Crystal Structure of Glycosomal Glyceraldehyde-3-phosphate Dehydrogenase from Leishmania mexicana: Implications for Structure-Based Drug Design and a New Position for the Inorganic Phosphate Binding Site

Abstract

The structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the trypanosomatid parasite Leishmania mexicana has been determined by X-ray crystallography. The protein crystallizes in space group P2(1)2(1)2(1) with unit cell parameters a = 99.0 A, b = 126.5 A, and c = 138.9 A. There is one 156,000 Da protein tetramer per asymmetric unit. The model of the protein with bound NAD+s and phosphates has been refined against 86% complete data from 10.0 to 2.8 A to a crystallographic Rfactor of 0.198. Density modification by noncrystallographic symmetry averaging was used during model building. The final model of the L. mexicana GAPDH tetramer shows small deviations of less than 0.5 degrees from ideal 222 molecular symmetry. The structure of L. mexicana GAPDH is very similar to that of glycosomal GAPDH from the related trypanosomatid Trypanosoma brucei. A significant structural difference between L. mexicana GAPDH and most previously determined GAPDH structures 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. A new inorganic phosphate position is observed in the L. mexicana GAPDH structure. Model building 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. Since crystals of L. mexicana GAPDH can be grown reproducibly and diffract much better than those of T. brucei GAPDH, L. mexicana GAPDH will be used as a basis for structure-based drug design targeted against trypanosomatid GAPDHs.

Protein structure-based design of anti-protozoal drugs

“…This proved to be a good strategy as our subsequent structure determination of the L. mexicana enzyme showed that their adenosine pockets are virtually identical, with a 0.2 Å rms deviation on backbone and 0.5 Å on side chain atom positions; the only amino acid difference is the replacement of Asn 39 of T.brucei GAPDH by a Ser in L.mexicana. 16 As expected, L.mexicana GAPDH inhibition by our new inhibitors was nearly identical to the T.brucei results.…”

Selective Tight Binding Inhibitors of Trypanosomal Glyceraldehyde-3-phosphate Dehydrogenase via Structure-Based Drug Design

“…Electrospray ionization (ESI) mass spectra were obtained on a Kratos Profile HV4 mass spectrometer. All target compounds were shown to be pure by reverse-phase HPLC on C 4 33,36 NAD + and glyceraldehyde-3-phosphate diethyl acetal were purchased from Sigma. Fresh glyceraldehyde-3-phosphate was prepared from its diethyl acetal according to the instructions provided by the manufacturer.…”

What Controls Glycolysis in Bloodstream Form Trypanosoma brucei?

“…Until now the design and synthesis of inhibitors of trypanosome glycolysis has been focused on the enzymes GAPDH (62)(63)(64)(65)(66)(67), PGK (68), and ALD (63). According to our model calculations, inhibition of these steps should be less effective than inhibition of glucose transport but far more effective than inhibition of HK, PFK, or PYK.…”