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 CO₂ reduction. Here, the truncated form of the Gossypium hirsutum fdh1 cDNA was cloned into pQE-2 vector, and overexpressed in Escherichia coli 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 T_m 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.

Keywords: Coenzyme regeneration; Formate dehydrogenase; Gossypium hirsutum; NADP+ specificity; Thermostability.

Fig. 1

Amino acid Blast analysis of GhFDH1: sequence alignments of GhFDH1 with other FDHs which have knowledge of crystallographical structure (Arabidopsis thaliana FDH (AtFDH), Pseudomonas 101 sp. FDH (PsFDH), Candida biodinii FDH (CbFDH), and Glycine max FDH (GmFDH) were determined). Conserved residues are highlighted in red and blue frames and indicate that more than 70% of the residues are similar according to physico-chemical properties

Fig. 2

Overexpression and His-tag purification of GhFDH1: expression of GhFDH1 in E. coli DH5αT1. 1. unstained protein marker, broad range (2-212 kDa; NEB); 2. crude extract from uninduced cells; 3. crude extract from induced cells (a). Purified protein with Ni–NTA resin, 1. Elute #3, 2. Elute #2, 3. Elute #1; 4. precision plus unstained protein standards (10-250 kDa; Bio-Rad)

Fig. 3

Heat inactivation of GhFDH1: the midpoint of thermal inactivation (T_m) value was determined from the plot of residual activity versus temperature and determined as 53.3 °C. 100% residual activity value is 1.06 ± 0.015 U mg⁻¹

Fig. 4

Homology model of truncated GhFDH1: apo-form of GhFDH1 constructed using AtFDH (pdb code: 3NAQ_A) as template (a). Important residues that are involved in substrate binding at the active site of GhFDH1 are shown on the model constructed using ligand bound AtFDH NAD⁺–azide ternary complex (pdb code: 3N7U) as template. Azide mimics the substrate (formate) in the transition state of the reaction (b)