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

• Published in: Angewandte Chemie International Edition Vol. 54; no. 42; pp. 12410 - 12415
• Main Authors: Sun, Zhoutong, Lonsdale, Richard, Kong, Xu-Dong, Xu, Jian-He, Zhou, Jiahai, Reetz, Manfred T.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 12.10.2015; WILEY‐VCH Verlag; Wiley Subscription Services, Inc
• Edition: International ed. in English
• Subjects: amino acid alphabet; Amino acids; Amino Acids - chemistry; Amino Acids - metabolism; Binding; Biotechnology; Cyclohexenes - chemistry; Cyclohexenes - metabolism; directed evolution; Enzymes; epoxide hydrolases; Epoxide Hydrolases - chemistry; Epoxide Hydrolases - genetics; Epoxide Hydrolases - metabolism; Models, Molecular; Molecular Structure; Mutagenesis; Oxides; Pocket; Randomization; Saturation; saturation mutagenesis; Stereoisomerism; Stereoselectivity; Strategy; Terpenes - chemistry; Terpenes - metabolism; epoxide hydrolases; amino acid alphabet; stereoselectivity; saturation mutagenesis; directed evolution
• ISSN: 1433-7851; 1521-3773; 1521-3773
• DOI: 10.1002/anie.201501809

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. Emil Fischer’s lock‐and‐key hypothesis has been revisited in experiments to reshape the binding pocket of limonene epoxide hydrolase (see picture) by using a single amino acid building block in the saturation mutagenesis of a 10‐residue site. Catalytic variants selective for the formation of both (R,R)‐ and (S,S)‐1,2‐cyclohexanediol in the hydrolytic desymmetrization of cyclohexene oxide were identified in one and the same mutant library.