Study On The Structure And Function Of Inorganic Pyrophosphatase From Schistosoma Japonicum

Background：Inorganic pyrophosphatase (PPase) is an enzyme catalyzing the hydrolysis of pyrophosphate(PPi) given two phosphates (Pi). As a byproduct of biosynthesis of macromolecules, the hydrolysis of PPiprovides a thermodynamic pull for synthesis reactions of DNA, RNA, protein and glycogen. In addition,PPi is also one of product from sulfate reduction. The hydrolysis of PPi is resulted in the increasing of thereducing sulfate for these synthesises of cysteine, homocysteine and methionine.Objectives：To establish a set of methods for recombinant expression and purification of PPase fromSchistosoma japonicum, and further to know the enzyme activity of SjPPase and obtain its spatial structureby crystallography, which will provide the molecular basis for function investigation of SjPPase.Methods：The gene encoding PPase from Schistosoma japonicum was constructed in pET-28a plasmid, andthe recombinant plasmid were transformed into E.coli strain BL21(DE3). Bacteria harboringSjPPase/pET-28a were cultured in LB and induced by IPTG. The expression product were lysed byultrasonic wave, then the supernatant was primary purified by Ni-NTA column and secondary purified byDEAE column. The activity of pure protein catalyzing the hydrolysis of PPi was determinated bycolorimetric analysis under various conditions. We investigated the PPase state in solution usingcross-linking experiment，and surveyed its interactions with some small molecules with Native-PAGEmethod, and observed the sensitivity and specificity of SjPPase regarded as a diagnostic antigen forschistosomiasis by ELISA. The screening of crystal growth conditions was conducted by gas-liquiddiffusion using96-well sitting-drop plates and the optimization was performated by changing the additionand precipitant concentration or pH value. The structures were resolved by X-ray diffraction and dataanalysis.Results：The purify of recombinant SjPPase we obtained is higher than95%and the enzyme activity underdifferent temperature and pH is investigated. The optimized temperature and optimum pH are about60℃and7.0, respesctively. The IC50of the three inhibitors (NaF, MDP and IDP) are1.92mM,3.72mM and4.97mM in turn. SjPPase is multimer in solution and the multimer is consisted by some dimers. Divalentmetal ion Mg2+and cysteines in SjPPase are contribute to improving the stability of the structure ofSjPPase. There are else subtrate-binding sites in SjPPase except active sites. Finally, we solved the structureby X-Ray diffraction.