................................. 29 pH dependence .................................................................................... 29 Enzyme substrate selectivity ................................................................ 30 Structure-function relationship ............................................................ 32 Aim of present study ................................................................................ 38 Alcohol dehydrogenases .......................................................................... 38 Common structural elements ............................................................... 39 Reaction ............................................................................................... 39 Applications in industry ....................................................................... 40 Alcohol dehydrogenase A from Rhodococcus ruber ........................... 42 Present investigations .................................................................................... 43 Characterization of Alcohol dehydrogenase A, ADH-A (Paper I) ........... 44 Aims ..................................................................................................... 44 Stability of ADH-A.............................................................................. 44 Substrate selectivity ............................................................................. 45 pH dependence .................................................................................... 48 Kinetic mechanism .............................................................................. 48 Conclusions ......................................................................................... 52 A look into enantioselectivity of ADH-A (Paper II) ................................ 54 Aims ..................................................................................................... 54 Reiteration of cofactor binding and an update of the mechanism ....... 54 Decision of libraries and their design .................................................
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