Structural studies of NAD+-dependent methanol dehydrogenase 1 from Bacillus methanolicus MGA3.

Methanol, a sustainable and cost-effective C1 compound, has been considered as a promising substrate for the biosynthesis of fuels and value-added chemicals. Synthetic methylotrophs have been developed by integrating natural methanol-assimilation pathways into non-native microbial hosts, with NAD+-dependent methanol dehydrogenases (MDHs) serving as attractive candidates for methanol oxidation. NAD+-dependent MDH1 from the methylotrophic bacterium Bacillus methanolicus MGA3 (BmMDH1) is one of the extensively studied MDHs. Although structural models of BmMDH1 had been proposed, its crystal structure had not been experimentally determined. In this study, the crystal structure of BmMDH1 is reported at 3.0 Å resolution. BmMDH1 forms a decamer made up of five dimers, stabilized by ionic and hydrogen-bonding interactions. Each monomer exhibits a conserved fold which is typical of the type III alcohol dehydrogenase family, comprising an N-terminal α/β dinucleotide-binding domain and a C-terminal all-α helical domain. Similar to other enzymes in this family, it has an NAD+-binding site formed by a Rossmann fold. As a metalloenzyme, BmMDH1 features a metal ion in its active site, coordinated by three histidine residues (His197, His262 and His276) and one aspartate residue (Asp193). Enzyme-activity assays identified Mn2+ as the most effective metal ion for supporting in vitro enzymatic activity. These findings provide essential structural insights for the rational engineering of methanol-utilizing biocatalysts, thereby advancing sustainable microbial biomanufacturing.