Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1484 Extending the Reach of Computational Approaches to Model Enzyme Catalysis BEAT ANTON AMREIN ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-554-9816-0 UPPSALA urn:nbn:se:uu:diva-314686 2017 Dissertation presented at Uppsala University to be publicly examined in A1:111a, BMC, Husargatan 3, Uppsala, Friday, 24 March 2017 at 09:15 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Prof. Adrian Mulholland (University of Bristol). Abstract Amrein, B. A. 2017. Extending the Reach of Computational Approaches to Model Enzyme Catalysis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1484. 67 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9816-0. Recent years have seen tremendous developments in methods for computational modeling of (bio-) molecular systems. Ever larger reactive systems are being studied with high accuracy approaches, and high-level QM/MM calculations are being routinely performed. However, applying high-accuracy methods to large biological systems is computationally expensive and becomes problematic when conformational sampling is needed. To address this challenge, classical force field based approaches such as free energy perturbation (FEP) and empirical valence bond calculations (EVB) have been employed in this work. Specifically: 1. Force-field independent metal parameters have been developed for a range of alkaline earth and transition metal ions, which successfully reproduce experimental solvation free energies, metal- oxygen distances, and coordination numbers. These are valuable for the computational study of biological systems. 2. Experimental studies have shown that the epoxide hydrolase from Solanum tuberosum (StEH1) is not only an enantioselective enzyme, but for smaller substrates, displays enantioconvergent behavior. For StEH1, two detailed studies, involving combined experimental and computational efforts have been performed: We first used trans-stilbene oxide to establish the basic reaction mechanism of this enzyme. Importantly, a highly conserved and earlier ignored histidine was identified to be important for catalysis. Following from this, EVB and experiment have been used to investigate the enantioconvergence of the StEH1-catalyzed hydrolysis of styrene oxide. This combined approach involved wildtype StEH1 and an engineered enzyme variant, and established a molecular understanding of enantioconvergent behavior of StEH1. 3. A novel framework was developed for the Computer-Aided Directed Evolution of Enzymes (CADEE), in order to be able to quickly prepare, simulate, and analyze hundreds of enzyme variants. CADEE’s easy applicability is demonstrated in the form of an educational example. In conclusion, classical approaches are a computationally economical means to achieve extensive conformational sampling. Using the EVB approach has enabled me to obtain a molecular understanding of complex enzymatic systems. I have also increased the reach of the EVB approach, through the implementation of CADEE, which enables efficient and highly parallel in silico testing of hundreds-to-thousands of individual enzyme variants. Keywords: epoxide hydrolase, enantioselectivity, regioselectivity, enantioconvergence, biocatalysis, empirical valence bond, computational directed evolution Beat Anton Amrein, Department of Cell and Molecular Biology, Box 596, Uppsala University, SE-75124 Uppsala, Sweden. © Beat Anton Amrein 2017 ISSN 1651-6214 ISBN 978-91-554-9816-0 urn:nbn:se:uu:diva-314686 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-314686) /LVW RI SDSHUV 7KLV WKHVLV LV EDVHG RQ WKH IROORZLQJ SDSHUV ZKLFK DUH UHIHUUHG WR LQ WKH WH[W E\ WKHLU 5RPDQ QXPHUDOV , 'XDUWH ) %DXHU 3 %DUUR]R $ $PUHLQ % $ 3XUJ 0 cTYLVW - DQG .DPHUOLQ 6 & / )RUFH )LHOG ,QGHSHQGHQW 0HWDO 3DUDPHWHUV 8VLQJ D 1RQERQGHG 'XPP\ 0RGHO - 3K\V &KHP % ± ,, $PUHLQ % $ %DXHU 3 'XDUWH ) -DQIDON &DUOVVRQ c 1DZRU\WD $ 0RZEUD\ 6 / :LGHUVWHQ 0 DQG .DPHUOLQ 6 & / ([SDQGLQJ WKH &DWDO\WLF 7ULDG LQ (SR[LGH +\GURODVH DQG 5HODWHG (Q]\PHV $&6 &DWDO ± ,,, %DXHU 3 -DQIDON &DUOVVRQ c $PUHLQ % $ 'REULW]VFK ' :LGHUVWHQ 0 DQG .DPHUOLQ 6 & / &RQIRUPDWLRQDO 'LYHUVLW\ 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