Structure-guided alteration of coenzyme specificity of formate dehydrogenase by saturation mutagenesis to enable efficient utilization of NADP+.
Formate dehydrogenase from Candida boidinii (CboFDH) catalyses the oxidation of formate anion to carbon dioxide with concomitant reduction of NAD(+) to NADH. CboFDH is highly specific to NAD(+) and virtually fails to catalyze the reaction with NADP(+). Based on structural information for CboFDH, the loop region between beta-sheet 7 and alpha-helix 10 in the dinucleotide-binding fold was predicted as a principal determinant of coenzyme specificity. Sequence alignment with other formate dehydrogenases revealed two residues (Asp195 and Tyr196) that could account for the observed coenzyme specificity. Positions 195 and 196 were subjected to two rounds of site-saturation mutagenesis and screening and enabled the identification of a double mutant Asp195Gln/Tyr196His, which showed a more than 2 x 10(7)-fold improvement in overall catalytic efficiency with NADP(+) and a more than 900-fold decrease in the efficiency with NAD(+) as cofactors. The results demonstrate that the combined polar interactions and steric factors comprise the main structural determinants responsible for coenzyme specificity. The double mutant Asp195Gln/Tyr196His was tested for practical applicability in a cofactor recycling system composed of cytochrome P450 monooxygenase from Bacillus subtilis, (CYP102A2), NADP(+), formic acid and omega-(p-nitrophenyl)dodecanoic acid (12-pNCA). Using a 1250-fold excess of 12-pNCA over NADP(+) the first order rate constant was determined to be equal to k(obs) = 0.059 +/- 0.004 min(-1).
Andreadeli
Aggeliki
A
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Greece.
Platis
Dimitris
D
Tishkov
Vladimir
V
Popov
Vladimir
V
Labrou
Nikolaos E
NE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002175:Candida / Q000201:enzymology; D002846:Chromatography, Affinity; D003067:Coenzymes / Q000378:metabolism*; D017931:DNA Primers; D005560:Formate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009249:NADP / Q000378:metabolism*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D003067:Coenzymes; D017931:DNA Primers; D009249:NADP; C037212:formate dehydrogenase (NADP+); D005560:Formate Dehydrogenases