Conformational Dynamics‐Guided Loop Engineering of an Alcohol Dehydrogenase: Capture, Turnover and Enantioselective Transformation of Difficult‐to‐Reduce Ketones

Abstract

Directed evolution of enzymes for the asymmetric reduction of prochiral ketones to produce enantio‐pure secondary alcohols is particularly attractive in organic synthesis. Loops located at the active pocket of enzymes often participate in conformational changes required to fine‐tune residues for substrate binding and catalysis. It is therefore of great interest to control the substrate specificity and stereochemistry of enzymatic reactions by manipulating the conformational dynamics. Herein, a secondary alcohol dehydrogenase was chosen to enantioselectively catalyze the transformation of difficult‐to‐reduce bulky ketones, which are not accepted by the wildtype enzyme. Guided by previous work and particularly by structural analysis and molecular dynamics (MD) simulations, two key residues alanine 85 (A85) and isoleucine 86 (I86) situated at the binding pocket were thought to increase the fluctuation of a loop region, thereby yielding a larger volume of the binding pocket to accommodate bulky substrates. Subsequently, site‐directed saturation mutagenesis was performed at the two sites. The best mutant, where residue alanine 85 was mutated to glycine and isoleucine 86 to leucine (A85G/I86L), can efficiently reduce bulky ketones to the corresponding pharmaceutically interesting alcohols with high enantioselectivities (∼99% ee). Taken together, this study demonstrates that introducing appropriate mutations at key residues can induce a higher flexibility of the active site loop, resulting in the improvement of substrate specificity and enantioselectivity.

The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes

“…Subsequently we turned to Tw ist Bioscience,abio-company that sells custom genes and also offers the possibility of preparing mutant libraries.T he Tw ist technique is based on ap roprietary DNAs ynthesis platform which utilizes smallscale solid phase gene synthesis on silicon-chips.Uptoatleast 100,000 unique oligonucleotide sequences can be synthesized fairly rapidly within af ew days.I nc ollaboration with the company,w et ested the method in combinatorial SM library construction. [36a] Following the separate synthesis of the designed mutagenic primers,t hey are extracted from the Alcohol dehydrogenase TbSADH substrate specificity,activity,and stereoselectivity CAST Computer-guided SM at CAST sites [134,141] Multidrug resistance regulator LmrR activity CAST NNK codonsperformed on eight CAST sites [142] Rma cytochrome ca ctivity,enantioselectivity CAST/ISMI SM, 22c trick [143] cytochrome P450 enzymes CÀHand BÀHbond functionalization CAST CAST/22c trick [144] respective plates.T he combination of efficient chemistry and molecular biology ensures uniform mixture of high-fidelity mutagenic primers applied in the modification of sequenceperfect scaffold fragments and in the assembly with formation of full-length constructs that can then be cloned (Figure 20). [36a] Limonene epoxide hydrolase (LEH) from Rhodococcus erythropolis was used as the catalyst in the model reaction (Scheme 11).…”

Reversal of Regioselectivity in Zinc‐Dependent Medium‐Chain Alcohol Dehydrogenase from Rhodococcus erythropolis toward Octanone Derivatives

“…However, the W296A variant could possibly show reversed enantioselectivity for the smaller substrates accepted by the WT, as the variant has a larger substrate binding pocket that allows the substrate to explore pro‐ R conformations with catalytically active hydride transfer distances. This phenomenon was observed in almost all previously engineered ketoreductases such as cpADH5 and TbSADH …”

“…This phenomenon was observed in almost all previously engineered ketoreductases such as cpADH5 [25] and TbSADH. [21,27]…”

Harnessing Conformational Plasticity to Generate Designer Enzymes

“…As another example, Fraser and co-workers performed directed evolution on a catalytically impaired variant of cyclophilin A (CypA), and were able to partially restore the catalytic activity of the enzyme through the introduction of two second-shell mutations that “rescued” activity through modulation of conformational dynamics. 181 For several more examples, we refer the readers to refs ( 26 , 181 , and 182 ). Taken together, these successful examples of modulating enzyme activity through targeting conformational dynamics, either deliberately or serendipitously, indicate their importance and further highlight the vast opportunity still present in the field.…”

“… 207 , 208 The insights gained from MD simulations can be directly applied toward the (semi-)rational design of variants with altered conformational dynamics. 182 , 203 , 209 Dodani et al utilized extensive MD simulations to identify a single residue that was responsible for controlling the conformational dynamics of the F/G loop of a nitrating cytochrome P450 TxtE, with point variants ultimately able to switch substrate regioselectivity. 203 Extensive MD simulations can be used to construct MSMs, which can provide thermodynamic and kinetic characterization of conformational substates.…”