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Formate dehydrogenase from Candida boidinii (CbFDH) reversibly catalyzes formate to CO2 with the redox couple NAD+/NADH. While many studies on CbFDH-catalyzed formate oxidation in the presence of NAD+ are reported, there are few studies on the detailed mechanism of the CbFDH-catalyzed CO2 reduction in the presence of NADH. In this study, the detailed mechanism of formate production by CbFDH with NADH in GTA buffer solution (consisting of 3,3-dimethylglutaric acid, tris(hydroxymethyl)aminomethane and 2-amino-2-methyl-1,3-propanediol) with different proportions of CO2, bicarbonate and carbonate was studied. A good correlation was found between the pH of the buffer solution, the CO2 concentration and the apparent initial rate of formate production with CbFDH. Under constant conditions of total carbonate concentration in the buffer solution, the process of the CbFDH-catalyzed reduction of CO2 to formate followed the Michaelis–Menten kinetics. Particularly, the reaction rate of formate production with CbFDH increased with increasing CO2 concentration under constant conditions of total carbonate concentration in the solution. As the apparent formate production rate with CbFDH increases under higher total carbonate concentration, bicarbonate or carbonate does not act as a competitive inhibitor of CO2, and it is predicted that the CO2 concentration equivalent consumed in the CbFDH-catalyzed formate production is supplied by bicarbonate or carbonate by the pH control function of the GTA buffer solution. Thus, we have clarified for the first time that these carbonates do not function as any inhibitor for the CbFDH-catalyzed CO2 reduction. [ABSTRACT FROM AUTHOR]

Formate dehydrogenase from Candida boidinii (CbFDH) reversibly catalyzes formate to carbon dioxide with the redox couple NAD+/NADH. There have been many studies on CbFDH-catalyzed oxidation of formate to carbon dioxide in the presence of NAD+. On the other hand, there are few studies on the detailed mechanism of the carbon dioxide reduction to formate catalyzed by CbFDH in the presence of NADH. In addition, it is not clear whether CbFDH can reduce carbon dioxide, bicarbonate or carbonate to formate. In this study, formate production with CbFDH in the presence of NADH was investigated in solutions with different ratios of carbon dioxide, bicarbonate and carbonate. The reaction rate of formate production with CbFDH increased in proportion to the concentration of carbon dioxide in the reaction solution. On the other hand, the formate production with CbFDH was suppressed in proportion to the concentration of bicarbonate or carbonate in the reaction solution. Thus, CbFDH was found to catalytically reduce only carbon dioxide to formate among the three types of carbonate species. [ABSTRACT FROM AUTHOR]