Biochemical and Structural Analysis of Thermostable Esterases Mark Levisson Thesis supervisors: Prof. dr. J. van der Oost Personal chair at the Laboratory of Microbiology Wageningen University Prof. dr. W.M. de Vos Professor of Microbiology Wageningen University Thesis co-supervisor: Dr. S.W.M. Kengen Assistant Professor at the Laboratory of Microbiology Wageningen University Other members: Prof. dr. ir. H. Gruppen Wageningen University Dr. M.C.R Franssen Wageningen University Dr. T. Sonke DSM, Geleen Prof. dr. B.W. Dijkstra University of Groningen Dr. A.M.W.H Thunnissen University of Groningen This research was conducted under the auspices of the Graduate School VLAG Biochemical and Structural Analysis of Thermostable Esterases Mark Levisson Thesis submitted in partial fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Doctorate Board to be defended in public on Monday 28 September 2009 at 4 PM in the Aula Levisson, M. Biochemical and Structural Analysis of Thermostable Esterases PhD Thesis, Wageningen University, Wageningen, The Netherlands (2009) 170 pages - With references, and with summaries in English and Dutch ISBN: 978-90-8585-459-3 TABLE OF contents Aim and outline 1 Chapter 1 Introduction 5 Chapter 2 Characterization and structural modeling of a new type of 21 thermostable esterase from Thermotoga maritima Chapter 3 Crystallization and preliminary crystallographic analysis of 39 an esterase with a novel domain from the hyperthermophile Thermotoga maritima Chapter 4 Crystal structure and biochemical properties of a novel 45 thermostable esterase containing an immunoglobulin-like domain Chapter 5 The crystal structures of a thermostable acetyl esterase / 65 cephalosporin C deacetylase from Thermotoga maritima in complex with PMSF and paraoxon reveal an altered conformation of the catalytic serine Chapter 6 Purification and partial characterization of a thermostable 87 esterase from Thermotoga maritima Chapter 7 Characterization of a lipase from the hyperthermophilic archaeon 99 Archaeoglobus fulgidus Chapter 8 Summary and general discussion 115 References 127 Appendix I: Co-author affiliations 141 Appendix II: Color figures 143 Nederlandse samenvatting 155 Dankwoord 161 Curriculum vitae 165 Publication list 166 Overview of completed training activities 167 Aim and outline 1 Aim and outline AIM AND OUtlINE This thesis describes the results of biochemical and structural analyses of thermostable esterases. The aim of this research project was to isolate and produce new esterases from hyperthermophilic microorganisms, to gain insight in their catalytic properties and to investigate their potential as biocatalysts in food-related conversions. Many processes in industry are operated under conditions, which are unfavorable for many biocatalysts. In this regard, enzymes from hyperthermophiles are promising candidates since they generally have a high intrinsic stability. The initial project aimed also at investigating the behavior of thermostable enzymes under high pressure or microwave irradiation. These methods may open new ways for the tuning of enzyme reactions, and thus offers the possibility of expanding the area of biocatalysis in the food industry. However, after disappointing pilot experiments, technical problems and better insight into high pressure theory, this approach was abandoned. Therefore, the main focus in this thesis is on the isolation, and biochemical and structural characterization of distinct esterases from the bacteriumThermotoga maritima (chapter 2-6) and a lipase from the archaeon Archaeoglobus fulgidus (chapter 7). The information obtained in this study provides fundamental knowledge, which may be useful for an industrial application. Chapter 1 - Introduction The first chapter gives a general overview of all currently characterized carboxylic ester hydrolases from hyperthermophilic bacteria and archaea. The biochemical properties, structures and classification of these enzymes are discussed. In addition, approaches for the discovery of new carboxylic ester hydrolases are described. Chapter 2 - Characterization and structural modeling of a new type of thermostable esterase from Thermotoga maritima The second chapter describes the identification, heterologous production, purification and biochemical characterization of an esterase (EstD) from T. maritima. A structural model was constructed based on threading and provided insight into the active site and substrate binding. Residues involved in catalysis were verified by site-directed mutagenesis and inhibition studies. Phylogenetic analysis of EstD suggested a new family of esterases. Chapter 3 - Crystallization and preliminary crystallographic analysis of an esterase with a novel domain from the hyperthermophile Thermotoga maritima The third chapter describes the cloning, purification, crystallization and preliminary X-ray analysis of an esterase (EstA) from T. maritima. Native and selenomethionine-substituted EstA was crystallized by the hanging-drop vapour-diffusion method. Multiple wavelength anomalous data sets were collected to 2.6 Å resolution and an initial analysis is described. 2 Aim and outline Chapter 4 - Crystal structure and biochemical properties of a novel thermostable esterase containing an immunoglobulin-like domain After obtaining its preliminary crystallographic data, as described in Chapter 3, EstA was investigated further, in order to provide insight into the function of the immunoglobulin-like domain and reveal the molecular basis of substrate recognition and catalysis. The structure of EstA, native and in complex with the competitive inhibitor paraoxon, is described and the quaternary structure was investigated using dynamic light scattering, mass-spectrometry and electron microscopy. At present, this is the only esterase that has been described to have an immunoglobulin-like domain. Chapter 5 - The crystal structures of a thermostable acetyl esterase / cephalosporin C deacetylase from Thermotoga maritima in complex with PMSF and paraoxon reveal an altered conformation of the catalytic serine Chapter five reports on the crystal structure of an acetyl esterase, presumably involved in xylan degradation. Insight into substrate binding was obtained from co-crystal structures with the inhibitors PMSF and paraoxon. Various biochemical properties and the positional specificity of the esterase was investigated. Chapter 6 - Purification and partial characterization of a thermostable esterase from Thermotoga maritima The sixth chapter describes the cloning, purification, crystallization and partial biochemical characterization of an esterase (EstB) from T. maritima. EstB was crystallized by hanging-drop vapour-diffusion and a dataset was collected to ~2.8 Å resolution. Its structure solution is ongoing. Chapter 7 - Characterization of a lipase from the hyperthermophilic archaeonArchaeoglobus fulgidus No true lipases, hydrolyzing long chain fatty acid esters, have thus far been identified in hyperthermophiles. The seventh chapter describes the identification, cloning, purification and partial characterization of a lipase (LipA) from A. fulgidus. Lipase activity on triacylglycerol esters was determined using qualitative plate assays. LipA was crystallized by hanging-drop vapour-diffusion and a dataset was collected to ~2.6 Å resolution. Its structure solution is ongoing. Chapter 8 - Summary and general discussion This final chapter is a brief summary of the findings described in this thesis. Discussed are the physiological role of esterases, the use of microwave irradiation and high pressure for biocatalysis, and the general aspects of the enzymes described in this thesis, with some concluding remarks. 3 4 1Chapter Introduction This chapter has been adapted from: Levisson, M., van der Oost, J. & Kengen, S.W.M. (2009) Carbohydrate ester hydrolases from hyperthermophiles. Extremophiles 13 (4), p. 567 - 581. 5 1 Introduction INTRODUctION The synthesis of specific products by enzymes is a fundamental aspect of modern biotechnology. This biocatalytic approach has several advantages over traditional chemical engineering, such as higher product purity, fewer waste products, lower energy consumption and more selective reactions due to the high regio- and stereo-selectivity of enzymes 1. One of the industrially most exploited and important groups of biocatalysts are the carboxylic ester hydrolases (EC 3.1.1.X) 2; 3. Carboxylic ester hydrolases are ubiquitous enzymes, which have been identified in all domains of life (Bacteria, Archaea and Eukaryotes), and in some viruses. In the presence of water, they catalyze the hydrolysis of an ester-bond resulting in the formation of an alcohol and a carboxylic acid. However, in an organic solvent they can catalyze the reverse reaction or a trans-esterification reaction (Figure 1.1)4 . Most carboxylic ester hydrolases belong to the α/β- hydrolase family and share structural and functional characteristics, including a catalytic triad, an α/β-hydrolase fold, and a co-factor independent activity. The catalytic triad is conserved and is usually composed of a nucleophilic serine in a GXSXG pentapeptide motif (where X is any residue), and an acidic residue (aspartate or glutamate) that is hydrogen-bonded to a histidine residue 5; 6; 7; 8. Figure 1.1: Reactions catalyzed by carboxylic ester hydrolases: a) hydrolysis, b) esterification and c) transesterification.