MASARYK UNIVERSITY FACULTY OF SCIENCE DEPARTMENT OF EXPERIMENTAL BIOLOGY LOSCHMIDT LABORATORIES Molecular modeling of enzymes’ substrate specificity Doctoral dissertation Lukáš Daniel Supervisors: Prof. Mgr. Jiří Damborský, Dr. Mgr. Jan Brezovský, Ph.D. Brno 2016 Poděkování Rád bych poděkoval svému školiteli Jiřímu Damborskému nejen za umožnění studia v Loschmidtových laboratořích, ale především za odborné vedení během mého akademického dozrávání, povzbuzení a za ochotu a čas věnovaných do četných diskuzí. Velmi děkuji i Janu Brezovskému za trpělivost a cenné rady poskytované během mého studia. Děkuji také všem současným i minulým kolegům, kteří formovali Loschmidtovy laboratoře po vědecké i lidské stránce v místo, které je velmi přívětivé a inspirující pro každodenní práci. Největší poděkování však patří mým rodičům a blízkým za neutuchající podporu a důvěru v mém studijním i osobním životě. Bibliographic entry Author: Mgr. Lukáš Daniel Loschmidt laboratories Department of Experimental Biology Faculty of Science Masaryk University Title of dissertation: Molecular modeling of enzymes’ substrate specificity Study programme: Biology Supervisor: prof. Mgr. Jiří Damborský, Dr. Supervisor-specialist: Mgr. Jan Brezovský, Ph.D. Year of defence: 2016 Keywords: biocatalysis, substrate specificity, haloalkane dehalogenases, virtual screening, protein engineering, protein tunnel Bibliografický záznam Autor: Mgr. Lukáš Daniel Loschmidtovy laboratoře Ústav experimentální biologie Přírodovědecká fakulta Masarykova univerzita Název disertace: Molekulové modelování substrátové specificity enzymů Studijní obor: Biologie Školitel: prof. Mgr. Jiří Damborský, Dr. Školitel specialista: Mgr. Jan Brezovský, Ph.D. Rok obhajoby: 2016 Klíčová slova: biokatalýza, substrátová specifita, halogenalkandehalogenasy, virtuální screening, proteinové inženýrství, proteinový tunel Zvítězí, kdo vytrvá. © Lukáš Daniel, Masaryk University 2016 CONTENTS MOTIVATION 1 SUMMARY 2 SHRNUTÍ 4 INTRODUCTION 7 1. Enzyme biotechnology 7 2. Haloalkane dehalogenases 8 3. Engineering compounds – virtual screening 13 4. Engineering enzymes – design of specificity 28 SYNOPSIS OF RESULTS 42 CHAPTER 1 Mechanism-based discovery of novel substrates of haloalkane dehalogenases using in silico screening 45 Supplementary information 63 CHAPTER 2 Discovery of novel haloalkane dehalogenase inhibitors 93 Supplementary information 111 CHAPTER 3 Structural and functional analysis of a novel haloalkane dehalogenase with two halide-binding sites 121 Supplementary information 147 CHAPTER 4 CAVER Analyst 1.0: Graphic tool for interactive visualization and analysis of tunnels and channels in protein structures 155 Supplementary information 161 CHAPTER 5 Structural basis of paradoxically thermostable dehalogenase from psychrophilic bacterium 171 Supplementary information 193 CHAPTER 6 Enzyme tunnels and gates as relevant targets in drug design 201 REFERENCES 236 CURRICULUM VITAE 250 LIST OF PUBLICATIONS 252 MOTIVATION Haloalkane dehalogenases (HLDs) emerged in enzyme biotechnology more than twenty-five years ago. Despite they catalyze a reaction of environmental, pharmaceutical, and industrial importance, the known range of compounds able to bind into them has not changed much since then. Although HLDs were found in many different organisms, the natural substrate of these enzymes remains mostly elusive. The catalysis of HLDs is mediated by a network of access tunnels connecting the deeply buried active site with the surrounding solvent. Therefore, the in silico screening of chemical databases and modulation of the access tunnels represent an efficient strategy for enhancing the scope of enzymes’ binders and might decipher novel entities utilized by HLDs. The objectives of the Ph.D. project: 1. Development of the in silico screening platform for substrate identification 2. Systematic identification of novel chemical scaffolds binding into haloalkane dehalogenases 3. Rational engineering of access tunnels and active sites in haloalkane dehalogenases 4. Development of advanced tools for tunnel analysis 5. Critical review of the importance of access tunnels in pharmaceutical targets 1 SUMMARY This Thesis describes an application of molecular modeling tools to study and modify the substrate specificity of haloalkane dehalogenases (HLDs), hydrolytic enzymes cleaving the carbon-halide bonds in a variety of halogenated hydrocarbons. HLDs are industrially attractive biocatalysts with a well-understood reaction mechanism that often serve as a benchmark for testing of different molecular modeling protocols. The Introduction of the Thesis provides an overview of structure, function and industrial applications of HLDs as well as two sets of methods broadening the substrate scope of enzymes: (i) identification of novel substrates or inhibitors of natural enzymes by virtual screening and (ii) rational engineering of the enzymes’ active sites and their access pathways. Despite the broad interest in the identification and characterization of novel HLDs, the reported substrate range covers only short-chain aliphatic linear or cyclic haloalkanes and their derivatives. To further explore the substrate scope, an in silico method tested by screening more than 40,000 halogenated compounds against the previously uncharacterized HLD DmmA was newly developed (Chapter 1). The correct prediction of 50 % substrates and 100 % binders from 16 compounds proposed for experimental testing was observed. The new substrates comprised aromatic moieties and on average 50 % higher molecular weight than the common substrates of HLDs. DmmA transformed these molecules with the comparable or higher catalytic activity then other substrates. Three novel substrates were also converted by three other HLDs suggesting a catalytic robustness of these enzymes. Derivatives of the identified substrates possess the highest affinity ever observed in this protein family. HLDs have recently been discovered in a number of bacteria, including symbionts and pathogens of both plants and humans. However, the biological roles of HLDs in these organisms are unclear. The development of efficient HLD inhibitors serving as molecular probes to explore their function would represent an important step toward a better understanding of these interesting enzymes. The Chapter 2 describes the first systematic search for HLD inhibitors using two different approaches. The first built on the structures of the enzymes’ known substrates and led to the discovery of less potent nonspecific HLD inhibitors. The second approach involved the virtual screening of 140,000 potential inhibitors against the crystal structure of HLD from the human pathogen Mycobacterium tuberculosis H37Rv. The best inhibitor exhibited high specificity for the target structure, with an inhibition constant of 3 µM and a molecular architecture that clearly differs from those of all known HLD substrates. The new inhibitors will be used to study the natural functions of HLDs in bacteria, to probe their mechanisms, and to achieve their stabilization. 2 The active site engineering was used to assess the role of a unique second halide- binding site identified in the crystal structure of HLD DbeA (Chapter 3). Since the determination of the structure of the two-point mutant (DbeA ΔCl) lacking this site was not successful, disruption of the second-halide binding site was studied by molecular modeling. The modeling suggested that the binding of a chloride into the second-halide binding site was more favorable in the wild-type enzyme than in DbeA ΔCl which was subsequently confirmed by stopped-flow fluorescence measurement. The elimination of the second-halide binding site in DbeA ΔCl shifted the substrate specificity, lowered the catalytic activity and thermostability and eliminated the substrate inhibition. The changes of the catalytic activity studied by molecular modeling and kinetic experiments were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine in DbeA ΔCl. Fine-tuning of substrate specificity of many enzymes has been achieved by forcing the incoming substrate to pass through an access tunnel before reaching the deeply buried active site. This newly proposed „lock-keyhole-key” model introduces in the traditional models of catalysis the geometry and physicochemical properties of the access tunnels to assure the selection of substrates. The Chapter 4 describes a development of CAVER Analyst 1.0, a software tool enabling analysis and visualization of protein tunnels to facilitate the study of molecular transportation and the design of novel catalysts or effective drugs. The concept of tunnel engineering was applied in the structure-function studies of a psychrophilic HLD DmxA originating from a bacterium naturally occurring in an Antarctic lake (Chapter 5). DmxA possesses paradoxically the highest thermostability (Tm = 65.9 °C) ever observed in any wild-type HLD. Analysis of the DmxA’s crystal structure revealed narrow access tunnels of this protein. To open the main tunnel, four residues forming the tunnel bottleneck were in silico mutated to smaller amino acids. The effect of two substitutions was predicted as destabilizing while the effect of the other two was predicted as neutral. Experimental characterization of a mutant with introduced destabilizing mutations revealed improved overall activity, shifted substrate specificity and lower thermostability (Tm = 56.9 °C) than the wild-type DmxA. The Chapter 6 describes the importance