Engineering of formate dehydrogenase: synergistic effect of mutations affecting cofactor specificity and chemical stability

Abstract

Formate dehydrogenases (FDHs) are frequently used for the regeneration of cofactors in biotransformations employing NAD(P)H-dependent oxidoreductases. Major drawbacks of most native FDHs are their strong preference for NAD(+) and their low operational stability in the presence of reactive organic compounds such as α-haloketones. In this study, the FDH from Mycobacterium vaccae N10 (MycFDH) was engineered in order to obtain an enzyme that is not only capable of regenerating NADPH but also stable toward the α-haloketone ethyl 4-chloroacetoacetate (ECAA). To change the cofactor specificity, amino acids in the conserved NAD(+) binding motif were mutated. Among these mutants, MycFDH A198G/D221Q had the highest catalytic efficiency (k cat/K m) with NADP(+). The additional replacement of two cysteines (C145S/C255V) not only conferred a high resistance to ECAA but also enhanced the catalytic efficiency 6-fold. The resulting quadruple mutant MycFDH C145S/A198G/D221Q/C255V had a specific activity of 4.00 ± 0.13 U mg(-1) and a K m, NADP(+) of 0.147 ± 0.020 mM at 30 °C, pH 7. The A198G replacement had a major impact on the kinetic constants of the enzyme. The corresponding triple mutant, MycFDH C145S/D221Q/C255V, showed the highest specific activity reported to date for a NADP(+)-accepting FDH (v max, 10.25 ± 1.63 U mg(-1)). However, the half-saturation constant for NADP(+) (K m, NADP(+) , 0.92 ± 0.10 mM) was about one order of magnitude higher than the one of the quadruple mutant. Depending on the reaction setup, both novel MycFDH variants could be useful for the production of the chiral synthon ethyl (S)-4-chloro-3-hydroxybutyrate [(S)-ECHB] by asymmetric reduction of ECAA with NADPH-dependent ketoreductases.

Cofactor engineering for advancing chemical biotechnology

“…Formate dehydrogenase, which oxidizes formate into carbon dioxide, is widely used to regenerate reduced cofactors. The FDH from Mycobacterium vaccae N10 was mutated on the conserved NAD + binding motif to give a NADPH dependent activity [58] more resistant to haloketones.…”

“…The synthetic biology revolution has allowed easier ways to construct combinations of redox complexes and connect them to give a focused controllable circuit that can be directed to a desired metabolite [18]. Advances in protein engineering also have helped direct cell redox metabolism toward desired ends [6,[19][20][21][22][23]. These advances intertwine with the theme of generating effective bioprocesses for fuels and chemicals from biomass and more recently from H 2 /CO 2 or electrodes [24,25].…”

The role of formate in combatting oxidative stress

“…These enzymes have been reported with differing cofactor requirements, electron acceptors, substrates and cellular locations (Hourton Cabassa et al, 1998). The FDHs NAD-dependent which have been extensively studied in both bacteria, yeast and plants, have been widely utilized in industry for NADH regeneration (Alekseeva, Savin & Tishkov, 2011;Hoelsch et al, 2013;Suzuki et al, 1998) …”

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

“…1). This indication derived from the sequencing of mutants displaying improvement in overall catalytic efficiency with NADP + , obtained by saturation mutagenesis or other mutagenesis approaches in different laboratories (Serov et al 2002;Andreadeli et al 2008;Wu et al 2009b;Hoelsch et al 2013;Ihara et al 2013).…”

“…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).…”