Richard Lonsdale

Reshaping an Enzyme Binding Pocket for Enhanced and Inverted Stereoselectivity: Use of Smallest Amino Acid Alphabets in Directed Evolution

Directed evolution based on saturation mutagenesis at sites lining the binding pocket is a commonly practiced strategy for enhancing or inverting the stereoselectivity of enzymes for use in organic chemistry or biotechnology. However, as the number of residues in a randomization site increases to five or more, the screening effort for 95 % library coverage increases astronomically until it is no longer feasible. We propose the use of a single amino acid for saturation mutagenesis at superlarge randomization sites comprising 10 or more residues. When used to reshape the binding pocket of limonene epoxide hydrolase, this strategy, which drastically reduces the search space and thus the screening effort, resulted in R,R- and S,S-selective mutants for the hydrolytic desymmetrization of cyclohexene oxide and other epoxides. X-ray crystal structures and docking studies of the mutants unveiled the source of stereoselectivity and shed light on the mechanistic intricacies of this enzyme.

Whole-Cell-Catalyzed Multiple Regio- and Stereoselective Functionalizations in Cascade Reactions Enabled by Directed Evolution

Biocatalytic cascade reactions using isolated stereoselective enzymes or whole cells in one-pot processes lead to value-added chiral products in a single workup. The concept has been restricted mainly to starting materials and intermediate products that are accepted by the respective wild-type enzymes. In the present study, we exploited directed evolution as a means to create E. coli whole cells for regio- and stereoselective cascade sequences that are not possible using man-made catalysts. The approach is illustrated using P450-BM3 in combination with appropriate alcohol dehydrogenases as catalysts in either two-, three-, or four-step cascade reactions starting from cyclohexane, cyclohexanol, or cyclohexanone, respectively, leading to either (R,R)-, (S,S)-, or meso-cyclohexane-1,2-diol. The one-pot conversion of cyclohexane into (R)- or (S)-2-hydroxycyclohexanone in the absence of ADH is also described.

Reduction of α,β-Unsaturated Ketones by Old Yellow Enzymes: Mechanistic Insights from Quantum Mechanics/Molecular Mechanics Calculations

Enoate reductases catalyze the reduction of activated C═C bonds with high enantioselectivity. The oxidative half-reaction, which involves the addition of a hydride and a proton to opposite faces of the C═C bond, has been studied for the first time by hybrid quantum mechanics/molecular mechanics (QM/MM). The reduction of 2-cyclohexen-1-one by YqjM from Bacillus subtilis was selected as the model system. Two-dimensional QM/MM (B3LYP-D/OPLS2005) reaction pathways suggest that the hydride and proton are added as distinct steps, with the former step preceding the latter. Furthermore, we present interesting insights into the reactivity of this enzyme, including the weak binding of the substrate in the active site, the role of the two active site histidine residues for polarization of the substrate C═O bond, structural details of the transition states to hydride and proton transfer, and the role of Tyr196 as proton donor. The information presented here will be useful for the future design of enantioselective YqjM mutants for other substrates.

Bioorthogonal Enzymatic Activation of Caged Compounds

Engineered cytochrome P450 monooxygenase variants are reported as highly active and selective catalysts for the bioorthogonal uncaging of propargylic and benzylic ether protected substrates, including uncaging in living E. coli. observed selectivity is supported by induced-fit docking and molecular dynamics simulations. This proof-of-principle study points towards the utility of bioorthogonal enzyme/protecting group pairs for applications in the life sciences.

Biocatalytic Route to Chiral Acyloins: P450-Catalyzed Regio- and Enantioselective α-Hydroxylation of Ketones

P450-BM3 and mutants of this monooxygenase generated by directed evolution are excellent catalysts for the oxidative α-hydroxylation of ketones with formation of chiral acyloins with high regioselectivity (up to 99%) and enantioselectivity (up to 99% ee). This constitutes a new route to a class of chiral compounds that are useful intermediates in the synthesis of many kinds of biologically active compounds.

Comparing Different Strategies in Directed Evolution of Enzyme Stereoselectivity: Single‐ versus Double‐Code Saturation Mutagenesis

Saturation mutagenesis at sites lining the binding pockets of enzymes constitutes a viable protein engineering technique for enhancing or inverting stereoselectivity. Statistical analysis shows that oversampling in the screening step (the bottleneck) increases astronomically as the number of residues in the randomization site increases, which is the reason why reduced amino acid alphabets have been employed, in addition to splitting large sites into smaller ones. Limonene epoxide hydrolase (LEH) has previously served as the experimental platform in these methodological efforts, enabling comparisons between single‐code saturation mutagenesis (SCSM) and triple‐code saturation mutagenesis (TCSM); these employ either only one or three amino acids, respectively, as building blocks. In this study the comparative platform is extended by exploring the efficacy of double‐code saturation mutagenesis (DCSM), in which the reduced amino acid alphabet consists of two members, chosen according to the principles of rational design on the basis of structural information. The hydrolytic desymmetrization of cyclohexene oxide is used as the model reaction, with formation of either (R,R)‐ or (S,S)‐cyclohexane‐1,2‐diol. DCSM proves to be clearly superior to the likewise tested SCSM, affording both R,R‐ and S,S‐selective mutants. These variants are also good catalysts in reactions of further substrates. Docking computations reveal the basis of enantioselectivity.

In pursuit of an accurate spatial and temporal model of biomolecules at the atomistic level: a perspective on computer simulation

The current computational techniques available for biomolecular simulation are described, and the successes and limitations of each with reference to the experimental biophysical methods that they complement are presented.

Structural and Computational Insight into the Catalytic Mechanism of Limonene Epoxide Hydrolase Mutants in Stereoselective Transformations.

Directed evolution of limonene epoxide hydrolase (LEH), which catalyzes the hydrolytic desymmetrization reactions of cyclopentene oxide and cyclohexene oxide, results in (R,R)- and (S,S)-selective mutants. Their crystal structures combined with extensive theoretical computations shed light on the mechanistic intricacies of this widely used enzyme. From the computed activation energies of various pathways, we discover the underlying stereochemistry for favorable reactions. Surprisingly, some of the most enantioselective mutants that rapidly convert cyclohexene oxide do not catalyze the analogous transformation of the structurally similar cyclopentene oxide, as shown by additional X-ray structures of the variants harboring this slightly smaller substrate. We explain this puzzling observation on the basis of computational calculations which reveal a disrupted alignment between nucleophilic water and cyclopentene oxide due to the pronounced flexibility of the binding pocket. In contrast, in the stereoselective reactions of cyclohexene oxide, reactive conformations are easily reached. The unique combination of structural and computational data allows insight into mechanistic details of this epoxide hydrolase and provides guidance for future protein engineering in reactions of structurally different substrates.

Chapter 11:QM/MM Studies of Cytochrome P450 Systems: Application to Drug Metabolism

The cytochromes P450 (CYPs) are an important family of enzymes involved in the metabolism of drugs. Prediction of the selectivity of CYP-mediated metabolism will assist in the avoidance of adverse drug reactions. Hybrid quantum mechanical/molecular mechanical (QM/MM) methods enable modelling of the complex chemistry that takes place at the active site of these enzymes, to a high degree of accuracy, while including effects of the protein environment. QM/MM methods have contributed significantly to understanding of CYPs at the molecular level, providing information that both complements and adds to experimental findings. In this chapter some current methods used in CYP metabolism prediction will be summarized, followed by some recent QM/MM studies of CYPs, and a discussion of the potential of these methods to contribute to drug development.

Methodology Development in Directed Evolution: Exploring Options when Applying Triple-Code Saturation Mutagenesis

Directed evolution of stereo‐ or regioselective enzymes as catalysts in asymmetric transformations is of particular interest in organic synthesis. Upon evolving these biocatalysts, screening is the bottleneck. To beat the numbers problem most effectively, methods and strategies for building “small but smart” mutant libraries have been developed. Herein, we compared two different strategies regarding the application of triple‐code saturation mutagenesis (TCSM) at multiresidue sites of the Thermoanaerobacter brockii alcohol dehydrogenase by using distinct reduced amino‐acid alphabets. By using the synthetically difficult‐to‐reduce prochiral ketone tetrahydrofuran‐3‐one as a substrate, highly R‐ and S‐selective variants were obtained (92–99 % ee) with minimal screening. The origin of stereoselectivity was provided by molecular dynamics analyses, which is discussed in terms of the Bürgi–Dunitz trajectory.