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Computational Protein Evolution Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1922 Computational Protein Evolution Modeling the Selectivity and Promiscuity of Engineered Enzymes KLAUDIA SZELER ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-0918-7 UPPSALA urn:nbn:se:uu:diva-407494 2020 Dissertation presented at Uppsala University to be publicly examined in Room C4:301, BMC, Husargatan 3, Uppsala, Monday, 18 May 2020 at 13:15 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Maria Ramos (University of Porto). Abstract Szeler, K. 2020. Computational Protein Evolution. Modeling the Selectivity and Promiscuity of Engineered Enzymes. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1922. 81 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0918-7. Enzymes are biological catalysts that significantly increase the rate of all biochemical reactions that take place within cells and are essential to maintain life. Many questions regarding their function remain unknown. Experimental techniques, such as kinetic measurements, spectroscopy, and site-directed mutagenesis, can provide relevant information about enzyme structure, key residues, active site conformations, and kinetics. However, they struggle to provide a full picture of enzyme catalysis. Combining experiments with computational techniques gives the possibility to generate a complete explanation with atomistic resolution. Computational modeling offers an incredibly robust toolkit that can provide detailed insight into the reactivity and dynamics of biomolecules. Compounds that contain phosphate and sulfate groups are essential in the living world. They are present as i.e., a biological source of energy (ATP), signaling molecules (GTP), coenzymes, building blocks (DNA, RNA). Furthermore, phosphate esters can be used as insecticides, herbicides, flame retardants, and as chemical weapons. Cleavage of the phosphate bond involves an extremely low rate of spontaneous hydrolysis, nevertheless it is common reaction in living organisms. Phosphatases (enzymes catalysing cleavage of phosphate bond) are crucial in both physiological regulation as well as serious pathological conditions including asthma, immunosuppression, cardiovascular diseases, diabetes. Understanding the basis of phosphoryl and sulfuryl transfer reactions is crucial for medical, biological, and biotechnological industries in order to i.e., create and improve existing drugs, modify enzyme structures, understand the development of some diseases. However, despite decades of both experimental and computational studies, mechanistic details of these reactions remain controversial. These reactions can occur via multiple different mechanisms involving intermediate steps or transition state structures. To solve these puzzles, we performed computational studies to verify the reaction pathway of diaryl sulfate diesters hydrolysis. We suggest that the reaction proceeds through a concerted mechanism with a loose (slightly dissociative) transition state. Serum paraoxonase 1 (PON1) is calcium-dependent lactonase, which is bound to high-density lipoprotein (HDL) with apolipoprotein A-I (ApoA-I). The enzyme is highly promiscuous and catalyzes the hydrolysis of multiple, different types of chemical compounds, such as lactones, aromatic esters, oxons, and organophosphates. We performed several, complex studies on PON1’s reaction mechanism, promiscuity, PON1-HDL interactions, and evolutionary trajectories. One of the most extensively used approaches in this thesis was the empirical valence bond (EVB) method. Our models reproduce essential experimental observables and provide mechanistic insights and a better understanding of the enzymes role and its evolutionary derived promiscuity. Keywords: enzymes, kinetics, catalysis, density functional theory, empirical valence bond, diesters, evolution, promiscuity Klaudia Szeler, Department of Chemistry - BMC, Biochemistry, Box 576, Uppsala University, SE-75123 Uppsala, Sweden. © Klaudia Szeler 2020 ISSN 1651-6214 ISBN 978-91-513-0918-7 urn:nbn:se:uu:diva-407494 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-407494) I was taught that the way of progress is neither swift nor easy. Maria Skłodowska-Curie To my beloved husband, kids and parents. List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Szeler K., Williams N. H., Hengge A. C., Kamerlin S. C. L., Modeling the alkaline hydrolysis of diaryl sulfate diesters: A mechanistic study. J. Org. Chem., Manuscript submitted II Ben-David M., Sussman J. L., Maxwell C. I., Szeler K., Kamer- lin S. C. L. And Tawfik D. S. Catalytic stimulation by restrained active-site floppiness – The case of high-density lipoprotein bound serum paraoxonase 1. J. Mol. Biol. 2015, 427, 1359. III Blaha-Nelson D., Krüger D. M., Szeler K., Ben-David M. and Kamerlin S. C. L. Active site hydrophobicity and the convergent evolution of paraoxonase activity in structurally divergent en- zymes: The case of serum paraoxonase 1. J. Am. Chem. Soc. 2017, 139, 1155. IV Ben-David M., Soskine M., Dubovetskyi A., Cherukuri K., Dym O., Sussman J. L., Liao Q., Szeler K., Kamerlin S. C. L., Tawfik D. S. Enzyme evolution: An epistatic ratchet versus a smooth re- versible transition. Mol. Biol. Evol. 2019, 37, 4, 1133. Reprints were made with permission from the respective publishers. Additional Publications I Ma H., Szeler K., Kamerlin S. C. L. and Widersten M. Linking coupled motions and entropic effects to the catalytic activity of 2-deoxyribose-5-phosphate aldolase (DERA). Chem. Sci. 2016, 7, 1415. II Carvalho T. P., Szeler K., Vavitsas K., Aqvist J. and Kamerlin S. C. L. Modelling the mechanisms of biological GTP hydroly- sis. Arch. Biochem. Biophys. 2015, 582. III Petrović D., Szeler K. and Kamerlin S. C. L. Challenges and ad- vances in the computational modeling of biological phosphate hydrolysis. ChemComm 2018, 54, 377. Contents 1. Introduction .............................................................................................. 11 2. Enzymes ................................................................................................... 13 2.1. Enzyme kinetics ................................................................................ 14 2.1.1. Michaelis-Menten kinetics ......................................................... 14 2.2. Enzyme catalysis ............................................................................... 17 2.3. Enzymes dynamics, promiscuity and evolution ................................ 20 3. Computational approaches ....................................................................... 23 3.1. Quantum mechanical approaches ...................................................... 23 3.1.1. Born-Oppenheimer approximation ............................................ 24 3.1.2. Basis sets .................................................................................... 25 3.1.3. Hartree-Fock method ................................................................. 26 3.1.4. Density functional theory ........................................................... 27 3.2. Classical methods .............................................................................. 28 3.2.1. Force fields ................................................................................. 29 3.2.2. Molecular dynamics ................................................................... 30 3.2.3. Free energy calculations............................................................. 31 3.3. Multiscale models ............................................................................. 32 3.3.1. The combined quantum mechanics/molecular mechanics (QM/MM) approach ............................................................................. 32 3.3.2. The empirical valence bond method .......................................... 33 3.4. Analysis of the results ....................................................................... 36 3.4.1. Clustering algorithms ................................................................. 36 3.4.2. Principal component analysis ..................................................... 38 4. Phosphate and sulfate esters ..................................................................... 40 4.1. Mechanisms of phosphate and sulfate ester hydrolysis .................... 41 4.2. Linear free energy relationship ......................................................... 43 4.3. Kinetic isotope effects ....................................................................... 45 4.4. The nature of the transition states in involved in phosphate and sulfate ester hydrolysis (Paper I) ......................................................................... 46 5. Understanding the mechanism and evolution of enzymes that catalyze phosphoryl and sulfuryl transfer. ................................................................. 49 5.1. Catalytic mechanism and properties of serum paraoxonase 1 .......... 49 5.2. Serum paraoxonase 1 ........................................................................ 50 5.3. Catalytic floppiness and the activity of PON1 (Paper II) ................. 52 5.4. Influence of solvation on PON1 activity (Paper III) ......................... 56 5.5. An epistatic ratchet in PON1 (Paper IV) .......................................... 62 6. Conclusions .............................................................................................
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