CRYSTAL STRUCTURE OF GLYCOSOMAL GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM LEISHMANIA MEXICANA: MECHANISTIC IMPLICATIONS AND POTENTIAL DRUG BINDING SITES. Hidong Kim^1,2, Christophe L.M.J. Verlinde¹, Alexander Aronov³, Michael H. Gelb³, Piet Herdewijn⁴, and Wim G. J. Hol^1,2, ¹Departments of Biological Structure and Biochemistry, and Biomolecular Structure Center, University of Washington, Seattle, Washington, ²Howard Hughes Medical Institute, University of Washington, Seattle, Washington, ³Department of Chemistry, University of Washington, Seattle, Washington, ⁴Laboratory of Medicinal Chemistry, Rega Institute, Catholic University of Leuven, Leuven, Belgium

The structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with bound NAD+ from the trypanosomatid parasite Leishmania mexicana has been determined by X-ray crystallography. The protein crystallizes in space group P2₁2₁2₁ (a = 99.0 Å, b = 126.5 Å, c = 138.9 Å) with one 156 kDa protein tetramer per asymmetric unit. Density modification by four-fold noncrystallographic symmetry averaging was used during model building and refinement. The overall structure of L. mexicana GAPDH is similar to previously determined structures of GAPDHs from other species, including the closely related trypanosomatid Trypanosoma brucei, the causative agent of African sleeping sickness. A significant structural difference between L. mexicana GAPDH and most other GAPDHs occurs in a loop region located at the active site. This unusual loop conformation in L. mexicana GAPDH occludes the inorganic phosphate binding site which has been seen in previous GAPDH structures, and results in a new inorganic phosphate position in L. mexicana GAPDH. Modelling studies indicate that this new anion binding site is well situated for nucleophilic attack of the inorganic phosphate on the thioester intermediate in the GAPDH-catalyzed reaction. In addition, L. mexicana GAPDH is being used as a target for structure-based design of trypanocidal compounds. The structures of GAPDHs from L. mexicana and T. brucei have been used to develop a new inhibitor which is nearly four orders of magnitude more potent than its lead compound.