THE CRYSTAL STRUCTURE OF T.cruzi GLYCOSOMAL GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE: IMPLICATIONS FOR THE CATALYTIC MECHANISM AND NEW POTENTIAL TARGET SITES FOR SELECTIVE INHIBITION. 1Oliva, G.,1,2Souza, D.H.F, 1Araújo, A.P.U., 1Jesus,W.D.P. 1Instituto de Física de São Carlos, USP, Cx.P.369, 13560, São Carlos, SP, Brazil; 2Instituto de Química de São Carlos, USP.
Aiming at the design of specific inhibitors of the enzyme GAPDH from the parasite Trypanosoma cruzi, causative agent of Chagas' disease, the crystallization and structure determination of this enzyme was undertaken. We report here the structure of crystal form I, obtained from wild-type recombinant protein overexpressed in E.coli: protein concentration 9mg/ml in 25mM Tris pH 7.8, 0.5M Ammonium Sulphate, 2mM NAD, 1mM Azide, 1mM EDTA and 1mM DTT, and reservoir solution 18% PEG 8000, 0.2M calcium acetate and 0.1 M cacodylate, at pH 6.5. Space group P1, a=88.23 Å, b=124.49 Å, c=85.35 Å, [[alpha]]=101.32¡, [[beta]]=112.82¡, [[gamma]]=83.65¡, two tetramers in the asymmetric unit, diffraction data collected to 2.8 Å, completeness 67%. The structure was solved by molecular replacement and refined by simulated annealing with strict 8-fold non-crystallographic symmetry. The final model included 92 water molecules per monomer and resulted in an Rfactor of 20.1%, Rfree=22.3%, with good geometry indicators. The structure is the first of the GAPDH family solved without any ions at the active site. This feature has resulted in a large change in the side chain conformation of active site Arg249, which salt-bridges to Asp210 in our structure. We propose that this could be important for the reaction mechanism, possibly a common feature of all other 7 known GAPDH structures. Comparison with the human enzyme structure also indicate new potential target sites for specific inhibitor design.
Acknowledgements: WHO (TDR grant 940854), PADCT/SBIO , CNPq, FAPESP and FINEP.