人二氢乳清酸脱氢酶蛋白的表达、纯化及其与新型抑制剂的晶体结构

Human dihydroorotate dehydrogenase (hDHODH) is a key enzyme that catalyzes the de novo pyrimidine biosynthesis. Recent studies have shown that inhibition of the enzyme can relieve the symptoms of rheumatoid arthritis. Currently there are few inhibitors of the enzyme, so it is of great significance to find an efficient inhibitor of the enzyme. In this study, the hDHODH gene was amplified by PCR. The recombinant plasmid pET-19b-hDHODH was constructed and expressed in Escherichia coli (E.coli) BL21 (DE3) to produce soluble proteins. The protein was purified by Ni²⁺-NTA affinity chromatography to obtain high purity (90%) hDHODH proteins. The hDHODH protein and inhibitor 3-(5-(ethylthio)-1H-1, 2, 4-triazol-3-yl) benzoic acid were mixed and incubated with substrate DHO. The crystal was screened by the Hampton kit and optimized by the chessboard method to obtain a hDHODH protein co-crystal with a perfect crystal shape and strong diffraction ability. The crystal structure was studied by X-ray diffraction, and the structure were analyzed by CCP4 and Coot softwares. Based on the structural analysis, it can be seen that the inhibitor has a very high affinity with protein. The inhibitor formed a hydrogen bond network with the protein at Tyr356 and Tyr147 through the hydrophilic carboxyl terminus and the five-membered ring of the inhibitor interacted with Leu359 and Thr360 of the protein, making the inhibitor firmly bind to the protein. The structural analysis of hDHODH complex crystallization provided an important basis for the development of new specific anti-rheumatoid arthritis drugs.