Quantum Chemical Studies of Epoxide- Transforming Enzymes

Kathrin H. Hopmann

Department of Theoretical Chemistry Royal Institute of Technology Stockholm, Sweden, 2007 ii

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.

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.

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

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 Regioselectivity of limonene-1,2-epoxide hydrolysis ...... 30 5.2 The soluble epoxide hydrolase (Papers II and III) ...... 31 5.2.1 The reaction mechanism of sEH ...... 31 5.2.2 Tyrosine mutants of sEH ...... 36 5.2.3 Studies on the regioselectivity of sEH ...... 38 5.3 The haloalcohol dehalogenase HheC (Paper IV) ...... 40 5.3.1 The epoxide-opening mechanism of HheC ...... 40 5.3.2 Regioselectivity of wild type HheC ...... 43 5.3.3 Regioselectivity of HheC mutants ...... 45

6 Conclusions ...... 51 6.1 Mechanistic strategies of epoxide-transforming enzymes ...... 51 6.2 Regioselectivities of epoxide-transforming enzymes ...... 52 6.3 Final Conclusions ...... 53

References ...... 55 Papers I – IV ...... 59

Chapter 1 Introduction

Computational chemistry has grown into a versatile tool for exploring enzymatic catalysis. Mechanistic proposals can be analyzed in depth, and optimization of intermediate structures and transition states enables detailed characterization of the reaction pathways employed by enzymes. Earlier limitations of quantum chemistry, only allowing accurate calculations of small systems, have been surpassed. Evolution of quantum mechanical methods and improvement of computational performance now allow treatment of increasingly larger models and complex systems at a relatively high level of theory. Especially the Density Functional Theory (DFT) method has proven to be superior in treatment of enzymatic systems. DFT, and in particular the B3LYP functional, exhibits high accuracy in regard to structure optimizations and energy related properties, in many cases comparable to highest-quality wave-function-based methods. At the same time, relatively large systems can be treated at a reasonable speed. With DFT, convenient treatment of quantum chemical models of up to 150 atoms is now possible. Although this is quite large for a quantum chemical model, at first sight it still appears to be a rather crude approximation to an enzyme containing thousands of atoms. However, enzymatic reactions usually take place within a small area of the enzyme only, the so-called active site. Here, the chemical reaction will be mediated by a limited number of functional groups. With models of the above size, it is usually possible to include the catalytic residues as well as other groups important for stabilization and binding of intermediate structures, thus arriving at a reasonable approximation to the enzyme active site.

In this thesis, B3LYP has been used to study the reaction mechanism of several epoxide-transforming enzymes. Conversion of epoxides is a useful route in organic chemistry to arrive at various chiral compounds. However, epoxide transformation in 2

solution often suffers from drawbacks in regard to selectivity and reaction conditions. Epoxide-converting enzymes are of great aid here, as they can act as biocatalysts for selective formation of desired products under ambient reaction conditions. Detailed understanding of the reaction mechanism and selectivities of these enzymes is not only of general scientific interest, but can also help to improve reaction conditions, indicate beneficial mutational modifications and aid in design of specific inhibitors.

In the following, a brief outline will be given of the theoretical approach employed in this thesis (Chapter 2). The protocol for quantum chemical modelling of enzyme active sites is discussed in Chapter 3. A presentation of enzymatic and non-enzymatic epoxide conversion is given in Chapter 4. The results of our theoretical investigations of three different epoxide-transforming enzymes are presented in Chapter 5. For each enzyme, the mechanism of action is studied and the regioselectivity of epoxide opening is analyzed. Finally, in Chapter 6, the results are summarized. The papers, in which our work was published, are attached at the end of this thesis.

Chapter 2 Computational chemistry

All calculations presented in this thesis have been performed using the density functional theory (DFT) approach, in particular the hybrid functional B3LYP. A short introduction of DFT and B3LYP is given below.a

2.1 Density functional theory Conventional quantum chemical methods are based on obtaining the wave function for a given molecule. Once the wave function is known, all properties of the molecule can be obtained. However, since the wave function depends on 3 spatial and 1 spin variables for each electron, a total of 4N variables enter the system. This becomes an unmanageable approach for models with many atoms, such as those studied in this thesis. A different approach to wave-function based methods is to use the electron density as variable. The density only depends on three spatial variables, independently of the number of electrons. However, it is not straightforward to see that a density- based approach can give us a similar amount of information as a wave-function-based approach. To begin with, it is not even clear that there is a unique relationship between the density of a system and its properties. In 1964, Hohenberg and Kohn published a paper, in which they present a functional for the ground state energy of an electron gas.1,2 The variable of the functional is the density, ρ(r). In their paper, Hohenberg and Kohn prove that the ground state density of a system uniquely defines all molecular properties. It is also shown that the density-dependent functional obeys the variational principle, which states that any calculated energy is always higher than or equal to the true ground state energy.2

aFor a more comprehensive discussion of quantum chemical methods and DFT in particular, please refer to Koch and Holthausen1 or Jensen4. 4

In the density functional theory approach, the electronic energy of the ground state of a molecule can be divided into different terms, each with the density ρ as the input variable1:

(2.1) Etot[ρ] = T[ρ] + Eee[ρ] + ENe[ρ]

where T is the kinetic energy, Eee is the electron-electron repulsion, and ENe is the nuclear-electron attraction. The later term is often referred to as the external potential and is system dependent. However, the first two parts are universally valid and can be grouped together into the density functional F[ρ]:

(2.2) Etot[ρ] = F[ρ] + ENe[ρ]

If F[ρ] was known, one would operate with exact DFT. However, exact determination of F[ρ] is not straightforward. As Hohenberg and Kohn stated:

“If F[ρ] were a known and sufficiently simple function of n, the problem of determining the ground-state energy and density in a given external potential would be rather easy since it requires merely the minimization of a functional of the three-dimensional density function. The major part of the complexities of the many-electron problems are associated with the determination of the 2, b universal functional F[ρ].”

Early attempts to approximate F[ρ] showed rather bad results. Kohn and Sham realized that the failure of certain density functionals in large part is due to the way the kinetic energy of the electrons is calculated.1 They developed instead an orbital-based scheme in which the total kinetic energy (T) is divided into two parts, the kinetic energy (TS) of a non-interacting system of N electrons (with the same density as the 3 real interacting system) and the residual part, TC :

(2.3) T[ρ] = TS[ρ] + TC[ρ]

With this approach, the F[ρ] functional can be written as: bIn the original notation by Hohenberg and Kohn, the density was referred to as n. This has been replaced with ρ here, for sake of clarity. 5

(2.4) F[ρ] = TS[ρ] + J[ρ] + EXC[ρ]

where J is the classical Coulomb interaction and EXC is the exchange-correlation energy defined as :

(2.5) EXC [ρ] = (T[ρ] - TS[ρ]) + (Eee[ρ]- J[ρ]) = TC[ρ] + Encl[ρ]

where T, Ts, and J are as defined above and Encl is the non-classical part containing correlation and exchange. The total energy can now be written as:

(2.6) Etot = ET + EJ + EV + EXC

where ET is the kinetic energy of the non-interacting system, EJ is the classical

Coulomb repulsion, EV the nuclear-electron energy, and EXC the exchange-correlation energy as defined above. It should be noted that all contributions to (2.6) can be calculated explicitly, except the exchange-correlation energy. The exchange- correlation functional EXC[ρ] is unknown and can currently only be approximated.

One approach to calculate the exchange-correlation energy involves the Local Density Approximation (LDA), where it is assumed that the density only varies slowly and locally can be treated as a uniform gas.4 A popular correlation functional developed using the uniform electron gas model is the correlation functional VWN introduced by Vosko, Wilk, and Nusair.5 However, the error in exchange energy using the LDA scheme is in general about 10%, making this approach less promising.4 The assumed uniform electron distribution works well for certain systems, but is not useful for most molecules, where the electron distribution is far from uniform. An improvement to LDA was the introduction of gradient corrected methods (GGA), which consider not only the density in a given point, but also the derivative of the density. Becke introduced a gradient-corrected functional for the exchange energy referred to as B88.6 A popular gradient-corrected functional for the correlation energy has been introduced by Lee,Yang, and Parr, which is referred to as LYP.7 The combination of B88 and LYP results in the widely used BLYP functional.4

2.2 B3LYP Becke argued that further improvement to the GGA scheme could be achieved if a certain amount of Hartree-Fock exchange also is included in the functional.8 This leads to the so-called hybrid functionals. One hybrid functional that incorporates B88, LYP, VWN and exact exchange is the hybrid functional B3LYP, which is defined as:

B3LYP LDA HF B88 LYP VWN (2.7) XC X X X C −++++−= c)E(1cEbEaEa)E(1E C

The coefficients a = 0.20, b = 0.72 and c = 0.81 were taken from the B3PW91 hybrid functional (having PW91 instead of LYP).1,8 The values of the coefficients were determined empirically for B3PW91 by a linear least-squares fit to 116 experimentally determined energies.8

2.2.1 Accuracy of B3LYP Evaluation of functionals is usually done by comparing to experimentally determined geometries and energies. The accuracy of B3LYP in regard to structural parameters has been evaluated on 53 molecules from the G2 test set.9 The set includes 71 bond lengths, 26 bond angles and 2 dihedral angles. Calculations at the B3LYP/6-31G(d) level of theory yielded average absolute errors of 0.013 Å for bond lengths, 0.62º for angles, and 0.35º for dihedral angles.9 The error in bond lengths and angles was only slightly reduced if a larger basis set was used, i.e. for calculations at the B3LYP/6- 311+G(3df,2p) level of theory, the average absolute errors were 0.008 Å for bond lengths and 0.61º for angles. For dihedral angles, the error was increased to 3.66º with the large basis set, but this result should be taken with caution, as only two dihedral angles are present in the test set.9 Overall, it can be concluded that the accuracy of B3LYP in regard to geometrical parameters is highly satisfactory.

The accuracy of B3LYP in determining different types of energies has also been tested. For example, atomization energies were calculated for 41 molecules, 26 diatomics from the G2 test set plus additional 15 other molecules, yielding an average error of 2.2 kcal/mol at the B3LYP/6-311+G(3df,2p) level (with the 6-31G(d) basis set, an error of 5.18 kcal/mol is observed).9 Atomization energies calculated with 7

Table 2.1 Mean Absolute Deviation of B3LYP-energies calculated on the G3/05 set.11 Energies (# of energies) MAD (kcal/mol) Enthalpies of formation (270) 4.63 Non-hydrogens (79) 5.77 Hydrocarbons (38) 5.40 Substituted hydrocarbons (100) 4.52 Inorganic hydrides (19) 2.16 Radicals (34) 2.87 Ionization energies (105) 3.83 Electron affinities (63) 2.99 Proton affinities (10) 1.39 Hydrogen-bond strength (6) 1.19 All (454) 4.11

B3LYP/6-311+G(3df,2p) for the entire G2 set (55 molecules) resulted in the same average error, 2.2 kcal/mol regardless if geometries were optimized with 6-31G(d) or with the 6-311+G(3df,2p) basis set.10 These results indicate that B3LYP is able to approach so-called chemical accuracy, which is considered to lie within 1-2 kcal/mol of error. However, the above test set of molecules is relatively small. A much bigger evaluation has recently been done by Curtis et al., who tested a number of density functionals on the G3/05 test set.11 This set includes 454 energies, all of which have experimental uncertainties less than ±1 kcal/mol. The computed results are based on single-point B3LYP/6-311+G(3df,2p) energies at MP2/6-31G(d) geometries with scaled (0.89) HF/6-31G(d) zero-point energies. Table 2.1 shows that B3LYP achieves chemical accuracy for certain energies in the G3/05 set such as proton affinities, while it performs less well for enthalpies of formation of for example hydrocarbons.

For our approach it is of particular interest to establish what error B3LYP usually displays in calculations of reaction barriers. In general, DFT is often quoted for underestimating barriers. For example for the SN2 attack of chlorine or bromide at a saturated carbon atom, the barriers are underestimated with B3LYP by at least 3-5 kcal/mol.12 An investigation of 12 small organic reactions with various DFT 8

functionals also showed a tendency to underestimate reaction barriers.13 For 9 pericyclic reactions, the enthalpies of activation calculated at the B3LYP/6-31G level had a mean absolute deviation from predictedc ΔH‡ values of 1.7 kcal/mol.14 For enzymatic reactions, Siegbahn concludes that using B3LYP, the error in relative energies in general is about 3 kcal/mol for molecules containing first- and second-row atoms.15 For systems involving transition metals, the error is up to 5 kcal/mol.15 Some sources of errors are discussed below.

2.2.2 Sources of error: self-interaction and near-degeneracy In Hartree-Fock theory, an artificial term is observed, which corresponds to the Coulomb repulsion of an electron with itself. This is obviously non-physical and is referred to as the self-interaction error. Fortunately, the magnitude of this term corresponds exactly to the size of the exchange term, and as these enter the equation 1 with opposite signs, the self-interaction is cancelled. In exact DFT, the exact EXC corrects for the error introduced by EJ. However, in approximate DFT functionals, a certain amount of self-interaction error remains.1 One consequence of the self- + interaction error can be illustrated with the dissociation of H2 . If the H-H bond is stretched, the system converges to a delocalized H0.5+ + H0.5+ state, instead of the correct localized H + H+ situation. The artificial stabilization of the former state occurs, because the delocalized electron exhibits less self-interaction. The error in dissociation becomes as large as 55 kcal/mol.16 This effect can also be assumed to be observed in transition states of certain reactions. In the reactants, the separate fragments have an integer number of electrons, but as the reaction proceeds to the transition state, some electron transfer occurs, resulting in the formation of systems with a fractional number of electrons.17 The self-interaction error artificially stabilizes the delocalized transition state, leading to an underestimation of the barrier. For example, with B3LYP the barrier height for the hydrogen abstraction reaction H2 + H

→ H + H2 is calculated to be 4.1 kcal/mol, which is 5.6 kcal/mol lower than the experimental value.18 If the functional is corrected for the self-interaction, a barrier of 11.1 kcal/mol is obtained, which is only 1.4 kcal/mol in error.18 Also for a set of nine cThe predicted ΔH‡ values at 0 Kelvin were derived from experimental ΔH‡ by subtracting theoretically derived thermal corrections. 9

reactions including proton transfer, hydrogen abstraction and radical dissociation reactions, the average absolute deviation from experimental and accurate ab initio energies was found to be as much as 12-14 kcal/mol for different density functionals.19 Including self-interaction corrections reduced the average absolute deviation to 3-5 kcal/mol.19 It can be noted that reaction energies in general are unaffected by the self-interaction error and no improvement is observed upon inclusion of self-interaction corrections.19

Siegbahn has pointed out that an underestimation of the barrier due to the self- interaction error usually only is observed for reactions involving proton or hydrogen transfer.15 Other reactions often exhibit an overestimation of barriers, because the dominating error here is the near-degeneracy effect, also referred to as non-dynamical correlation error.15 Non-dynamical correlation is a long-range effect that for example becomes of importance if a single bond such as in H2 is stretched to give two non- interacting atoms, each with one electron.1 Two determinants are required to describe the dissociation of H2 correctly, each with equal weight. If only one determinant is used, a wrong potential energy curve emerges, at least if a restricted approach is used.1 The energetic problem can often be solved by using an unrestricted approach.1,15 10

Chapter 3

Modelling enzyme active sites

Building of the model is a critical step in quantum chemical calculations. For modelling of enzymatic mechanisms, careful considerations about what to include in the model are necessary. Often a number of models are built to asses the effect and importance of various groups or residues on the investigated reaction mechanism.

3.1 Modelling of active site residues Usually, the protein X-ray crystal structure serves as a basis for the model. The structure is analyzed and the active site residues that are assumed to be involved in substrate conversion and stabilization are extracted from the PDB file. The identification of important residues is usually facilitated if mutational studies have been performed on the enzyme in question. In certain cases, there is no hint about the identity of the catalytic residues and different possibilities have to be tested. The residues that were extracted from the PDB file are usually truncated to reduce the model size. Often only parts of the side-chains are used in the quantum model (Figure 3.1). The truncated residues are normally fixed at the atom of truncation to preserve the spatial arrangement of residues. In general, only the functional groups of the side chains are included, extended by 1-2 carbons. For example aspartate and glutamate are often modelled as acetic acid, tyrosine and phenylalanine as phenol and phenyl, respectively, arginine as N-methyl-guanidine, serine as ethanol, and histidine as imidazole. In certain cases, the whole side chain and sometimes even the backbone part is included, if it is evaluated that increased flexibility of the residue in question is crucial for the mechanism. In some cases the backbone part is involved in explicit interactions with the catalytic residues or the substrate, and it is thus included in the model. 11

X-ray crystal structure Active site residues Final QM model *

Arg99 Asp101 * Asp101

Arg99 Substrate Inhibitor

Wat Wat

Asp132 Asp132 * * Asn55 Asn55 Tyr53 Tyr53 Active site *

Figure 3.1 Building a quantum chemical active site model. Important active site residues are extracted from the X-ray crystal structure and truncated. Points of truncation (denoted with asterisks) are fixed in calculations. An appropriate substrate is manually modelled in place of an inhibitor (or substrate analogue/product).

Current protein crystal structures mainly exhibit resolutions of 2-3 Å, which does not allow assignment of hydrogen atoms. These are therefore added manually to the extracted residues. The low resolution also does not allow identification of the protonation state of the residue side chains. In some cases, the pKa value of the residue in question has been established experimentally and can be used as a guideline for its protonation state. Otherwise, models with different protonation states might be build to asses the effect on the reaction mechanism.

3.2 Modelling of substrates Some enzymatic structures are determined in presence of products, substrate analogues or inhibitors, which serve as a good starting point for manual modelling of a substrate. If this is not the case, a suitable substrate is modelled from scratch and added to the model. The substrate is chosen based on the known substrate specificity of the enzyme. The substrate might be truncated if it is concluded that certain parts are not of importance for the reaction. In some cases, different substrate orientations are tested to establish if the position is critical for the mechanism or energetics of the reaction.

3.3 Computational strategy The chemical model is now used to investigate the reaction mechanism with the chosen substrate. The computational strategy comprises calculations done in separate steps. First, geometries are optimized. For minima, this is usually straight forward. For transition state structures, a linear transit scan along a chosen reaction coordinate might be employed to locate the transition state structures. The optimized geometries corresponding to the reaction pathway of interest are then subjected to different single point calculations to calculate solvation effects and big basis set corrections to the energy. Finally, frequency calculations are performed, which are used to obtain zero- point vibrational (ZPV) effects. Details of the computational protocol employed in this thesis are given below.

3.3.1 Geometry optimizations Calculations are performed using B3LYP as implemented in the Gaussian03 program package.20 Geometry optimizations are done using the split-valence double-ζ basis set 6-31G(d,p). Frequency calculations are performed on the optimized geometries at the same level of theory to confirm the nature of the stationary points. Minima should only exhibit positive eigenfrequencies, while transition states exhibit one negative (imaginary) eigenfrequency. Freezing some atoms to their crystallographically observed positions introduces a few small negative eigenvalues for the optimized structures, but this are usually only on the order of -10 to -20 cm-1, and do not affect the results significantly.

3.3.2 Modelling of the surroundings The conductor-like polarizable continuum model21 (CPCM) is used to calculate the solvation effect that the surrounding protein would have on the energetics. Single point calculations are performed on the optimized structures at the same level as optimizations. In the CPCM model, the solvent is represented by a homogenous dielectric medium surrounding a cavity containing the solute. The dielectric constant of the medium is set to ε = 4, which is the standard value used in modelling protein surroundings.22 This value corresponds roughly to a mixture of a protein medium (dielectric constant of 2-3) and water (dielectric constant of 80). Estimating the effects 13

of the surrounding as single point calculations includes the implicit assumption that the gas-phase-optimized geometries will not differ substantially from the geometries that would be optimized in presence of solvent. This assumption is usually valid, except in certain cases, for example for geometries with a high degree of charge separation, where the solution structure might differ substantially from the gas-phase- optimized geometry. Another implicit approximation in the CPCM approach is the assumption that the heterogeneous protein environment can be modelled with a homogenous medium. This approach might seem crude but has nonetheless shown to be a reasonably accurate method in determining enzymatic reaction mechanisms.22 The explicit effect of various surrounding groups can of course not be assessed with this method. However, care is usually taken to ensure that explicit interactions between the catalytic groups and the surrounding protein are included in the quantum chemical model. The solvation corrections calculated for the systems considered in this thesis are around 1-4 kcal/mol. In some cases, if a much bigger effect is observed, this can indicate that the model size is insufficient. In general, as the size of quantum chemical models tends to increase, the calculated solvation corrections on the reaction energetics become less, since most of the solvation effects are already present in the quantum model itself.

3.3.3 Final energies Normally, single-point big basis set calculations are performed on the optimized geometries at the B3LYP/6-311+G(2d,2p) level to obtain more accurate energies. For the systems considered here, the difference in small basis set (6-31G(d,p)) energies and the big basis set calculations are typically less than 2 kcal/mol. Zero-point vibrational (ZPV) effects, which are obtained from frequency calculations, are added to the final energies. These effects are often less than 1 kcal/mol, but can in some cases be 2-3 kcal/mol. If very large models are employed, the computation of ZPV effects can become too demanding and the effect is instead estimated from a smaller model describing the same reaction. The final energies are composed of the big basis set energies, corrected for solvation effects and for ZPV effects. 14

3.4 Comparison with experiment In order to compare theoretically determined energies with experimentally determined reaction rates, classical transition state theory can be used. The relationship between the rate constant k of a reaction and the free energy of activation (∆G≠) can be expressed as:4

Tk ⎛ − ΔG≠ ⎞ (3.1) k = B exp ⎜ ⎟ h ⎜ RT ⎟ ⎝ ⎠

-1 −23 where k is the rate constant (s ), kB is Boltzmann’s constant (1.38 × 10 J/K), T is the temperature (in Kelvin, 298.15 at room temperature), h is Planck’s constant (6.626 × 10-34 Js), ΔG≠ is Gibbs free energy difference between the reactant and the transition state, and R is the gas constant (8.314 J/K).

With the above equation, experimentally derived rate constants can be converted into activation energies and compared to the computationally determined reaction barriers. It is important to note the exponential relationship between the rate and the barrier in (3.1). For every 1.4 kcal/mol increase in barrier, the rate changes with a factor of 10. Since the error in computed barriers often is 2-3 kcal/mol, it is not possible to predict reaction rates from computed barriers.

It should also be realized that the barriers calculated in this work only correspond to the enthalpy part (∆H≠) of the free energy of activation (∆G≠ = ∆H≠ - T∆S≠). The entropic part could not be calculated, due to the freezing scheme employed in geometry optimizations. The small negative eigenvalues that are generated in this approach would generate an artificial effect on the entropic contribution to the energy. It is therefore assumed here that ∆G≠ ≈ ∆H≠. This is in many cases a valid assumption, since the change in entropy (∆S≠) in going from the reactant to the transition state often is small. 15

Chapter 4

Epoxide chemistry

Epoxides are versatile intermediates in organic chemistry. They generally exhibit high reactivity due to the strain of the three-membered ring and can relatively easily be converted into various substituted alcohols.23 Regioselectivity is an important issue in epoxide chemistry, since epoxides are ambident substrates, i.e. they can react at different carbon centers (Scheme 4.1). Opening of asymmetric epoxides will thus in many cases lead to multiple products.23 Another issue concerns the enantioselectivity of epoxide conversion (Scheme 4.1). Epoxides are often obtained in racemic mixtures, and methods for kinetic resolution and enantioselective reaction with one of the two epoxide isomers are thus of high interest.

Regioselectivity Enantioselectivity

O O O R R R αC Cβ C C C C

Nu Nu Scheme 4.1 Regioselectivity and enantioselectivity of epoxide conversion.

4.1 Regioselectivity of epoxide opening The regioselectivity of epoxide opening with a given nucleophile depends on various factors such as the acidity of the medium, the size of the nucleophile and the substitution pattern on the epoxide. These factors can in general be divided into steric and electronic effects. Under conditions with neutral or basic pH values, steric factors usually dominate and the reaction occurs through an SN2 attack of the nucleophile on the less substituted epoxide carbon23 (attack at the less substituted carbon is 16 sometimes referred to as anti-Markovnikov-type epoxide opening24). Under acidic conditions, the reaction occurs either through an SN1 mechanism (with protonation of the epoxide and ring opening prior to nucleophilic attack) or through an SN2 mechanism (opening and attack occur concertedly). During epoxide opening, a positive charge evolves at the epoxide carbon (Scheme 4.2), which is better stabilized at the more substituted carbon, due to electron donating effects. Polarization of the oxygen-carbon bond through protonation or hydrogen bonding enhances the preference for the more substituted carbon. Under acidic conditions, electronic effects can thus reverse the regioselectivity of epoxide opening and lead to attack at the more substituted carbon.23

A H δ-O R δ+ C C

Scheme 4.2 At the transition state for epoxide-opening, a build-up of positive charge occurs at the oxirane carbon.

4.2 Enantioselectivity of epoxide conversion Epoxides are often obtained as racemates, i.e. equal mixtures of two enantiomers. The two isomers can in principle be viewed as two different compounds that will exhibit different properties and functionalities. It is of high interest to develop methods that can lead to cheap separation of the racemates. The principle of kinetic resolution is based on finding conditions that will mediate conversion of only one of the enantiomers, while the other remains untouched and can be isolated in pure form. Since the reactivities of the epoxide enantiomers with a free nucleophile often are identical, this is a difficult task. However, the use of chiral catalysts can result in enantioselective conversion of epoxides. Enzymes are especially useful for this approach, since they provide a chiral environment and often exhibit large affinity differences for epoxide enantiomers. 17

4.3 Non-enzymatic transformation of epoxides In solution, epoxide ring opening can occur with a large variety of nucleophiles, such as water, azide, cyanide, halides, alcohols, or amines.25 Regioselectivities for reactions involving these nucleophiles depend on the reaction conditions as well as on the epoxide. Enantioselectivity is in general poor or absent. One epoxide of interest in our studies of enzymatic reactions is the phenyl-substituted epoxide styrene oxide (Scheme 4.3). Different protocols for conversion of epoxides, in particular styrene oxide, are discussed below.

*C C α β

Scheme 4.3 The terminal epoxide styrene oxide. One chiral center is present (denoted with *). The benzylic and terminal carbons are referred to as α and β, respectively.

4.3.1 Hydrolysis Hydrolysis of epoxides results in the formation of vicinal diols. The reaction can be catalysed by acid or by base.23 Regioselectivities of attack depend on the conditions, as described above. Basic and neutral conditions usually lead to attack at the more accessible carbon, acidic conditions to attack at the more substituted carbon.23 However, for aryl-substituted epoxides, substantial attack at the benzylic position might occur even under basic conditions. For example, in a 3M KOH solution, styrene oxide exhibits 51% attack at Cα and 49% attack at Cβ.26 Under acidic conditions, styrene oxide is exclusively opened at the benzylic carbon (α).26,27

4.3.2 Azidolysis If nucleophiles other than water/hydroxy are used, the opening of epoxides will lead to the formation of substituted alcohols. Azidolysis of epoxides under different conditions has been reported. Epoxides react with NaN3, for example in presence of acid or metal salts.28 Asymmetric 1,2-epoxides will mainly react at the terminal 18 carbon (β), but regioselectivity is not complete.28 Aryl substituted epoxides are different and exhibit preferred (but not exclusive) attack at the benzylic position under almost all conditions. Regioselectivities of 81-83% for the benzylic position are 28 reported for the reaction of styrene oxide with NaN3 in presence of metal salts. With diiodosamarium as catalyst, trimethylsilylazide can be used to convert styrene oxide into the protected azidoalcohol, with 90% attack at the benzylic carbon.29 In aqueous medium containing NaN3, a preference of 97% for the benzylic carbon of styrene oxide is observed under both acidic and basic conditions (respective pH values of 4.2 and 9.5).30 Interestingly, high enantiomer-selective azidolysis of various epoxides can 31 be achieved with a (salen)Cr(III)N3 complex. However, using this catalyst, very poor regioselectivity is obtained for styrene oxide.31

4.3.3 Cyanolysis Introduction of a cyanide group into a compound is often useful, because the cyanide can be transformed into various other functional groups such as amines and carboxylic acids. Epoxides can be converted into cyanoalcohols with for example trimethylsilyl cyanide, TMSCN. In presence of solid base (CaO or MgO), TMSCN will donate a cyanide group to the β-carbon of styrene oxide with 93-96% regioselectivity.32 With TMSCN and lithiumperchlorate, styrene oxide exhibits 92% attack at the terminal (β) carbon.33 Also diiodosamarium can be used as a catalyst for the TMSCN mediated reaction, however, regioselectivities for styrene oxide are poor (58-77% attack at Cα).29 The use of TMSCN is generally associated with two major drawbacks. CN is an ambident nucleophile, and using TMSCN, both isonitriles (covalent bond to N) and nitriles (covalent bond to C) are often formed.34,35 Another drawback is that the TMS group will attach to the alcohol group, generating trimethylsilyl O-protected alcohols, which need to be cleaved to release the desired product.32,35 Opening of epoxides can instead be performed with NaCN or KCN. If KCN is used in presence of metals salts, no isonitriles are formed and preferred attack at the terminal position is observed for most epoxides.35 However, for aryl substituted epoxides a mixture of products is 35 observed with 77% to 84% attack at the β-carbon. With NH4Cl as acid and KCN, 92% attack at the β-carbon of styrene oxide occurs.35 In presence of β-cyclodextrin, conversion of various 1,2-epoxides with NaCN leads to complete regioselectivity for 19

36 the β-carbon, also for styrene oxide. With Ce(OTf)4 as catalyst in water, styrene oxide will react with NaCN, but the reaction exhibits very poor regioselectivity.37 Addition of sodium dodecyl sulfate (SDS) to the mixture increases the regioselectivity for the benzylic carbon to 92%.37 Under mildly basic conditions using triethylamine in THF, acetone cyanohydrin can be used to convert terminal epoxides to the corresponding cyano alcohols, with exclusive attack at the terminal (β) carbon, also for styrene oxide.38

4.3.4 Haloalcohol formation Haloalcohols (also known as halohydrins) can be formed from epoxides with for example lithium halides such as LiI, LiBr, or LiCl.39 Asymmetric 1,2-epoxides mainly react at the terminal carbon, while reaction with styrene oxide results in a mixture of products with the major haloalcohol regioisomer exhibiting addition to the benzylic 39 carbon. In aqueous media containing SDS and Ce(OTf)4 as catalyst, the reaction of 37 styrene oxide with NaCl or NaBr will lead to halide attack at the benzylic position.

4.3.5 Conclusions on non-enzymatic epoxide transformation The above section describes some of the many different ways epoxides can be transformed in solution. Multiple protocols exist, but they often exhibit important drawbacks such as low regioselectivity, formation of protected alcohols that need to be cleaved subsequently, or involvement of harmful compounds. If one was to list the desired criteria for an epoxide-converting process, this would include mild reaction conditions, non-toxic catalysts, formation of pure products, high regioselectivity, high chemoselectivity (i.e. reaction occurs only at the desired site and not with other functional groups), and preferably also high enantioselectivity. Interestingly, epoxide conversion using enzymes as biocatalysts seem to fulfil many of the wishes on the above list.

4.4 Enzymatic transformation of epoxides Enzymes usually react in aqueous solutions around physiological pH values at ambient temperatures, thus providing mild reaction conditions. However, what makes them truly attractive as biocatalysts is the high selectivity displayed in many reactions. 20

This regards regioselectivity, chemoselectivity, and in particular enantioselectivity. Since enzymes are chiral catalysts, different binding modes and different reaction rates are often observed for enantiomers of the same compound. This makes enzymes extremely useful for the kinetic resolution of racemic mixtures of epoxides.40

We have studied epoxide-converting enzymes from three different classes in this thesis. All three classes exhibit enzymes that display biocatalytic potential. The two classes of epoxide hydrolases are useful for transforming epoxides into vicinal diols, while the haloalcohol dehalogenases can be used for formation of substituted alcohols, including azido- and cyanoalcohols. An overview of the usefulness of different members of these three enzyme classes in biocatalytic applications will be given here.

4.4.1 The soluble epoxide hydrolases The soluble epoxide hydrolases (sEHs) catalyze conversion of epoxides into vicinal diols. Homologues of sEH are found in most organisms, including plants, fungi, mammals, and insects. The different sEH display different substrate specificities, regio- and eantioselectivities, i.e. a multitude of epoxide hydrolysis reactions can be performed. One sEH with biocatalytic potential is the soluble epoxide hydrolase EchA, which was isolated from the bacterium Agrobacterium radiobacter AD1.41,42 EchA has relatively broad substrate specificity and reacts with for example styrene oxide, which is converted into the corresponding diol. Attack occurs exclusively at the terminal carbon (β) of styrene oxide.43 The enantioselectivity of the wild-type EchA with styrene oxide is low, displaying an enantiomeric ratiod of 17.43 However, protein engineering of EchA resulted in an I219F mutant with an enantiomeric ratio of 91 for styrene oxide transformation.44

It has been shown that conversion of epoxides in presence of two sEHs with different regio- and enantioselectivities can result in formation of very pure products in high yields. In conventional kinetic resolutions, the yield of optically pure product cannot exceed 50%, however, in this approach a product yield of up to 100% can be dThe enantiomeric ratio is defined as the ratio of the reaction rates for the two enantiomers, E = (kcat/KM)R / (kcat/KM)S (where kcat is the rate constant and KM is the Michaelis–Menten constant, and R/S refers to the enantiomers). 21 achieved. For example, styrene oxide conversion in simultaneous presence of both the EchA-I219F mutant and the StEH from potato (Solanum tuberosum) resulted in enantioconvergent production of (R)-1-phenyl-1,2-ethanediol from a racemic mixture of styrene oxide, resulting in almost 100% yield with 98% enantiomeric excess (ee)e.45 This is possible since EchA prefers to convert (R)-styrene oxide, attacking exclusively the β-carbon, while StEH prefers (S)-styrene oxide, attacking almost only the α-carbon. In both cases, the product is (R)-1-phenyl-1,2-ethanediol.45 Also the sEHs from Aspergillus niger and Beauveria sulfurescens has been utilized with the same purpose, yielding (R)-1-phenyl-1,2-ethanediol with 92% yield and 89% ee.46

4.4.2 Limonene epoxide hydrolase The limonene epoxide hydrolase (LEH) originates from the bacterium Radiobacter erythropolis DCL14.47,48 Structural and biochemical studies revealed that it belongs to a different class than the sEHs introduced above. LEH is therefore considered to be the founding member of a new protein class, with only few putative members so far.47 The biocatalytic potential of LEH is in principle low, since LEH has narrow substrate specificity and accepts only few substrates, these include limonene-1,2-epoxide, 1- methyl cyclohexene oxide and indene oxide.48 However, LEH displays high enantioselectivity and regioselectivity in conversion of the four stereoisomers of the natural substrate, limonene-1,2-epoxide (LE). For example, conversion of the diastereomeric mixture of (1S,2R,4R)-LE and (1R,2S,4R)-LE lead to enatioconvergent formation of (1S,2S,4R)-limonene-1,2-diol.48 Conversion of (1S,2R,4S)-LE and (1R,2S,4S)-LE lead to enatioconvergent formation of (1R,2R,4S)-limonene-1,2-diol. The carbon skeleton of limonene is similar to several important flavour and medicinal compounds, making formation of optically pure products of interest.

4.4.3 The haloalcohol dehalogenases Haloalcohol dehalogenases catalyze the reversible dehalogenation of vicinal haloalcohols to form epoxides. They have exclusively been identified in microbes, 49 50,51 51,52 including Corynebacterium sp., Arthrobacter sp., Agrobacterium sp., and

eEnantiomeric excess, ee, is defined as: ee (%) = ([R] - [S])/([R] +[S]) * 100 22

51 Mycobacterium sp. . In addition to dehalogenation, haloalcohol dehalogenases also catalyze the irreversible ring opening of various epoxides with the nucleophiles CN¯, 53,54,55,56,57 N3¯, and NO2¯. One haloalcohol dehalogenase that has been studied intensively is HheC, which originates from the bacterium A. radiobacter AD1.51,51 HheC displays high enantioselectivity for both aromatic and non-aromatic halohydrins. The native enzyme displays an E value of 150 for conversion of p-nitro-2- bromo-1-phenylethanol.58 For a W249F-mutant of HheC, the E value of this reaction is 900.58 W249F-HheC has been used successfully in kinetic resolution of racemic 4- chloro-3-hydroxybutyric acid methylester, yielding the (S)-enantiomers of 4-cyano-3- hydroxybutanoate methylester with 97% ee and 4-chloro-3-hydroxybutyric acid methylester with 95% ee.59 Kinetic resolution on preparative scale has been reported with various 3-Alkenyl and heteroaryl chloroalcohols with ee values of >99%.60 In the epoxide opening reaction, HheC mediates high β-regioselectivity for different 1,2-epoxides.54,55,57 This regioselectivity is opposite to that observed for azidolysis of aryl-substituted epoxides in water, where for example styrene oxide and p-chloro- styrene oxide display a regioselectivity of 98(α):2(β) and 97(α):3(β), respectively.57 Using HheC, this regioselectivity is changed to 21(α):79(β) for styrene oxide and 11(α):89(β) for p-chloro-styrene oxide.57 HheC has broad substrate specificity and can be used to prepare a large variety of β–substituted aliphatic and aromatic azido- and cyano-alcohols.53 With nitrite as nucleophile, the final product is a diol, since the formed nitrite ester is unstable in water.55 However, nitrite can be used in for example kinetic resolution of racemic p-nitro-styrene oxide, since HheC displays high enantioselectivity for the (R)-enantiomer of this substrate.55

4.4.4 Conclusions on enzymatic epoxide transformation The above section illustrates how enzymes can be used for epoxide-transforming reactions. High enantioselectivities and regioselectivities can be achieved, making formation of optically pure products possible. However, for several of these enzymes, the detailed reaction mechanism is not known and the causes for the displayed selectivities are not identified. A theoretical study of epoxide-transforming enzymes helps to understand their selectivity and reaction mechanisms, providing valuable information that can be used for design of new epoxide-transforming reactions. 23

Chapter 5 Quantum chemical studies of epoxide-transforming enzymes

In the present thesis, one member from each of the three classes of epoxide-converting enzymes introduced above was studied computationally. The first enzyme studied is the Limonene Epoxide Hydrolase (LEH), which is found in the bacterium Rhodococcus erythropolis DCL14.47 The second enzyme studied here is the human soluble Epoxide Hydrolase (sEH), which belongs to the superfamily of α/β-fold hydrolases.61 The third enzyme is the haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1.51 We analyzed different aspects of enzymatic epoxide conversion. The reaction mechanisms employed by the above enzymes were studied in detail, and the role of different active site residues was analyzed. The importance of certain residues for a given reaction step was investigated through in silico mutations. Another major topic of our study was the regioselectivity of epoxide opening. Factors that influence the regioselectivity were identified and quantified.

OH O OH 1 H O 1 2 2 2 4 4

Limonene-1,2-epoxide Limonene-1,2-diol

Scheme 5.1 Hydrolysis reaction catalyzed by LEH.

5.1 Limonene epoxide hydrolase (Paper I) The limonene epoxide hydrolase (LEH) catalyzes the conversion of limonene-1,2- epoxide (LE) to limonene-1,2-diol (LD, Scheme 5.1).47,62 LEH is part of a degradation pathway for limonene in Rhodococcus erythropolis DCL14.63 The crystal structure of LEH revealed a novel fold and a novel putative active site.64 Due to the fundamental differences between LEH and other known epoxide hydrolases, LEH was suggested to be the founding member of a new protein family.47

Arg99 Arg99

NH N O O 2NH HN + + Asp101 2 Asp101 NH 2 NH O OH 2 - O O O Hydrolysis O OH - H R Asp132 O Asp132 OH OH H R OH OH O O

Tyr53 Tyr53 2NH Asn55 2NH Asn55

Scheme 5.2 Epoxide hydrolysis mechanism of LEH.

5.1.1 Elucidating the mechanism of LEH LEH constitutes a novel enzyme and clues about its mechanism were thus limited. Mutational studies identified several potentially important residues, including Asp132, Asp101, Tyr53, and Asn55.64 Also Arg99 was suggested to be of importance, but it was not clear if its function is only structural or also catalytical. Based on the above results and the X-ray crystal structure of LEH, a possible Asp132-Arg99-Asp101 catalytic triad was suggested (Scheme 5.2).64 Asp132 was proposed to activate the nucleophilic water molecule, which attacks the oxirane ring. Asp101 was suggested to donate a proton to the emerging oxyanion of the substrate. Tyr53 and Asn55 are involved in binding of the nucleophilic water molecue.64 We studied the putative mechanism of LEH to establish if the proposal is energetically feasible. The quantum chemical model was based on the crystal structure of LEH in complex with the 25 inhibitor valpromide (PDB 1NU3)64. The model was composed of the putative catalytic triad Asp132-Arg99-Asp101, Tyr53 and Asn55, as well as a crystallographically observed water molecule (Figure 5.1). Conversion of different substrates was studied with this model. The LEH-mediated hydrolysis of one stereoisomer of LE, (1R,2S,4R)-LE, is shown in Figure 5.1. For formation of the diol, (1S,2S,4R)-LD, a barrier of 14.9 kcal/mol was obtained, and a reaction energy of -9.7 kcal/mol. The experimental activation energy for LEH-mediated conversion of (1R,2S,4R)-LE is 12.4 kcal/molf, which is close to the computed value.62

Our results show that the suggested catalytic triad indeed is able to catalyze the conversion of limonene-1,2-epoxide to limonene-1,2-diol. As proposed, Asp132 abstracts a proton from the nucleophilic water molecule, which attacks the epoxide. Asp101 donates a proton to the epoxide oxygen, in concert with epoxide opening. A step-wise mechanism was also investigated, but was found to be unlikely based on our results (Paper I). Arg99 stabilizes and orients the two aspartate residues. It could also play a pivotal role in transferring a proton from Asp132 to Asp101 in a subsequent step to restore the active site for the next catalytic cycle. Tyr53 and Asn55 bind the catalytic water molecule and position it for attack on the substrate. The same non- covalent general acid/general base mechanism was observed for all studied substrates (Paper I).

5.1.2 Analysis of the regioselectivity of LEH LEH accepts only few substrates, among these are the four stereoisomers of limonene- 1,2-epoxide and both enantiomers of 1-methylcyclohexene oxide. Experimental studies with the two enantiomers of 1-methylcyclohexene oxide (1 and 2, Scheme 5.3) showed preferred attack at the more substituted oxirane carbon, C1.65 The regioselectivity for 1-methylcyclohexene oxide was reported to be 85(C1):15(C2).65 However, conversion of limonene-1,2-epoxide exhibited conflicting results. Exclusive attack at the less substituted carbon (C2) is observed for stereoisomers 3 and 6, while f The activation energy was determined with a diastereomeric mixture of (1R,2S,4R)- and (1S,2R,4R)- LE. However, as their hydrolysis occurs sequentially, it is assumed that the determined value only is based on the substrate that is converted first, that is, (1R,2S,4R)-LE.

A * Arg99 1.99 Asp101

1.99 1.01 * 1.65 1.68 1.59 1.47 O 12 1.70 CH3 Asp132 1.48 half-chair, P-helicity * Water Tyr53 1.62 1.82 Substrate 1.99 (1R,2S,4R)-LE * Asn55 *

B * 1.83 Arg99 Asp101

1.79 * 1.37 1.72 1.78 1.08 H 1.98 O 1.56 C1 CH3 Asp132 1.02 chair 2.31 * 1.75 Tyr53 1.99 1.71

* Asn55

C * 1.77 Arg99 Asp101

1.62 * 1.64 1.95 2.01 0.99 OH 1.00 1.72 CH3 Asp132 chair 1.47 OH * 1.91 Tyr53 1.72 1.96

Asn55 Product * (1S,2S,4R)-LD *

Figure 5.1 LEH-mediated conversion of (1R,2S,4R)-limonene-1,2-epoxide (helicity 3,4 P) to (1S,2S,4R)-limonene-1,2-diol. A) Reactant, B) Transition state, C) Product. Insets show the substrate conformation. Asterisks indicate atoms, which in calculations were fixed to their crystallographically observed position. 27

1-methylcyclohexene oxide limonene-1,2-epoxide O O O O O O 1 1 1 1 1 2 2 2 2 1 2 2 4 4 4 4

(1R,2S) (1S,2R) (1R,2S,4S) (1S,2R,4S) (1R,2S,4R) (1S,2R,4R) 1 2 3456

OH OH OH OH OH 1 OH OH OH 2 1 2 1 1 2 2 4 4

(1S,2S) (1R,2R) (1R,2R,4S) (1S,2S,4R) 7 8 9 10 1-methylcyclohexane-1,2-diol limonene-1,2-diol

Scheme 5.3 Conversion of the two enantiomers of 1-methylcyclohexene oxide and the four stereoisomers of limonene-1,2-epoxide by LEH.

exclusive attack at the more substituted carbon (C1) is seen for the stereoisomers 4 and 5 (Scheme 5.3).48,63 The differences in regioselectivity are intriguing, since in each case the two limonene-1,2-epoxide stereoisomers with the same stereochemistry at the oxirane ring, (1R,2S) for 3 and 5 and (1S,2R) for 4 and 6, exhibit attack at opposite carbons (Scheme 5.3). A suggested explanation for the differences was differential binding of the substrates in the active site, which would lead to attack at different carbons.64

We investigated the hydrolysis of all stereosiomers of limonene-1,2-epoxide and 1- methylcyclohexene oxide with our QM model. Interestingly, this relatively small model is able to reproduce the experimentally observed regioselectivity for all studied substrates (Paper I). In our model, the orientations of the different stereosiomers are very similar, as the substrate only interacts with Asp101 (Figure 5.1). These results indicated that the differences in epoxide opening is not due to differential binding of the substrate in the active site but has other causes.

Cyclohexane Cyclohexene oxide

O O O = =

chair half-chair half-chair 3,4 M helicity 3,4 P helicity

Scheme 5.4 Preferred conformations of cyclohexane and cyclohexene oxide.

The regioselectivity for conversion of the different cyclohexene oxides is instead caused by the conformation of the substrate. A cyclohexane ring typically prefers to be in a chair conformation, but presence of the oxirane ring in cyclohexene oxides forces the epoxide into a half-chair conformation (Scheme 5.4). The half-chair conformation can exist in two different forms, which are best described by the helicity about the 3,4 bond. These will here be referred to as the P and the M helicity respectively (Scheme 5.4). The two helicities are in equilibrium and are present in ratios depending on the energy difference between the two conformers. For example, if a large substituent is present on the ring, one helicity might be significantly preferred over the other (see below).

The presence of the two different helicity forms is crucial for understanding the regioselectivity of the above substrates. Equally important is it to realize that in a given helicity, the two carbons will display very different reactivities. Depending on which epoxide carbon is attacked, a chair or a twist-boat is formed in the transition state and in the subsequent product (Scheme 5.5). The chair-like conformation is energetically preferred and it can be assumed that attack exclusively occurs at the carbon leading to a chair-like transition state (Paper I). This is independent of the substitution at the oxirane carbons, i.e. the preferred carbon can be the more or the less substituted carbon. The above observations can be summarized as follows: Cyclohexene oxides are half-chairs that can exist in two different helicities. Each helicity can be attacked at the two oxirane carbons, but the one that will lead to a chair-like transition state will be preferred. The different regioselectivities reported for limonene-1,2-epoxide and 1-methyl-cyclohexene oxide can be fully explained considering these observations. 29

3,4 M helicity 3,4 M helicity OH OH O O

1 2 1 2 chair twist-boat H O OH H O 2 2 OH Scheme 5.5 Attack on an epoxide half-chair in a given helicity leads to products (and transition states) that have either a chair or a twist-boat conformation.

5.1.2.1 Regioselectivity of 1-methylcyclohexene oxide hydrolysis Of the two stereoisomers of 1-methylcyclohexene, the (1R,2S) isomer (1, Scheme 5.3), is the preferred substrate of LEH. The two helicities of 1 have almost the same energy and are assumed to exist in equal amounts (Paper I). For each helicity, attack occurs at the carbon that will lead to a chair transition state, for 1 in the M-helicity this is C2 and for the P-helicity this is C1 (Paper I). The two helicity forms of 1 are competing substrates of LEH, but since attack at the more substituted carbon C1 of the P-helicity has a lower barrier (14.9 kcal/mol, Table 5.1) than attack at C2 of the M- helicity (15.9 kcal/mol, Table 5.1), attack occurs preferentially, but not exclusively, at C1 of the P-helicity (Paper I). A mixture of products is thus expected, with preferred attack at C1 (of the P-helicity) and minor attack at C2 (of the M-helicity). The difference in barrier of 1 kcal/mol correspond very well to the experimentally observed regioselectivity of 85C1:15C2 for 1-methylcyclohexene oxide.65

Table 5.1 Calculated barriers and reaction energies (kcal/mol) for LEH–mediated conversion of 1-methylcyclohexene oxide to 1-methylcyclohexane-1,2-diol. Substrate a Attack at TS b Product c Barrier Reaction (1R,2S), 3,4 M C1b twist-boat (1S,2S) 17.5 -3.4 (1R,2S), 3,4 M C2 chair-like (1R,2R) 15.9 -9.9 (1R,2S), 3,4 P C1 chair-like (1S,2S) 14.9 -9.5 (1R,2S), 3,4 P C2 twist-boat (1R,2R) 19.2 -4.0 (1S,2R), 3,4 M C1 chair-like (1R,2R) 16.0 -9.0 (1S,2R), 3,4 M C2 twist-boat (1S,2S) 19.1 -3.2 (1S,2R), 3,4 P C1 twist-boat (1R,2R) 19.0 -2.8 (1S,2R), 3,4 P C2 chair-like (1S,2S) 15.7 -9.5

a Epoxide stereochemistry and helicity around the 3,4 bond. b Conformation of the substrate in the transition state. c Stereochemistry of resulting diol. 30

* * 1.83 Arg99 1.81 Arg99 Asp101 Asp101 1.79 * 1.78 1.37 1.76 * 1.72 1.42 1.78 1.08 1.78 1.98 H 1.06 O 1.56 C1 Asp132 1.51 H Asp132 1.03 C2 1.96 O 1.02 CH3 CH3 2.31 chair * 2.14 * 1.75 1.91 Tyr53 * twist-boat Tyr53 1.71 1.99 1.71 1.96 * * Asn55 Asn55

* *

Figure 5.2 Hydrolysis of (1R,2S,4R)-limonene-1,2-epoxide (helicity 3,4 P) with the LEH active site model. The TS for attack at C1 is shown to the left and for attack at C2 to the right. Insets show the substrate conformation. Asterisks indicate atoms, which in calculation were fixed to their crystallographically observed position.

5.1.2.2 Regioselectivity of limonene-1,2-epoxide hydrolysis For the natural substrate of LEH, limonene-1,2-epoxide, the situation is less complex. The isopropyl substituent on C4 of limonene-1,2-epoxide determines the preferred helicity of the half-chair, and for each stereoisomer, only the helicity with the substituent in an equatorial position will be observed (Paper I). For this helicity, attack occurs at the carbon that leads to a chair transition state. The transition states for attack at either C1 or C2 of the stereoisomer 5 are shown in Figure 5.2. Attack at C1 of 5 leads to a chair-like transition state and exhibits a barrier of 14.9 kcal/mol. Attack at C2 of 5 results in a twist-boat transition state and exhibits a barrier of 19.5 kcal/mol. The difference in barriers indicates that exclusive attack at C1 is expected, in perfect agreement with experimental results.48,62 Calculated regioselectivities for the other stereoisomers are also in agreement with experimental results, with 4 exhibiting preferred attack at C1, and 3 and 6 exhibiting preferred attack at C2 (Table 5.2).48,63 The regioselectivity of limonene-1,2-epoxide opening is thus not determined by binding of the substrate in the LEH active site, but by the conformation of the potential transition states.

Table 5.2 Calculated barriers and reaction energies (kcal/mol) for LEH–mediated conversion of limonene-1,2-epoxide to limonene-1,2-diol. Substrate a Attack at TS b Product c Barrier Reaction (1R,2S,4S), 3,4 M C1 twist-boat (1S,2S,4S) 17.6 -3.5 (1R,2S,4S), 3,4 M C2 chair-like (1R,2R,4S) 16.5 -9.7 (1S,2R,4S), 3,4 M C1 chair-like (1R,2R,4S) 16.1 -9.5 (1S,2R,4S), 3,4 M C2 twist-boat (1S,2S,4S) 19.0 -3.6 (1R,2S,4R), 3,4 P C1 chair-like (1S,2S,4R) 14.9 -9.7 (1R,2S,4R), 3,4 P C2 twist-boat (1R,2R,4R) 19.5 -4.1 (1S,2R,4R), 3,4 P C1 twist-boat (1R,2R,4R) 19.0 -2.8 (1S,2R,4R), 3,4 P C2 chair-like (1S,2S,4R) 16.3 -9.4 a Epoxide stereochemistry and helicity around the 3,4 bond. b Conformation of the substrate in the transition state. c Stereochemistry of resulting diol.

5.2 The soluble epoxide hydrolase (Papers II and III) Most known EHs are members of the superfamily of α/β-fold hydrolases. Only a handful of EHs do not belong to this family. These include the LEH enzyme discussed above and the mammalian Leukotriene A4 epoxide hydrolase.66 The α/β-fold hydrolase EHs comprise a large family of enzymes identified in almost all species, including bacteria, fungi, plants, insects, and mammals. One EH from the α/β-fold hydrolase family is the soluble epoxide hydrolase (sEH, also referred to as cytosolic EH, CEH). In humans, sEH is mainly found in the liver, where it converts various xenobiotic epoxides into their corresponding vicinal diols.66,67

5.2.1 The reaction mechanism of sEH The mechanism of α/β-fold hydrolase EHs differs substantially from the mechanism of LEH. In sEH (and the closely related microsomal EHs), a covalent mechanism is observed, where an active site aspartate becomes covalently bonded to the substrate (Scheme 5.6).68,69,70,71 The ester bond is subsequently hydrolyzed by water, which is activated by a His-Asp charge relay, similar to the charge relay in serine hydrolases.72,73,74,75,76,77 Additionally, the sEHs contain two active site tyrosines that seem to be of importance in stabilizing and protonating the emerging epoxide oxyanion.42,61,78,79,80,81,82 A conserved HGXP motif (X = any residue), which is involved in formation of the oxyanion hole, is also found in sEH.83 The oxyanion hole stabilizes the tetrahedral intermediate formed in the second part of the reaction. 32

Scheme 5.6 Hydrolysis mechanism of soluble epoxide hydrolase (residue numbering as in human sEH). Step A is in general referred to as the alkylation half-reaction, while step B and C comprise the hydrolytic half-reaction.

Although the mechanism of sEH has been studied quite well experimentally, a detailed theoretical analysis has been lacking. We studied the mechanism of sEH using a quantum chemical model based on the X-ray crystal structure of the human sEH in complex with the inhibitor cyclohexyl-N´-(iodophenyl) urea (CIU), PDB 1VJ561 (Paper II). The model is composed of parts of the residues Asp495, His523, Asp333, Tyr381, Tyr465, Trp334, Phe265, Gly264, and Val497. Also included are a crystallographically observed water molecule and parts of CIU, which was remodelled into the substrate (1S,2S)-β-methylstyrene oxide (MSO) (Paper II). The model was used to study the full reaction pathway for attack at C1 and C2 of MSO. The computed energetics are shown in Figure 5.3. 33

In general, our results confirm the suggested sEH mechanism (Scheme 5.6). The alkylation half-reaction seems to occur in two steps. In the first step, the Asp333 carboxylate attacks the epoxide carbon, resulting in formation of a covalent enzyme- substrate intermediate. At the TS for attack at C1 of MSO, the Asp333 oxygen is positioned at a distance of 2.29 Å to C1 (Figure 5.4A). The epoxide ring is partially opened with an O-C1 distance of 1.89 Å. The barrier for this step was calculated to 7.8 kcal/mol (Figure 5.3). The emerging epoxide oxyanion is stabilized by the two tyrosine residues, but a proton transfer from tyrosine to the formed intermediate occurs in a separate subsequent step. It was discussed that the stepwise epoxide opening and protonation might be an artefact of our model (Paper II). However, recent experimental results for a homologue of human sEH, the potato StEH, give support to a scenario in which alkylation and protonation do not occur concertedly.84 For StEH, the amount of tyrosinate formed during the alkylation step was measured to 0.4, indicating only partly ionization of one or both tyrosines.84 We compute a small energy difference between the protonated and the unprotonated intermediate, indicating that the two species will be in equilibrium. The high barrier for the subsequent hydrolytic step allows formation of such an equilibrium. The mixture of unprotonated and protonated intermediates would explain the experimentally observed partial ionization.

In the hydrolytic half-reaction, Asp495 and His523 act in concert to activate a water molecule, which attacks the Asp333-substrate bond (Figure 5.4B). At the TS for water attack, the water oxygen is positioned 1.74 Å from the Asp333 carbonyl carbon. One water proton is transferred to His523 and is located between Nε of His523 and the water oxygen with distances of 1.23 and 1.27 Å, respectively. The barrier for water attack was computed to 18.1 kcal/mol (for the C1 pathway). We also observe a proton transfer from Nδ of His523 to Asp495 in this step (Figure 5.4B). It is possible that this proton transfer is an artefact of the model that could be avoided if additional residues stabilizing the anionic Asp495 would be included in the model (Paper II). The formed tetrahedral intermediate is stabilized by the oxyanion hole composed of hydrogen bonds from the backbone nitrogens of Trp334 and Phe265. In our calculations, dissociation of the tetrahedral intermediate occurs in concert with proton 34 transfer from Nε of His523 to the forming diol (Figure 5.4C). The barrier for this step is computed to 5.5 kcal/mol (Figure 5.3). At the transition state, the scissile O-C bond has a length of 2.02 Å (Figure 5.4C). The His523 proton is positioned at 1.14 Å from His523-Nε and 1.42 Å from the emerging anion of the diol. In the overall mechanism, His523 does thus first act as a general base and then as a general acid. The energetics computed for conversion of MSO show that the hydrolytic half-reaction is rate- limiting (Figure 5.3). This is in line with experimental results for the sEH homologues EchA and StEH.43,85 For attack at C2 of MSO, we observe the same mechanism, but with slightly different energies (Figure 5.3). We also tested another mechanistic proposal, in which His523 is protonated at Nε during the first step of the reaction (Paper III). It has been suggested that such a protonation would be essential to orient and activate Asp333.85,86 Protonation of His523 during the alkylation half-reaction was found to be unlikely based on the calculated energetics (Paper III).

20 TS dissociation 15 TS TS 14.2 alkylation water attack 14.0 11.0 11.1 11.3 10 8.5 7.8 9.5 Tetrahedral 5 intermediate

0.0 0 Reactant

-5 -6.4

Relative (kcal/mol) energy -8.6 -9.2 -8.0 -10 -8.9 Covalent intermediate Covalent This difference is due (unprotonated) intermediate to differences in hydro- -15 (protonated) gen bonding patterns. Attack C2 of MSO -17.7

-20 Attack C1 of MSO Product

Figure 5.3 Computed energies for MSO hydrolysis in the wild type human sEH active site model (Paper II). 35

Tyr381 Tyr465 A * * * Tyr465 * O O Tyr381 1.00 H H1.01 1.66 1.61 * O Val497 CH3 1.89 Ph

CH3 2.29 CH3 O * Phe265 1.94 Asp333 O 1.95 O 1.06 ε N H Nδ H O H Val497 * Trp334 O1.65 Water 1.02 * H N *H His523 **CH Asp333 Asp495 1.78 3 * 2.29 Trp334 * O H O 1.03 Water Gly264 N H * H * * His523 Phe265 Gly264 * O * Asp495

B Tyr381 Tyr465 * * * Tyr381 Tyr465 * O O 1.00 H 1.52 * 1.69 H Val497 CH O1.36 3 Ph

CH3 1.46 CH * O 3 Phe265 1.41 1.27 Val497 1.14 1.27 1.23 1.74 O 1.72 O ε N H O H N δ H O 1.03 * Trp334 1.41 * H N H His523 * * * 2.04 1.69 CH3 * Asp495 Trp334 O H O * 1.03 Gly264 N H His523 * H * * Asp495* Phe265 Gly264 * Asp333 O

C Tyr381 Tyr465 Tyr465 * * * Tyr381 * O O 1.01 H 1.57 1.64 H * 1.02 Val497 CH3 O Ph 1.43 Phe265 CH3 1.41 CH3 1.42 O 2.02 1.25 * 1.14 H O 1.75 Trp334 O 1.43 ε N O Val497 * H N δ H O 1.03 * 1.09 * H N *H His523 2.32 * CH* Asp495 1.75 3 * Trp334 Gly264 O H O 1.03 Asp333 * N H * H * * His523 Phe265 Gly264 O * Asp495

Figure 5.4 Optimized transition states for hydrolysis of MSO in the wild type sEH model, attack at C1. A) Alkylation TS, B) TS for water attack, C) TS for dissociation. 36

Table 5.3 Calculated barriers (kcal/mol) for MSO hydrolysis with the wild type and mutant models of human sEH. TS TS TS water TS Overall Model alkylation Dealkylationa attack dissociation barrier WT 7.8 16.4 18.1 5.5 22.6 Y381F 15.7 15.7 16.1 6.2 20.9 Y465F 13.2 16.2 16.6 4.7 19.8 Y381F/Y465F 24.8 11.7 - - - a The reverse reaction of the reversible alkylation step

5.2.2 Tyrosine mutants of sEH The role of the two active site tyrosine residues in sEH was investigated by in silico mutations to phenylalanine (Paper III). Full reaction pathways were calculated for the single mutants Y381F and Y465F (Y = tyrosine, F = phenylalanine), while only the alkylation step was investigated for the double mutant Y381F/Y465F. The optimized alkylation transition states for the tyrosine mutants are shown in Figure 5.5. The barrier of 7.8 kcal/mol observed for attack at C1 of MSO in the wild type sEH model increased to 13.2 and 15.7 kcal/mol in the Y465F and Y381F models, respectively (Table 5.3). If both tyrosines are mutated, a barrier of 24.8 kcal/mol is obtained. The results indicate that the single mutants would remain catalytically active, while the double mutant would not. This is in agreement with experimental results for the mouse sEH and the EHs from bacteria and potato, EchA and StEH.80,81,85 It should be noted that the effect of the single mutations might be overestimated in our model. For EchA, the rate for alkylation of (R)-styrene oxide changed from 1100 s-1 in the wild type to 60 s-1 in the Y215F mutant (Y215 in EchA equals Y465 in human sEH).80 This corresponds to a barrier increase of about 1.8 kcal/mol, while in our calculations, we observe a barrier increase of 5.4 kcal/mol for mutation of Y465. It is possible that in the enzyme active site, other polarization effects might compensate for loss of the Tyr465 hydrogen bond and thus reduce the effect of the Y465F mutation.

The barriers for the subsequent steps of the reaction mechanism, i.e. for attack by water on the Asp333-ester and for dissociation of the ester bond, changed only 1-2 kcal/mol from the energies computed for the wild type model (Table 5.3). The major effect of the tyrosine residues is thus in the first step of the reaction, the alkylation. 37

Y381F Tyr381Phe Tyr465 Tyr465 Tyr381Phe * * * *

O 3.45 H 1.04 1.49 * O Val497 CH3 1.97 Ph Phe265 CH3 2.15 CH3 O * * 1.93 Asp333 O 1.89 O 1.06 ε N H N δ H O H Val497 * O1.65 Water 1.02 * H N Asp333 *H His523 * *CH * Asp495 1.79 3 2.23 Trp334 Water O H O Trp334 1.03 * Gly264 N H H * * His523 Phe265 Gly264 * O * Asp495

Y465F Tyr381 Tyr465Phe * * * Tyr465Phe * O 1.02 Tyr381 H 3.23 1.55 * O Val497 CH3 1.97 Ph

CH3 2.16 CH3 O * Phe265 1.93 Asp333 O 1.89 O 1.07 ε N H N δ H O H * Trp334 O1.64 Water 1.01 Val497 * H N *H His523 * CH* Asp333 Asp495 2.29 1.81 3 * O H O Trp334 * 1.03 Water N H * Gly264 H * * Phe265 Gly264 His523 O Asp495 * *

Y381F/Y465F Tyr381Phe Tyr465Phe Tyr465Phe * * * * Tyr381Phe 4.18 3.15

* O Val497 CH3 2.01 Ph CH3 Val497 CH3 1.97 Phe265 O * 2.01 Asp333 O 1.79 * O 1.08 ε N H N δ H O H Asp333 Gly264 O1.58 Water 1.02 * N *H His523 H * * * 1.86 CH3 Asp495 2.55 Trp334 * * O H O Water 1.03 Trp334 N H H * * Phe265 Gly264 His523 * O Asp495*

Figure 5.5 Optimized geometries for the alkylation TS in the Y381F, Y465F, and Y381F/Y465F models. Optimized distances (Å) are shown to the left. 38

5.2.3 Studies on the regioselectivity of sEH We used the sEH model to investigate the regioselectivity of epoxide opening for two substrates, (1S,2S)-β-methylstyrene oxide (MSO) and (S)-styrene oxide (SSO). Regioselectivities were explored with both the wild type and the tyrosine mutant models (Paper II and III). The results are summarized in Table 5.4 and 5.5. The substrate MSO is singly substituted at each oxirane carbon, with a methyl group on C2 and a phenyl group on C1. As discussed in section 4.1, opening of the epoxide ring leads to formation of a positive charge at the attacked carbon. Better stabilization of this charge is expected at the benzylic position of MSO. Indeed, in our calculations, attack is favoured at the C1 of MSO in all models, even for the double mutant, in which no general acid is present (Table 5.4). The differences in barriers for attack at C1 and C2 are 2.9 – 4.4 kcal/mol for the different models, indicating that attack exclusively occurs at the benzylic position of MSO, also in absence of the two tyrosine residues. For SSO, the results are somewhat different (Table 5.5). Removal of the methyl group from C2 reduces the steric hindrance for attack at this carbon and the barrier for opening of SSO at C2 is thus only 7.0 kcal/mol, compared to 11.0 kcal/mol for opening at C2 of MSO. However, the steric advantage for attack at C2 is still lower than the electronic advantage for attack at C1 of SSO, and attack at C1 remains preferred, albeit only with 0.7 kcal/mol. The small difference in barriers suggests that a mixture of products would be formed. The single tyrosine mutants also exhibit preferred attack at C1 of SSO, while in complete absence of a general acid, steric factors dominate, resulting in preferred attack at C2 for the double mutant (Table 5.5).

Table 5.4 Regioselectivity of MSO attack with different sEH models. Barriers (in kcal/mol) for the alkylation TS for attack at C1 or C2. Model Barrier C1 (benzylic) Barrier C2 (homo-benzylic) Regioselectivitya WT 7.8 11.0 +3.2 Y465F 13.2 16.9 +3.7 Y381F 15.7 20.1 +4.4 Y381F/Y465F 24.8 27.7 +2.9 aDefined as difference in barrier, (barrier C2 – barrier C1). 39

Table 5.5 Regioselectivity of SSO attack. Barrier (in kcal/mol) for the alkylation TS for attack at C1 or C2. Model Barrier C1 (benzylic) Barrier C2 (terminal) Regioselectivitya WT 6.3 7.0 +0.7 Y465F 14.0 14.8 +0.8 Y381F 13.7 15.5 +1.8 Y381F/Y465F 24.2 23.6 -0.6 aDefined as difference in barrier, (barrier C2 – barrier C1).

To our knowledge, the regioselectivities of MSO and SSO opening have not been studied experimentally with human sEH. However, studies have been performed with a number of homologues. Conversion of (1S,2S)-β-methylstyrene oxide by the Aspergillus terreus EH leads to 95% attack at the benzylic position.87 Conversion of the same substrate by the EH from Rhodotorula glutinis, the rabbit sEH or the mouse sEH yield virtually exclusive attack at the benzylic position.88,89,90 These regioselectivities are very much in line with the energetics calculated for MSO.

The regioselectivity of styrene oxide opening with various EHs has given a broad range of results. Several enzymes exhibit significant attack at both C1 and C2 of SSO, as suggested from the calculated energetics. For example conversion of SSO leads to regioselectivities of 45%(α):55%(β) for the rabbit sEH,87 15%(α):85%(β) for the Aspergillus niger EH91 and 64%(α):36%(β) for Rhodococcus NCIMB 11216 EH (although the latter seems to be slightly biased by non-enzymatic hydrolysis).92 Other enzymes exhibit almost exclusive attack at one carbon center, for example with EchA from Agrobacterium radiobacter AD1, attack occurs exclusively at the terminal carbon (β) of styrene oxide.43 For the EH from Beauveria sulfurescens and the potato (Solanum tuberosum) StEH, attack occurs respectively 99% and 98% at the benzylic position (α) of SSO.91,93 Thus, regioselectivities of styrene oxide opening vary significantly for different epoxide hydrolases. The experimental results seem understandable in light of the calculated energetics. For styrene oxide, there is a considerable steric advantage for attack at the terminal carbon. At the same time there is an electronic advantage for attack at the benzylic carbon. In our calculations, the 40 difference in barriers for attack at either carbon of SSO is only 0.7 kcal/mol. The results indicate that the regioselectivity of SSO opening can be influenced by differential binding in the active site. Binding that reduces the steric advantage for attack at C2 would increase the amount of benzylic attack, while binding that reduces the stabilization through the active site tyrosines would enhance attack at the terminal position.

5.3 The haloalcohol dehalogenase HheC (Paper IV) The third class of epoxide-transforming enzymes studied in this thesis comprises the bacterial haloalcohol dehalogenases, which are related to the superfamily of NAD(P)H-dependent short-chain dehydrogenase/reductases (SDRs).51 The haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1 was introduced above. It catalyzes the conversion of halohydrins into epoxides, but is also able to catalyze the reverse reaction, the transformation of epoxides into various substituted alcohols. In the epoxide opening reaction, a number of interesting 53,54,55,59 nucleophiles are accepted, such CN¯, N3¯, and NO2¯.

5.3.1 The epoxide-opening mechanism of HheC The SDRs exhibit a Lys-Tyr-Ser catalytic triad, and based on the similarities of HheC to SDRs, an Arg149-Tyr145-Ser132 catalytic triad was proposed for HheC (Scheme 5.7). Mutational studies confirmed the importance of these residues for the activity of HheC.51,52 In the dehalogenation mechanism, Tyr145 is suggested to act as a catalytic base, abstracting a proton from the halohydrin substrate. Arg149 activates Tyr145 by abstracting a proton from Tyr145 in concert with epoxide formation. The role of Ser132 seems to be primarily substrate binding.51,52 In the epoxide opening mechanism, the proposed catalytic triad can be expected to operate in the reverse fashion, with Arg149 donating a proton to Tyr145, which donates a proton to the emerging oxyanion of the alcohol. We studied the epoxide opening mechanism of HheC with the substrate (R)-styrene oxide (RSO, Paper IV). Two different nucleophiles were tested, azide and cyanide. The model was based on the crystal structure of HheC in complex with (R)-1-para-nitro-phenyl-2-azido-ethanol, (PDB code 1PXO).52 The final QM model with azide and RSO has a size of 130 atoms and 41

Tyr145 Tyr145

Arg149 Ser132 Arg149 Ser132

H O H N O N O O H H H H H H H N N H N O N + O H H H H R R - Cl Cl

halide binding site halide binding site

Scheme 5.7 Proposed dehalogenation mechanism of HheC.

will in the following be referred to as the wild type model. With this model, we find a one-step mechanism for the epoxide opening, where nucleophilic attack by azide and proton transfer from Tyr145 occur concertedly. The transition state for azidolysis at the β-carbon of RSO is shown in Figure 5.6A. Attack of azide at the terminal carbon occurs at a distance of 2.16 Å. The Cβ-O bond is elongated to 1.79 Å. A proton transfer from Arg149 to Tyr145 was not observed, and in the formed product geometry, a tyrosinate has been formed (Paper IV). Arg149 stabilizes the tyrosinate through hydrogen-bonding. The barrier for the concerted proton transfer from Tyr145 and epoxide opening at Cβ has a computed barrier of 8.1 kcal/mol. The overall reaction energy is calculated to -25.2 kcal/mol, which agrees well with the observed irreversibility of HheC-mediated azidolysis.57

The computed energies show that the proposed epoxide opening mechanism with azide is energetically feasible. For cyanolysis of RSO, the same mechanism is found. The TS for attack at the β-carbon of RSO by cyanide is shown in Figure 5.6C. The optimized distances are similar to the TS for azidolysis, with the main difference being the nucleophile-Cβ distance, which is somewhat longer (2.32 Å). The computed energies for cyanolysis are somewhat different than for azidolysis. The barrier for nucleophilic attack of cyanide at Cβ is calculated to 7.0 kcal/mol, while the overall reaction energy is -46.0 kcal/mol. 42

A * B * Arg149 Arg149 * Tyr145 1.03 * 1.03 1.77 Tyr145 1.74 * 1.03 1.99 Ser132 1.91 1.08 * 1.02 1.83 1.80 1.08 1.36 1.36 Phe12 Ser132 1.79 1.82 Pro175 * Pro175 *2.16 * 2.45 Phe186 * * Asn 176 * * Phe186 * * 2.09 * * 1.98 Phe12 * 2.18 1.95 * * Asn176 1.96 * Leu178 2.18 Leu178 2.08 Tyr187 2.10 * * Tyr177* * Tyr177 Tyr187 * * *

CDArg149 * Arg149 * * 1.03 * 1.79 Tyr145 1.73 Ser132 * 1.92 Tyr145 1.02 1.91 1.07 Ser132 1.13 1.82 1.39 * 1.82 1.28 Phe12 1.74 1.85 Phe12 * 1.87 * * * Pro175 2.68 2.32 Phe186 * * Asn176 1.92 * * Pro175 Phe186 1.96 1.93 Asn176 1.97 2.12 * * 2.13 2.13 Leu178 Leu178 * Tyr187 Tyr187 * * * * * Tyr177 Tyr177

Figure 5.6 Transition states for cyanolysis and azidolysis of RSO in the HheC active site model. A) Attack of azide at Cβ, B) Attack of azide at Cα, C) Attack of cyanide at Cβ, D) Attack of cyanide at Cα.

From the computed epoxide opening reactions it can be concluded that the role of Tyr145 is binding and protonation of the epoxide, with aid of Arg149. It is less clear what function Ser132 is fulfilling. An experimental Ser132Ala mutant of HheC exhibited complete loss of activity in the dehalogenation reaction, indicating that Ser132 is essential for catalysis.51 The interaction between Ser132 and the substrate might be important for substrate positioning. Additionally, Ser132 could have a role in stabilizing the emerging epoxide oxyanion, analogous to the role of the second tyrosine residue in sEH (Paper III). We prepared a Ser132Ala mutant and calculated the barriers for cyanolysis in this model (Paper IV). Mutation of Ser132 raises the barrier for attack at Cβ by 2.2 kcal/mol compared to the wild type model (Table 5.7). This shows that Ser132 plays a role in lowering the barrier for epoxide opening. However, the barrier increase in absence of Ser132 does not explain the loss of activity of the experimental Ser132Ala mutant.51 It is possible that Ser132 also is essential for substrate binding in the active site, which could explain the observed inactivity of the Ser132Ala mutant.

5.3.2 Regioselectivity of wild type HheC Experimental studies show that HheC mediates attack at the less substituted carbon of various epoxides with the nucleophiles azide, cyanide, and nitrite.53,54,55,57,59 For azidolysis of styrene oxide, the observed regioselectivity is different from that in solution. While chemical conversion of styrene oxide with NaCN results in 3% attack at the terminal carbon (Cβ), HheC-mediated azidolysis increases this value to 79%.57 We studied the regioselectivity for azidolysis of styrene oxide with two different models. The first model comprises only RSO, azide, and a phenol residue as acid (Figure 5.7). In this model, the calculated barriers for attack of azide at Cα and Cβ of RSO are 10.7 and 13.5. kcal/mol, respectively (Table 5.6). The observed regioselectivity is in line with the strong preference for the Cα in the chemical azidolysis of styrene oxide.57 With the HheC wild type active site model of 130 atoms, the barriers for azidolysis were computed to 8.6 and 8.1 kcal/mol, respectively, for attack at Cα and Cβ (Figure 5.6, Table 5.6). The regioselectivityg has thus changed

gThe regioselectivity is here defined as the difference in barriers, (Barrier Cβ) – (Barrier Cα). 44

A B

1.02 1.56 1.01 1.61 1.81 1.77 α 2.40 β 2.23

C D

1.01 1.60 1.00 1.73 1.60

1.70 α 2.49 2.40 β

Figure 5.7 Phenol-catalyzed azidolysis and cyanolysis of RSO. A) Attack of azide at Cα, B) Attack of azide at Cβ, C) Attack of cyanide at Cα, D) Attack of cyanide at Cβ.

significantly, from +2.8 kcal/mol in the phenol-catalyzed model to -0.5 kcal/mol in the HheC active site model. The slight preference for Cβ in the HheC active site model is in line with experimental results.57 The reported regioselectivity of HheC-mediated azidolysis of RSO is 79%(β):21%(α).57 This corresponds approximately to a difference in barriers of -0.8 kcal/mol. For cyanolysis of RSO, we calculated a similar pattern. With the phenol-catalyzed model (Figure 5.7), the barriers for attack at Cα and Cβ are 10.5 and 11.0 kcal/mol, i.e. a slight preference for the Cα is observed. With the HheC active site model (Figure 5.6), the barriers for cyanolysis are 10.6 and 7.0 kcal/mol respectively, for attack at Cα and Cβ. The regioselectivity has thus changed from +0.5 in the phenol- catalyzed model to -3.6 kcal/mol in the HheC active site model (Table 5.6). 45

Table 5.6 Calculated barriers (kcal/mol)a for azidolysis and cyanolysis of RSO. Model (# atoms) Nucleophile Barrier Cα Barrier Cβ Regioselectivity

Phenol-catalyzed (33) N3¯ 10.7 13.5 +2.8

HheC active site (130) N3¯ 8.6 8.1 -0.5

Phenol-catalyzed (32) CN¯ 10.5 11.0 +0.5 HheC active site (129) CN¯ 10.6 7.0 -3.6 a Solvation effects calculated with ε = 80 for the phenol-catalyzed and ε = 4 for HheC active site models.

For both the azidolysis and the cyanolysis of RSO, HheC promotes attack at Cβ with several kcal/mol compared to the phenol-catalyzed epoxide opening. We were interested in establishing what effects cause this change in regioselectivity. For this purpose, different functional groups in the active site model were mutated individually to establish their influence on the regioselectivity of RSO conversion (Paper IV).

5.3.3 Regioselectivity of HheC mutants Analysis of the optimized structures for the cyanolysis and azidolysis of RSO in the wild type HheC active site model indicate that there are multiple effects that might mediate the preference for attack at the Cβ. These include the position and orientation of the nucleophile and the substrate in the active site, steric effects mediated by surrounding residues, and electrostatic stabilization of attack at Cβ. These possible effects were analyzed in depths in the following.

One residue that might influence the regioselectivity of epoxide-opening is the backbone carbonyl of Pro175. The Pro175 CO appears to interact with the Cβ- hydrogen of RSO (Scheme 5.8), which might aid in stabilization of the positive charge that evolves at the β-carbon during epoxide opening. The optimized distances of the wild type model support this idea. In the reactant structure with azide, the distance from the Pro175 CO to the Cβ-hydrogen is 2.54 Å. At the transition state for attack of azide at Cα, this distance is 2.40 Å, while at the transition state for Cβ attack, the distance has been shortened to 2.06 Å, indicating an interaction. For cyanolysis, the 46

O H R H β α Pro175 O

N H Scheme 5.8 Interaction of the backbone CO of Pro175 with the Cβ hydrogen of RSO.

Pro175-CO to Cβ-hydrogen distance is 2.29 Å in the reactant structure, 2.31 Å at the transition state for Cα attack and 2.15 Å at the transition state for Cβ attack. The interaction of Pro175 with the Cβ-hydrogen of RSO might facilitate attack at Cβ, thereby reducing the preference for the Cα atom. We tested this hypothesis by mutating the backbone carbonyl of Pro175 into CH2. The transition states for cyanolysis at Cα and Cβ in the Pro175CO→CH2 model are shown in Figure 5.8. The barrier for attack at Cα is 10.8 kcal/mol, which is almost the same as in the wild type model (10.6 kcal/mol). However, the barrier for attack at Cβ is raised by 1.5 kcal/mol compared to the wild type model (Table 5.7). This decreases the preference for attack at Cβ by 1.3 kcal/mol, although it is not sufficient to reverse the regioselectivity.

Other effects that might influence the regioselectivity are the position and orientation of the substrate and the nucleophile. For example, in the optimized reactant of the wild-type model, the distance from the cyanide carbon to the Cα and Cβ carbons of RSO are respectively 4.44 Å and 3.47 Å. For a reaction in solution, where the nucleophile and the substrate are not restricted in their movements, this distance difference might be less critical. However, in the HheC active site, large movements of the substrate or the nucleophile might be energetically costly, as they could result in changes in the hydrogen bonding pattern or in steric interactions with surrounding residues. We tested what effect the hydrogen bonds to RSO and cyanide have on the regioselectivity of attack. The epoxide forms hydrogen bonds with the hydroxyl groups of Ser132 and Tyr145. Tyr145 can be considered essential for the reaction mechanism and it was thus only tried to investigate how mutation of Ser132 affects the regioselectivity of epoxide opening by mutating Ser132 into an alanine residue. 47

A Arg149 * B Arg149 * Tyr145 * Tyr145 * 1.80 1.71 1.02 1.02 Ser132 * 1.92 Ser132 1.05 2.00 1.07 * 1.43 1.82 1.38 1.82 Phe12 1.85 Pro175 * 1.72 Phe12 * * * Pro175 2.35 2.72 * Phe186 * Phe186 * 2.06 Asn176 2.00 * * * Asn176 * 2.09 * Tyr187 2.10 Tyr187 Leu178 * Leu178 * * * * * Tyr177 Tyr177

Figure 5.8 Transition states for RSO cyanolysis in the Pro175-CO→CH2 model, A) Attack at Cβ, B) Attack at Cα. The mutated backbone carbon atom is marked with ∆.

The barrier for attack at Cβ in the Ser132→Ala model was calculated to be 9.2 kcal/mol, while for nucleophilic attack of cyanide at Cα, we calculate a barrier of 12.1 kcal/mol. The regioselectivity is -2.9 kcal/mol, which is 0.7 kcal/mol less than in the wild type model (Table 5.7). Thus, the interaction of the substrate with Ser132 contributes little to the preferred β-regioselectivity.

Table 5.7 Calculated barriers (kcal/mol)a for cyanolysis of RSO with different models. Model (# atoms) Barrier Cα Barrier Cβ Regioselectivity Phenol-catalyzed (32) 10.5 11.0 +0.5 HheC wild type (129) 10.6 7.0 -3.6 HheC Ser132 → Ala (128) 12.1 9.2 -2.9

HheC Pro175-CO → CH2 (130) 10.8 8.5 -2.3 HheC Leu178NH → O (128) 9.1 6.5 -2.6 HheC RmTyr187 (116) 7.9 7.8 -0.1 a Solvation effects calculated with ε = 80 for the phenol-catalyzed and ε = 4 for HheC active site models. 48

The position of cyanide in the halide binding site might have an effect on the regioselectivity. In the HheC active site model, cyanide forms hydrogen bonds with the backbone nitrogen of Leu178 and the water molecule (Figure 5.6). A weak interaction is also observed with the backbone NH of Tyr177. We tested if the hydrogen bond from cyanide to the backbone nitrogen of Leu178 has a significant effect on the regioselectivity of epoxide opening (Paper IV). In the Leu178NH→O model, the backbone amide was mutated into an ester bond, i.e. the NH was replaced by oxygen. This eliminates the backbone hydrogen bond to cyanide. The barrier for attack at Cα is somewhat reduced in this model (9.1 kcal/mol, compared to 10.6 kcal/mol in the wild type, Table 5.7) and the regioselectivity is reduced to -2.6 kcal/mol. This indicates that the Leu178NH hydrogen bond has an effect on the regioselectivity of attack.

The position of the nucleophile could also be affected by steric hindrance caused by surrounding residues. One residue that might have an effect on the movement of the nucleophile is Tyr187. It has previously been suggested that Tyr187 contributes to the rigidity of the halide binding site, probably through its interaction with Asn176.52,58 We prepared an HheC active site model in which Tyr187 was removed (Figure 5.9).

In the optimized structures of the RmTyr187 model, the position of cyanide has changed slightly, resulting in a more favourable interaction with the backbone NH of Tyr177. In the reactant of the wild type model, the distance from the nitrogen of cyanide to the hydrogen of the Tyr177 amide is 2.73 Å, while in the RmTyr187 model, this distance is only 2.10 Å. This indicates that in the wild type model, Tyr187 hinders cyanide from positioning itself close to the Tyr177 backbone. In the

RmTyr187 model, the computed barriers are 7.9 and 7.8 kcal/mol for attack at Cα and Cβ, respectively (Table 5.7). Thus, the regioselectivity is reduced to only -0.1 kcal/mol, implying that cyanolysis would lead to a racemic mixture of cyanoalcohols. The regioselectivity of the RmTyr187 mutant is thus quite close to the regioselectivity of the phenol-catalyzed model.

We also tested the effect of Tyr187 on RSO azidolysis. The computed barriers for azidolysis in the RmTyr187 model are 5.6 kcal/mol for attack at Cα and 8.1 kcal/mol 49

A B Arg149 * Tyr145 * * Arg149 *

Tyr145 1.74 Ser132 1.81 * 1.03 1.86 * 1.87 Ser132 1.06 1.03 1.42 1.30 1.77 1.86 1.12 1.92 1.86 Phe186 Phe12 * Phe186 2.31 Phe12 * * * Pro175 * 2.65 * * * * * * Pro175 1.96 Asn176 2.14 Asn176 * 1.96 2.12 * 2.10 2.07 2.19 * 2.30 Tyr177 * * Leu178 Tyr177 * Leu178 *

Figure 5.9 Transition states for cyanolysis of RSO in the HheC RmTyr187 model, A) Attack at Cβ, B) Attack at Cα.

for attack at Cβ. The resulting regioselectivity is +2.5 kcal/mol, which is similar to the value obtained with the phenol-catalyzed model (+2.8 kcal/mol, Table 5.6). For both azidolysis and cyanolysis, Tyr187 is thus identified as being critical for the observed change in regioselectivity in going from the phenol-catalyzed to the HheC active site model. It can be argued that the effect of Tyr187 in the active site model is exaggerated due to the freezing scheme employed. In our model, Tyr187 is truncated and fixed at Cγ, while in the native enzyme, the side chain would be extended with Cβ and might exhibit larger flexibility. However, the interaction of Tyr187 with Asn176 (and also with Trp249) observed in the HheC crystal structure indicates that Tyr187 has little flexibility also in the native enzyme.52,58

From the above analysis, the factors governing the regioselectivity of HheC-mediated epoxide opening can be summarized as follows (Scheme 5.9):

Ser132 Tyr145

O R Pro175 O β α

Nu- Tyr187 H2O

Leu178 Tyr177

halide binding site

Scheme 5.9 Illustration of effects governing the regioselectivity of HheC-mediated epoxide opening.

• The nucleophile and the substrate are bound in the active site through multiple hydrogen bonds. This restricts their movement relative to each other. Removal of for example the hydrogen bond from cyanide to the Leu178 backbone reduces the regioselectivity of RSO cyanolysis by 1.0 kcal/mol.

• The backbone carbonyl of Pro175 stabilizes opening at Cβ. Mutation of the Pro175 carbonyl reduces the regioselectivity of RSO cyanolysis by 1.3 kcal/mol.

• Tyr187 imposes steric hindrance on the nucleophile, making attack at Cα less favourable. Removal of Tyr187 reduces the regioselectivity of RSO cyanolysis by 3.5 kcal/mol, resulting in equally preferred attack of cyanide at Cβ and Cα.

Chapter 6

Conclusions

The presented theoretical studies of epoxide-transforming enzymes give a deeper understanding of the mechanistic strategies employed in epoxide conversion. Several elements can be identified that are common for all three enzymes. Also, the theoretical results make it possible to evaluate the effects that contribute to the observed regioselectivities.

6.1 Mechanistic strategies of epoxide-transforming enzymes At first glance, the mechanisms and catalytic residues employed by the studied enzymes appear to be quite different. LEH employs a concerted general acid/general base mechanism mediated by an Asp-Arg-Asp triad. The nucleophile in LEH is an activated water molecule (Paper I). sEH exhibits an Asp-His-Asp catalytic triad in combination with two tyrosine residues and employs a covalent reaction mechanism. The nucleophile in sEH is the side chain carboxy of Asp333 (Paper II and III). HheC employs an Arg-Tyr-Ser catalytic triad to perform a concerted, acid-catalyzed reaction. A variety of nucleophiles are accepted by HheC, including azide, cyanide, and nitrite (Paper IV).

Comparison of the studied mechanisms reveals certain mechanistic features, which are shared by all three enzymes. For example, in all cases, epoxide opening takes place through an SN2 mechanism, in which epoxide opening and nucleophilic attack occur concertedly (Paper I, II and IV). An SN1 mechanism, in which epoxide opening and nucleophilic attack occur step-wise, is not observed for any of the studied enzymes or substrates. For all three enzymes, a general acid is present, which donates a proton to the emerging oxyanion. For HheC and LEH, proton donation occurs concertedly with epoxide opening. If the general acid is eliminated, the barriers for epoxide opening 52 increase substantially. For example, the ring opening of (1R,2S)-1-methylcyclohexene oxide (3,4 P) was studied with an LEH active site model in which Asp101 was deprotonated (Paper I). In that situation, the barrier for epoxide opening increases from 14.9 kcal/mol to 44.1 kcal/mol (Paper I). If both tyrosines in sEH are mutated to phenylalanines, the barrier for MSO opening increases from 7.8 kcal/mol to 24.8 kcal/mol (Paper III). In each case, removal of the general acid renders the enzyme catalytically inactive. The theoretical results are consistent with experimental studies of sEH and LEH mutants. If both tyrosines are mutated in the bacterial EchA, the mouse sEH or the potato StEH, virtually no catalytic activity is detected.80,81,85 Mutation of Asp101 to alanine or asparagine in LEH also results in a catalytically inactive enzyme.64 A feature shared by HheC and sEH is the presence of an additional hydrogen bond donor to the epoxide oxygen, besides the general acid. In HheC this is an optimally positioned serine residue, and in sEH a second tyrosine is present. In silico mutation of the second tyrosine in sEH to phenylalanine results in a barrier increase from 7.8 to 13.2 or 15.7 kcal/mol for the epoxide opening reaction, respectively, for the Tyr465Phe and the Tyr381Phe mutations (Paper III). In HheC, mutation of Ser132 to alanine results in a barrier increase from 7.0 to 9.2 kcal/mol for attack of cyanide at Cβ of RSO (Paper IV). The additional hydrogen bond donor does thus provide a barrier reduction of several kcal/mol for both sEH and HheC.

6.2 Regioselectivities of epoxide-transforming enzymes The regioselectivities of epoxide opening are substantially different for the studied enzymes. sEH mediates preferred attack at the more substituted, benzylic carbon of (1S,2S)-β-methylstyrene oxide (MSO) and (S)-styrene oxide (SSO) (Paper II), while HheC mediates preferred attack at the less substituted, terminal carbon of (R)-styrene oxide (Paper IV). LEH mediates preferred attack at either the more or the less substituted carbon, depending on the substrate (Paper I). In each case, very different factors govern the regioselectivity. The regioselectivity of epoxide opening is in general influenced by steric and by electronic factors. For LEH, the regioselectivity of limonene-1,2-epoxide and 1-methylcyclohexene oxide conversion is dominated by conformational factors (Paper I). The structure of the formed transition state 53 determines if attack occurs at either the more substituted (C1) or the less substituted (C2) carbon. In each case, attack will be favoured at the atom that leads to a chair-like transition state (Paper I). In sEH, electronic factors seem to dominate the opening of MSO and SSO. The stabilization provided by the phenyl substituent of the substrate results in attack at the benzylic carbon, a preference that is further enhanced by presence of the two tyrosine residues (Paper III). When both tyrosines in sEH are mutated to phenylalanine, steric factors play a more dominant role and preferred attack at the terminal carbon of SSO is observed (Paper III). For HheC, both steric and electronic factors can be identified that mediate preferred attack at the terminal carbon of RSO. For example, the backbone carbonyl of Pro175 interacts with the hydrogen on the terminal carbon of RSO, providing electronic stabilization to the emerging positive charge. In silico mutation of the Pro175 backbone carbonyl to a CH2 reduces the preference of cyanide for the β-carbon by 1.3 kcal/mol (from -3.6 kcal/mol in the wild type model to -2.3 in the Pro175-CO → CH2 model, Paper IV). In addition, steric factors reduce the advantage for attack at the benzylic position of RSO. For example Tyr187 seems to hinder the movement of the nucleophile (Paper IV). If Tyr187 is removed from the model, the regioselectivity is reduced to only -0.1 kcal/mol.

The observed effects on the regioselectivity of epoxide opening can be summarized in regard to their nature. Electronic effects include stabilization mediated by substituents on the substrate, the presence of hydrogen bond donors/general acids to the epoxide oxygen, and stabilization effects provided by the surroundings such as the backbone carbonyl of Pro175 in HheC. Steric effects on the regioselectivity are caused by the substitution on the substrate (attack at an less substituted carbon is sterically preferred), conformations of the formed transition states (such as for limonene-1,2- epoxide), and the orientations imposed on the substrates/nucleophiles, when they are bound in the active site.

6.3 Final Conclusions The presented results show how a theoretical approach can give valuable information about enzymatic mechanisms. For example, the detailed analysis of individual effects influencing the regioselectivity of epoxide opening in HheC would not be possible in 54 a similar fashion in an experimental setting. Many mutations which easily are introduced in silico would be almost impossible to introduce in vitro/in vivo. Also the characterization of short-lived transition states is not (yet) possible with conventional experimental methods. Only indirect strategies can be employed, such as structural characterization of enzymes in complex with transition state analogues. In the computational approach, it is possible to optimize transition state structures, which are of great aid in explaining reaction mechanisms. For example, the discrimination between different mechanistic pathways in LEH-mediated conversion of limonene- 1,2-epoxide can be explained with the conformations of the potential transition states. Overall, this thesis confirms the usefulness of a computational approach in investigating enzymatic reaction mechanisms.

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