Characterization of a novel thermostable NAD+-dependent formate dehydrogenase from Methylacidiphilum kamchatkense Kam1 (MkaFDH)

Abstract

Metal-independent NAD⁺-dependent formate dehydrogenases (FDHs) are enzymes responsible for catalyzing the conversion of formate (HCOO^–) to carbon dioxide (CO₂), a biological reaction involved in microbial carbon processing and cofactor regeneration. These enzymes show large potential for environmental bioremediation and biotechnological uses. However, FDHs applications are hampered by the enzymes’ limited stability under extreme conditions, such as high temperatures or extreme pH. Therefore, we aimed to identify and characterize novel metal-independent FDHs with improved activity and thermostability compared to known FDHs. By using four different FDH protein sequences, CtFDH (from Chaetomium thermophilum), MtFDH (from Myceliophthora thermophile), OpFDH (from Ogata parapolymorpha DL-1) and PseFDH (from Pseudomonas sp.101) we retrieved 18,850 FDHs sequences from the NCBI database and matched against the species present in the database of thermophilic bacteria, ThermoBase. Our phylogenetic analysis identified four distinct FDHs in thermophilic bacteria: Methylocaldum szegediense (MszFDH), Methylacidiphilum kamchatkense (MkaFDH), Mycobacterium arosiense (MarFDH) and Mycobacterium genavense (MgeFDH). We selected and characterized the MkaFDH as it was expressed in the thermophilic bacterium with the highest optimum growth (55 °C) among the four bacteria. The MkaFDH was cloned, and the recombinant protein was expressed in E. coli and purified. The conditions for the optimal catalytic activity for formate oxidations were screened and identified, revealing metal-independent, NAD⁺-restricted activity in phosphate buffer, pH 8. Importantly, the enzyme showed remarkable thermal stability and catalytic activity, showing a melting temperature (Tm) of 60.15 °C, as confirmed by far-UV circular dichroism (CD). Finally, the enzyme showed good thermostability for formate oxidation up to 57.5 °C, and its high catalytic efficiency (k_cat/K_m = 0.44 s⁻¹mM⁻¹) suggested its potential industrial application. Collectively, we describe here a novel FDH with relevant thermostability that can be exploited as a prototype for industrial applications.