Abstract
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic pathway that catalyzes the conversion of D-glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate. Here, the full-length GAPDH type 1 from Escherichia coli (EcGAPDH1) was cloned and overexpressed, and the protein was purified. Biochemical analyses found that the optimum reaction temperature and pH of EcGAPDH1 were 55°C and 10.0, respectively. The protein has a certain amount of thermostability. Crystals of EcGAPDH1 were obtained using the sitting-drop vapor-diffusion technique and X-ray diffraction data were collected to 1.88 Å resolution. Characterization of the crystals showed that they belonged to space group P4(1)2(1)2, with unit-cell parameters a = b = 89.651, c = 341.007 Å, α = β = γ = 90°. The structure of EcGAPDH1 contains four subunits, each of which includes an N-terminal NAD(+)-binding domain and a C-terminal catalytic domain. Analysis of the NAD(+)-bound form showed some differences between the structures of EcGAPDH1 and human GAPDH. As EcGAPDH1 shares 100% identity with GAPDH from Shigella sonnei, its structure may help in finding a drug for the treatment of shigellosis.