Purification and catalysis of choline dehydrogenase from Escherichia coli

Choline dehydrogenase (CHDH) is a membrane-bound enzyme belonging to the glucose-methanol-choline (GMC) oxidoreductase superfamily, which is characterized by a crucial FAD-binding domain essential for catalytic function. CHDH catalyzes the oxidation of choline to betaine aldehyde, which is further oxidized to betaine, a vital osmoprotectant and methyl donor for cellular physiology and metabolism. However, the detailed catalytic mechanism of CHDH still remains poorly understood. In our investigation, we gained purity E. coli CHDH samples in DDM (n-dodecyl-β-D-maltoside) and SMA (styrene maleic acid) copolymer respectively and examined their structural composition and catalytic activity separately. Our findings demonstrated the effectiveness of SMA, commonly employed for extracting transmembrane proteins and can preserve the natural bio-membrane environment surrounding the enzyme, in extracting peripheral membrane proteins like CHDH here, which lacks transmembrane helices. CHDH exhibited a trimeric conformation in SMA, whereas it existed as monomers in DDM, as determined by our negative staining analysis. Our experiments also revealed that highly pure E. coli CHDH could only oxidize choline to betaine aldehyde but failed to further oxidize betaine aldehyde to betaine as determined by the biochemical and enzymatic reaction kinetic assays. In addition, the enzyme in SMA displayed greater catalytic activity compared to that in DDM. Furthermore, we confirmed the crucial role of His473, which is hypothesized to be a critical site for substrate binding from our structural comparative analysis between CHDH and its highly homologous choline oxidase, in the catalytic activity of the enzyme through gene mutation. Our work also sheds light on CHDH's contribution to cellular osmotic tolerance through gene knockout. This research enhances our better understanding of CHDH within cellular biochemistry and metabolic pathways.