Engineered formate dehydrogenase from Chaetomium thermophilum, a promising enzymatic solution for biotechnical CO2 fixation.

OBJECTIVES: Formate dehydrogenases (FDHs) are NAD(P)H-dependent enzymes that catalyse the reversible oxidation of formate to CO2. The main goal was to use directed evolution to obtain variants of the FDH from Chaetomium thermophilum (CtFDH) with enhanced reduction activity in the conversion of CO2 into formic acid. RESULTS: Four libraries were constructed targeting five residues in the active site. We identified two variants (G93H/I94Y and R259C) with enhanced reduction activity which were characterised in the presence of both aqueous CO2(g) and HCO3-. The A1 variant (G93H/I94Y) showed a 5.4-fold increase in catalytic efficiency (kcat/KM) compared to that of the wild-type for HCO3- reduction. The improved biocatalysts were also applied as a coupled cofactor recycling system in the enantioselective oxidation of 4-phenyl-2-propanol catalysed by the alcohol dehydrogenase from Streptomyces coelicolor A3 (ScADH). Conversions in these reactions increased from 56 to 91% when the A1 variant was used instead of wild-type CtFDH. CONCLUSIONS: Two variants presenting up to five-fold increase in catalytic efficiency and kcat were obtained and characterised. They constitute a promising enzymatic alternative for CO2 utilization and will serve as scaffolds to be further developed in order to meet industrial requirements.

Functional effects of active site mutations in NAD+-dependent formate dehydrogenases on transformation of hydrogen carbonate to formate.

Conversion of hydrogen carbonate to formate by mutants of Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) formate dehydrogenases (FDHs) was studied. Hydrogen carbonate is not the primary substrate for the hydride transfer reaction in FDHs. The chosen mutations were selected so that enzyme activity could remain at an adequate level. In CtFDH, the mutation Asn120Cys in the active site inactivated the enzyme for formate (oxidation) but increased the specific activity for hydrogen carbonate (reduction) as a function of substrate concentration. The mutation Asn120Cys in CtFDH increased 6.5-fold the KM, indicating that substrate binding was weakened. A 6.5-fold increase of kcat compensated the lower affinity suggesting that product release was improved. The corresponding mutation Asn119Cys in CmFDH inactivated the enzyme for both substrates. Molecular dynamics simulations indicated that the active site dimensions change differently with different substrates after mutations, and in the mutant Asn120Cys of CtFDH, hydrogen carbonate adopted better reactive position than formate. With hydrogen carbonate, the active site enlarged enough for two hydrogen carbonate molecules to be placed there. The change of Asn119 to bulky Tyr or His in CmFDH requires changes in the active site to accommodate the substrate; activity with formate was retained but not with hydrogen carbonate. This study showed that the active site of FDHs can be modified radically, which gives possibilities for further enzyme engineering to improve the reaction with hydrogen carbonate or carbon dioxide for enzymatic fixing of carbon dioxide.

Chaetomium thermophilum formate dehydrogenase has high activity in the reduction of hydrogen carbonate (HCO3 -) to formate.

While formate dehydrogenases (FDHs) have been used for cofactor recycling in chemoenzymatic synthesis, the ability of FDH to reduce CO2 could also be utilized in the conversion of CO2 to useful products via formate (HCOO-). In this study, we investigated the reduction of CO2 in the form of hydrogen carbonate (HCO3-) to formate by FDHs from Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) in a NADH-dependent reaction. The catalytic performance with HCO3- as a substrate was evaluated by measuring the kinetic rates and conducting productivity assays. CtFDH showed a higher efficiency in converting HCO3- to formate than CmFDH, whereas CmFDH was better in the oxidation of formate. The pH optimum of the reduction was at pH 7-8. However, the high concentrations of HCO3- reduced the reaction rate. CtFDH was modeled in the presence of HCO3- showing that it fits to the active site. The active site setting for hydride transfer in CO2 reduction was modeled. The hydride donated by NADH would form a favorable contact to the carbon atom of HCO3-, resulting in a surplus of electrons within the molecule. This would cause the complex formed by hydrogen carbonate and the hydride to break into formate and hydroxide ions.