Computational Design Pipeline for Enhanced D-Lac Activity: A Novel CSFp Strategy for Enzyme Engineering

Vitamin B5 and its additives are widely employed in the food, pharmaceutical, animal feed, cosmetics and other industries,52 and enable acetyl-CoA to synthesize the neurotransmitter acetylcholine, which has attracted wide attention in the research of neurodegenerative diseases.53 However, the evolution of D-Lac proved to be a challenging task. Initially, researchers attempted error-prone (PCR) and DNA shuffling techniques54, which lacked rational design due to the absence of the crystal structure information on D-Lac. The catalytic mechanism of D-Lac remains poorly understood, and designing enzymes based on the transition states obtained by quantum mechanics is not yet feasible. Moreover, the substrate (D-pantolactone, D-PL) lacks large or polar groups, and a single amino acid alteration in the active pocket results in low complementarity between the active site and the overall shape of the ligand. Fortunately, a novel computational design pipeline has been developed, which has led to the discovery of combinatorial strategies for ultra-low throughput screening. The proposed CSFp strategy provides a viable generic solution to minimize experimental effort while maximizing the exploration of supernumerary effects in terms of additivity and/or synergy between mutant sets. Ultimately, an encouraging mutant (N96S/A271E/F274Y/F308G) with a 56-fold increase in activity towards D-PL compared to wild-type (WT) D-Lac was obtained. With this experimental example, we anticipate that the CSFp strategy will facilitate optimised computational enzyme engineering, rescuing various enzymes that lack crystal structure information and have relatively obscure catalytic mechanisms, and facilitating its application in situations where improved enzyme activity is required. Complementing many excellent enzyme engineering methods, this approach will provide ideas for computational enzyme modification.