Biocatalyzed asymmetric reduction of ketones is one of the most cost-effective and environmentally friendly approaches for preparing chiral alcohols. However, few natural ketone reductases (KREDs) ...

Stereochemistry in Asymmetric Reduction of Bulky–Bulky Ketones by Alcohol Dehydrogenases

The strategy of rational design to engineer enzymes is to predict the potential mutants based on the understanding of the relationships between protein structure and function, and subsequently introduce the mutations using the site-directed mutagenesis. Rational design methods are universal, relatively fast and have the potential to be developed into algorithms that can quantitatively predict the performance of the designed sequences. Compared to the protein stability, it was more challenging to design an enzyme with improved activity or selectivity, due to the complexity of enzyme molecular structure and inadequate understanding of the relationships between enzyme structures and functions. However, with the development of computational force, advanced algorithm and a deeper understanding of enzyme catalytic mechanisms, rational design could significantly simplify the process of engineering enzyme functions and the number of studies applying rational design strategy has been increasing. Here, we reviewed the recent advances of applying the rational design strategy to engineer enzyme functions including activity and enantioselectivity. Five strategies including multiple sequence alignment, strategy based on steric hindrance, strategy based on remodeling interaction network, strategy based on dynamics modification and computational protein design are discussed and the successful cases using these strategies are introduced.

Rational design of enzyme activity and enantioselectivity

Reshaping Substrate-Binding Pocket of Leucine Dehydrogenase for Bidirectionally Accessing Structurally Diverse Substrates

Synthesis of P-stereogenic cyclicphosphinic amide via electrochemical enabled cobalt-catalyzed enantioselective C–H annulation

Direct reductive amination of prochiral 2-oxo-4-phenylbutyric acid (2-OPBA) catalyzed by phenylalanine dehydrogenase (PheDH) is highly attractive in the synthesis of the pharmaceutical chiral building block l-homophenylalanine (l-HPA) given that its sole expense is ammonia and that water is the only byproduct. Current issues in this field include a poor catalytic efficiency and a low substrate loading. In this study, we report a structure-guided steric hindrance engineering of PheDH from Bacillus badius to create an enhanced biocatalyst for efficient l-HPA synthesis. Mutagenesis libraries based on molecular docking, double-proximity filtering, and a degenerate codon significantly increased catalytic efficiency. Seven superior mutants were acquired, and the optimal triple-site mutant, V309G/L306V/V144G, showed a 12.7-fold higher kcat value, and accordingly a 12.9-fold higher kcat/Km value, than that of the wild type. A paired reaction system comprising V309G/L306V/V144G and glucose dehydrogenase converted 1.08 M 2-OPBA to l-HPA in 210 min, and the specific space–time conversion was 30.9 mmol g−1 L−1 h−1. The substrate loading and specific space–time conversion are the highest values to date. Docking simulation revealed increases in substrate-binding volume and additional degrees of freedom of the substrate 2-OPBA in the pocket. Tunnel analysis suggested the formation of new enzyme tunnels and the expansion of existing ones. Overall, the results show that the mutant V309G/L306V/V144G has the potential for the industrial synthesis of l-HPA. The modified steric hindrance engineering approach can be a valuable addition to the current enzyme engineering toolbox.

/pdf/structure-guided-steric-hindrance-engineering-of-bacillus-489owiv375.pdf

Structure-guided steric hindrance engineering of Bacillus badius phenylalanine dehydrogenase for efficient L-homophenylalanine synthesis.

https://pubs.rsc.org/en/content/articlepdf/2020/CY/C9CY02444A

Engineering an alcohol dehydrogenase with enhanced activity and stereoselectivity toward diaryl ketones: reduction of steric hindrance and change of the stereocontrol element

Efficient Asymmetric Synthesis of Ethyl (S)-4-Chloro-3-hydroxybutyrate Using Alcohol Dehydrogenase SmADH31 with High Tolerance of Substrate and Product in a Monophasic Aqueous System

Manganese Catalyzed Cross-Coupling of Allylic Alcohols and Nitriles: An Elegant Route for Access of δ-Hydroxynitriles

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Papers

Engineering an alcohol dehydrogenase with enhanced activity and stereoselectivity toward diaryl ketones: reduction of steric hindrance and change of the stereocontrol element

Efficient Asymmetric Synthesis of Ethyl (S)-4-Chloro-3-hydroxybutyrate Using Alcohol Dehydrogenase SmADH31 with High Tolerance of Substrate and Product in a Monophasic Aqueous System

Manganese Catalyzed Cross-Coupling of Allylic Alcohols and Nitriles: An Elegant Route for Access of δ-Hydroxynitriles