Characterization of a novel thermotolerant NAD+-dependent formate dehydrogenase from hot climate plant cotton (Gossypium hirsutum L.)

Abstract

NAD-dependent formate dehydrogenases (FDH, EC 1.2.1.2), providing energy to the cell in methylotrophic microorganisms, are stress proteins in higher plants and the level of FDH expression increases under several abiotic and biotic stress conditions. They are biotechnologically important enzymes in NAD(P)H regeneration as well as CO reduction. Here, the truncated form of the cDNA was cloned into pQE-2 vector, and overexpressed in DH5α-T1 cells. Recombinant GhFDH1 was purified 26.3-fold with a yield of 87.3%. Optimum activity was observed at pH 7.0, when substrate is formate. Kinetic analyses suggest that GhFDH1 has considerably high affinity to formate (0.76 ± 0.07 mM) and NAD (0.06 ± 0.01 mM). At the same time, the affinity (1.98 ± 0.4 mM) and catalytic efficiency (0.0041) values of the enzyme for NADP show that GhFDH1 is a valuable enzyme for protein engineering studies that is trying to change the coenzyme preference from NAD to NADP which has a much higher cost than that of NAD. Improving the NADP specificity is important for NADPH regeneration which is an important coenzyme used in many biotechnological production processes. The value of GhFDH1 is 53.3 °C and the highest enzyme activity is measured at 30 °C with a half-life of 61 h. Whilst further improvements are still required, the obtained results show that GhFDH1 is a promising enzyme for NAD(P)H regeneration for its prominent thermostability and NADP specificity.

From the Amelioration of a NADP+‐dependent Formate Dehydrogenase to the Discovery of a New Enzyme: Round Trip from Theory to Practice

“…In comparison to Gra FDH, Gra FDH2 showed a 4.5‐fold higher biocatalytic efficiency for formate in presence of NAD + (K M 10.51±1.62 mM, k cat 3.84±0.11 s −1 ) and, as expected, better kinetic constants for NAD + (K M 0.22±0.01 mM, k cat 3.78±0.05 s −1 ). The kinetic characterization highlighted the better performances of Gra FDH2; in particular, its 10‐fold lower K M value for formate is very similar to the best reported recombinant FDHs, whose K M values range between 3 and 10 mM while those for NAD + range between 0.03 and 1.20 mM …”