Site-saturation mutagenesis of formate dehydrogenase from Candida bodinii creating effective NADP+-dependent FDH enzymes

The analysis of previous reported results envisioned that residues Asp195, Tyr196 and Gln197 of formate dehydrogenase from Candida bodinii (CboFDH) might play critical roles in determining the enzyme's cofactor specificity. With the aim to develop novel NADP+-dependent formate dehydrogenase enzymes, simultaneous site-saturation mutagenesis of residues Asp195 and Tyr196 of CboFDH coupled with screening resulted in two mutant enzymes, D195Q/Y196R and D195S/Y196P, which showed significant NADP+ specificity. The overall catalytic efficiencies (kcat/Km) toward NADP+ were 1.14 × 104 and 2.9 × 103 M−1 s−1, respectively, which are higher than the reported mutant CboFDHs obtained by sequential mutagenesis. The ratio of catalytic efficiencies (kcat/Km)NADP+/(kcat/Km)NAD+ of D195Q/Y196R and D195S/Y196P were 2.1 and 0.2, respectively. Mutation of residue Gln197 of D195Q/Y196R to Asn further increased the enzyme's overall catalytic efficiencies (kcat/Km) toward NADP+ to 29.1 × 103 M−1 s−1, with (kcat/Km)NADP+/(kcat/Km)NAD+ being 17.1, which are much higher than the reported data for a mutant enzyme of formate dehydrogenase from Pseudomanas sp. 101 (PseFDH). This study demonstrates that residues 195, 196 and 197 really play critical roles in determining the enzyme's cofactor specificity.