Quantum Chemical Studies of Epoxide- Transforming Enzymes Kathrin H. Hopmann Department of Theoretical Chemistry Royal Institute of Technology Stockholm, Sweden, 2007 ii © Kathrin H. Hopmann, 2007 ISBN 978-91-7178-640-1 ISSN 1654-2312 TRITA-BIO-Report 2007:3 Printed by Universitetsservice US-AB, Stockholm, Sweden. iii Abstract Density functional theory is employed to study the reaction mechanisms of different epoxide-transforming enzymes. Calculations are based on quantum chemical active site models, which are build from X-ray crystal structures. The models are used to study conversion of various epoxides into their corresponding diols or substituted alcohols. Epoxide-transforming enzymes from three different families are studied. The human soluble epoxide hydrolase (sEH) belongs to the α/β-hydrolase fold family. sEH employs a covalent mechanism to hydrolyze various epoxides into vicinal diols. The Rhodococcus erythrobacter limonene epoxide hydrolase (LEH) constitutes a novel epoxide hydrolase, which is considered the founding member of a new family of enzymes. LEH mediates transformation of limone-1,2-epoxide into the corresponding vicinal diol by employing a general acid/general base-mediated mechanism. The Agrobacterium radiobacter AD1 haloalcohol dehalogenase HheC is related to the short-chain dehydrogenase/reductases. HheC is able to convert epoxides using various nucleophiles such as azide, cyanide, and nitrite. Reaction mechanisms of these three enzymes are analyzed in depth and the role of different active site residues is studied through in silico mutations. Steric and electronic factors influencing the regioselectivity of epoxide opening are identified. The computed energetics help to explain preferred reaction pathways and experimentally observed regioselectivities. Our results confirm the usefulness of the employed computational methodology for investigating enzymatic reactions. iv Acknowledgements I would like to express my sincere gratitude to my supervisor Dr. Fahmi Himo. Thank you for giving me the opportunity to work in this field. This work would not have been possible without your enthusiasm and inspiration. Thanks to Professor Hans Ågren for accepting me in his group and for providing a stimulating working environment. I am also grateful that I was given many opportunities to travel and participate in conferences, winter schools, and collaborations. A big thanks to all my colleagues at the Department of Theoretical Chemistry in Stockholm. Special thanks to Emil, Peter, Robin, Polina, and Elias for all the good times together. Thanks to my dear roommates over the years, Viviane, Luca, Freddy, Oscar, Katia and Qiong. Thanks to Pawel for always being extremely helpful and for guiding a biochemist through the jungle of Linux. Also thanks to Laban, Emanuel, Prakash, Chen, Johanna, Cornel, Bin Gao, Yong Zeng, Liao, Mathias, YanHua, Yassen, Kai Liu, Na Lin, TT, JJ, Phoenix, Wenhua, Ke Zhao, Pekka, Haakan, and Luo. All of you contributed to making the department more than a place of work. Thanks for movie evenings, opera nights, poker games, Chinese New Year parties, and Friday pubs. Also thanks to our administrative staff, Pia and Lotta, for helping with many practical issues, and to Elizabeth, Cecilia, and Maria from the library for providing me with every literature reference I wished for. Most of this thesis was written during an extended stay in Calabria, Italy, and I would like to thank Professor Nino Russo for inviting me to his group. Thanks to Monica for a fruitful collaboration. And thanks also to all the wonderful friends I met in Calabria, especially Francesca, Fatima, Maria Fernanda, Tanja, and Rosa. Baci to all of you for making my stay in Italy such a wonderful experience. Danke auch an meinen lieben Zwilling David. Ich bin froh, dass es Dich gibt. Finally, a special thanks to Anders. I will never forget you. v Papers included in this thesis I. Catalytic Mechanism of Limonene Epoxide Hydrolase, a Theoretical Study. Kathrin H. Hopmann, B. Martin Hallberg, Fahmi Himo, J. Am. Chem. Soc. 2005, 127, 14339-347. II. Theoretical Study of the Full Reaction Mechanism of Human Soluble Epoxide Hydrolase. Kathrin H. Hopmann, Fahmi Himo, Chem. Eur. J. 2006, 12, 6898-909. III. Insights into the Reaction Mechanism of Soluble Epoxide Hydrolase from Theoretical Active Site Mutants. Kathrin H. Hopmann, Fahmi Himo, J. Phys. Chem. B. 2006, 110, 21299-310. IV. A Theoretical Study of the Azidolysis and Cyanolysis of Epoxides by Haloalcohol Dehalogenase. Kathrin H. Hopmann, Fahmi Himo, Manuscript Comments on my contributions to the included papers: I was responsible for all presented calculations, as well as for preparation of all manuscripts. vi Abbreviations CIU Cyclohexyl-N´-(Iodophenyl) Urea CPCM Conductor-like Polarizable Continuum Model DFT Density Functional Theory E enantiomeric ratio ee enantiomeric excess EchA Epoxide hydrolase from A.radiobacter AD1 EH Epoxide hydrolase GGA Generalized Gradient Approximation HF Hartree-Fock HheC Haloalcohol dehalogenase C from A.radiobacter AD1 LDA Local Density Approximation LD Limonene-1,2-diol LE Limonene-1,2-epoxide LEH Limonene Epoxide Hydrolase from R.erythropolis DCL14 MAD Mean Absolute Deviation MP2 Second order Møller Plesset perturbation theory MSO (1S,2S)-β-methylstyrene oxide PDB Protein Data Bank QM Quantum Mechanical RSO (R)-styrene oxide SDR Short-chain Dehydrogenase/Reductase SDS Sodium Dodecyl Sulfate sEH soluble Epoxide Hydrolase SSO (S)-styrene oxide StEH Epoxide hydrolase from potato (Solanum tuberosum) TMSCN Trimethylsilyl cyanide TS Transition State ZPV Zero Point Vibrational vii Amino acid symbols A/Ala Alanine C/Cys Cysteine D/Asp Aspartate E/Glu Glutamate F/Phe Phenylalanine G/Gly Glycine H/His Histidine I/Ile Isoleucine K/Lys Lysine L/Leu Leucine M/Met Methionine N/Asn Asparagine P/Pro Proline Q/Gln Glutamine R/Arg Arginine S/Ser Serine T/Thr Threonine V/Val Valine W/Trp Tryptophan X/Xaa Any residue Y/Tyr Tyrosine viii Contents Acknowledgements ....................................................................................... iv List of papers ............................................................................................... v Abbreviations ............................................................................................... vi Symbols for Amino Acids .............................................................................. vii 1 Introduction ........................................................................................... 1 2 Computational chemistry ........................................................................ 3 2.1 Density functional theory .................................................................... 3 2.2 B3LYP ............................................................................................. 6 2.2.1 Accuracy of B3LYP .................................................................. 6 2.2.2 Sources of error: self-interaction and near-degeneracy ................... 8 3 Modelling enzyme active sites ................................................................... 10 3.1 Modelling of active site residues .......................................................... 10 3.2 Modelling of substrates ....................................................................... 11 3.3 Computational strategy ....................................................................... 12 3.3.1 Geometry optimizations ............................................................. 12 3.3.2 Modelling of the surroundings .................................................... 12 3.3.3 Final energies .......................................................................... 13 3.4 Comparison with experiment ............................................................... 14 4 Epoxide chemistry ................................................................................. 15 4.1 Regioselectivity of epoxide opening ...................................................... 15 4.2 Enantioselectivity of epoxide conversion ............................................... 16 4.3 Non-enzymatic transformation of epoxides ............................................ 17 4.3.1 Hydrolysis ............................................................................... 17 4.3.2 Azidolysis ............................................................................... 17 4.3.3 Cyanolysis ............................................................................... 18 4.3.4 Haloalcohol formation ............................................................... 19 4.3.5 Conclusions on non-enzymatic epoxide transformation ................... 19 4.4 Enzymatic transformation of epoxides ................................................... 19 4.4.1 The soluble epoxide hydrolases ................................................... 20 4.4.2 Limonene epoxide hydrolase ....................................................... 21 4.4.3 The haloalcohol dehalogenases .................................................... 21 4.4.4 Conclusions on enzymatic epoxide transformation .......................... 22 ix 5 Quantum chemical studies of epoxide-transforming enzymes ...................... 23 5.1 Limonene epoxide hydrolase (Paper I) .................................................. 24 5.1.1 Elucidating the mechanism of LEH ............................................. 24 5.1.2 Analysis of the regioselectivity of LEH ........................................ 25 5.1.2.1 Regioselectivity of 1-methylcyclohexene oxide hydrolysis .. 29 5.1.2.2