Deciphering the Molecular Mechanism of Formate Dehydrogenase in the CO2-reducing Acetogen, Eubacterium callanderi KIST612

Abstract

Metal-dependent formate dehydrogenase (FDH) is a promising biocatalyst for bioelectrocatalytic CO2 reduction. Owing to its metabolic diversity, FDH exhibits various structural and functional types across various microorganisms. However, studying this enzyme remains challenging due to factors such as oxygen sensitivity and the complexity of its structure, which hinder detailed functional characterization and make it difficult to identify the specific molecules involved in its mechanism. In this study, we aimed to characterize FDH from Eubacterium callanderi KIST612 and to identify the key molecules and cofactors involved in its mechanism. As a result, we specifically identified two fdh genes (ELI_0994 and ELI_3306) that encode the characteristic sequence from the putative gene cluster and categorized them into different types based on three points of consideration. i.e., subunit composition, presence of chaperon in the operon, and cofactor. Evaluations classified ELI_0994 as type 1, while ELI_3306 falls under type 6. Gene-cluster analysis suggests that these two FDH systems may operate through distinct mechanisms. The major difference between them appears to lie in the location of the electron-bifurcation center: a hydrogenase in the case of ELI_0994 (supported by similarity to A.woodii), and FMN for ELI_3306 (tentatively proposed). Across both systems, the key molecules implicated in their function include NADH, ferredoxin, FMN, the molybdenum cofactor, and hydrogenase. These predicted mechanisms may aid in selecting suitable FDH candidates for future applications. Keywords: Formate dehydrogenase; CO2 reduction; Molecular mechanism; Eubacterium callanderi KIST612