Structure‐guided alteration of coenzyme specificity of formate dehydrogenase by saturation mutagenesis to enable efficient utilization of NADP⁺

Abstract

NAD+ -dependent formate dehydrogenase (FDH; EC 1.2.1.2) plays an important role in methylotrophic yeasts, catalysing the final step in the methanol oxidation pathway [1,2]. In general, FDH is a highly conservative enzyme. The primary sequence homology is no < 80-85% between enzymes of the same organismal group and 50-55% or more between enzymes from different groups. In addition, the catalytic amino acids, as well as the amino acids that contribute to the structural stability, are almost totally conserved (sequence homology of approximately 95%). Formate dehydrogenase from Candida boidinii (CboFDH) is a dimer with two identical subunits, each possessing an independent active centre [1,3].Detailed studies of FDH are justified by the considerable biotechnological potential of this enzyme. For example, it is used as a diagnostic enzyme for the determination of formic acid [4] . Based on structural information for CboFDH, the loop region between b-sheet 7 and a-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 · 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 x-(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 .Abbreviations 12-pNCA, x-(p-nitrophenyl)dodecanoic acid; CboFDH, Candida boidinii FDH; CYP102A2, cytochrome P450 monooxygenase from Bacillus subtilis; FDH, formate dehydrogenase; G6PDH, glucose 6-phosphate dehydrogenase; PTDH, phosphite dehydrogenase.

Structural basis for double cofactor specificity in a new formate dehydrogenase from the acidobacterium Granulicella mallensis MP5ACTX8

“…The latter preference is the main drawback for the systematic utilization of available FDHs for the in situ regeneration methods since several dehydrogenases used in industrial biocatalysis, e.g., to synthesize ketones or to resolve racemic mixtures of alcohols or amines, are strictly NADP + dependent. Attempts to circumvent the problem have been tried by protein engineering (through rational design, sitedirected or site saturation mutagenesis) NAD + -dependent FDHs from different sources, both prokaryotic and eukaryotic (Gul-Karaguler et al 2001;Serov et al 2002;Andreadeli et al 2008;Wu et al 2009a;Hoelsch et al 2013). Nevertheless, their application has not yet caught on as a routine method in organic synthesis practice, whereas the systems exploiting glucose dehydrogenases (Wong et al 1985;Kaswurm et al 2013) and phosphite dehydrogenases (Woodyer et al 2003;Johannes et al 2007) are largely preferred at both the laboratory and industrial scale (for a recent review, see Weckbecker et al 2010).…”

Protein Engineering Applications on Industrially Important Enzymes: Candida methylica FDH as a Case Study

“…Engineered FDH is also a favourite enzyme to regenerate NADPH among other investigated enzymes such as ADH and glucose-6 phosphate dehydrogenase because of its cheap substrate. Engineered FDH that can accept NADP+ and it is preferred over glucose-6 phosphate dehydrogenase given the expense of its substrate (Andreadeli et al, 2008;Wu et al, 2009). The number of studies on FDH and its application for coenzyme regeneration in the processes of chiral synthesis with NAD(P)Hdependent enzymes is getting larger year by year (Holbrook et al, 2000;Tishkov & Zaitseva, 2008).…”

“…Many attempts using both rational design and site saturation mutagenesis approaches have been made to change the coenzyme specificity of FDH from different sources (Andreadeli et al, 2008;Karaguler et al, 2001;Serov et al, 2002b;Wu et al, 2009) In our previous experiments we achieved cmFDH to use NADP + by the single mutation Asp195Ser (Karaguler et al, 2001). However, this mutant binds NADP + weakly and we suggested that cmFDH possesses an aspartic acid residue (195) which binds the hydroxyl groups of the adenine ribose moiety of NAD + , in common with many NAD + -dependent dehydrogenases containing the Rossmann fold.…”

“…Site saturation mutagenesis has been successfully used to improve several enzymatic properties as well as thermostability (Andreadeli et al, 2008;Reetz et al 2006;Wu et al, 2009;Zheng, 2004).…”

Alteration of coenzyme specificity of malate dehydrogenase from Streptomyces coelicolor A3(2) by site-directed mutagenesis

“…As a result, when k cat /K m coenzyme value was again considered as a measure of the catalytic efficiency of the enzyme, ScMDH-T4 increased 423.7-fold for NADPH and decreased 5.3-fold for NADH, compared with the case of wild-type ScMDH. When a preference for NADH over NADPH was defined by ( (Tomita et al, 2006b;Andreadeli et al, 2008), it showed 218.67 for wild-type ScMDH and 0.098 for ScMDH-T4 (Table 3). This means that the four-amino-acid replacement shifted the coenzyme specificity of ScMDH 2231.3-fold toward NADPH, which is significantly larger than the shift in TfMDH alteration of coenzyme specificity, namely 1562.6-fold (Tomita et al, 2006b), as shown in Table 3.…”