Clostridium beijerinckii glyceraldehyde-3-phosphate dehydrogenase GAPDH: purification, crystallization, and X-ray crystallographic analysis

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous and highly abundant glycolytic enzyme.It plays a pivotal role for the energy and carbon metabolism of most organisms including industrial bacteria.It catalyzes the two step oxidative phosphorylation of D-glyceraldehyde-3-phosphate into 1,3-bisphosphoglycerate using inorganic phosphate and nicotinamide adenine dinucleotide (NAD + ) as cofactor.In this study, GAPDH from C. beijerinckii (CbGAPDH) was successfully expressed and purified using Ni-NTA affinity, Q anion-exchange, and gel-filtration chromatography.The protein crystal was obtained and diffracted to a resolution of 1.60 Å.The crystal belonged to the hexagonal space group P6 2 22, with unit-cell parameters of a = 120.6,b = 120.6,and c = 122.1 Å.The Matthews coefficient and solvent content were estimated to be 3.50 Å 3 Da -1 and 64.90%, respectively, assuming that the asymmetric unit contained only one recombinant protein molecule.