Identification of catalysis, substrate, and coenzyme binding sites and improvement catalytic efficiency of formate dehydrogenase from Candida boidinii

Abstract

Formate dehydrogenases (FDHs) are continually used for the cofactor regeneration in biocatalysis and biotransformation with hiring NAD(P)H-dependent oxidoreductases. Major weaknesses of most native FDHs are their low activity and operational stability in the catalytic reaction. In this work, the FDH from Candida boidinii (CboFDH) was engineered in order to gain an enzyme with high activity and better operational stability. Through comparing and analyzing its spatial structure with other FDHs, the catalysis, substrate, and coenzyme binding sites of the CboFDH were identified. To improve its performance, amino acids, which concentrated on the enzyme active site or in the conserved NAD⁺ and substrate binding motif, were mutated. The mutant V120S had the highest catalytic efficiency (k _cat/K _m ) with COONH₄ as it enhanced the catalytic velocity (k _cat) and k _cat/K _m 3.48-fold and 1.60-fold, respectively, than that of the wild type. And, the double-mutant V120S-N187D had the highest k _cat/K _m with NAD⁺ as it displayed an approximately 1.50-fold increase in k _cat/K _m . The mutants showed higher catalytic efficiency than other reported FDHs, suggesting that the mutation has achieved good results. The single and double mutants exhibited higher thermostability than the wild type. The structure-function relationship of single and double mutants was analyzed by homology models and site parsing. Asymmetric synthesis of L-tert-leucine was executed to evaluate the ability of cofactor regeneration of the mutants with about 100 % conversion rates. This work provides a helpful theoretical reference for the evolution of an enzyme in vitro and promotion of the industrial production of chiral compounds, e.g., amino acid and chiral amine.