title: 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.
authors.affiliation: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.|The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, 361005, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.|The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, 361005, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Lin
authors.affiliation: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.|The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, 361005, China.
authors.forename: Ruonan
authors.initials: R
authors.lastname: Yang
authors.affiliation: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China. fbs@xmu.edu.cn.|The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, 361005, China. fbs@xmu.edu.cn.|The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen, Fujian, 361005, China. fbs@xmu.edu.cn.
authors.forename: Baishan
authors.initials: B
authors.lastname: Fang
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002175:Candida / Q000201:enzymology*; D020134:Catalytic Domain; D003067:Coenzymes / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D015202:Protein Engineering
chemical_list: D003067:Coenzymes; D050505:Mutant Proteins; D009243:NAD; D005560:Formate Dehydrogenases
keywords: Biocatalysis; Biocatalyst; Chiral compounds; Cofactor regeneration; Molecular modeling