Protozoan parasite Cryptosporidium parvum
is a common cause of water-borne diseases throughout the world [1
]. There is no effective anti-cryptosporidial therapy [2
]. Cryptosporidium infection can be lethal in immunocompromised individuals, and therefore it poses a particularly serious problem in patients with AIDS [4
]. Despite the urgent need for identification of novel drug targets, very little is known about the metabolic enzymes of this relatively newly recognized human pathogen. The parasite apparently relies mainly on the anaerobic oxidation of glucose for energy production [5
]. Therefore, glycolytic enzymes are of considerable interest as possible targets for anti-cryptosporidial drugs. One glycolytic enzyme, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), has been considered a candidate for inhibition of protozoan parasites [9
]. By exploiting the differences in the structure of the parasitic and human GAPDH, specific inhibitors of trypanosomatid GAPDH have been designed [11
]. However, the Cryptosporidium enzyme has not been characterized previously.
GAPDH plays an essential role in glycolysis by catalyzing the reversible two step oxidative phosphorylation of D-glyceraldehyde 3-phosphate (D-G3H) into 1,3-diphosphoglycerate using NAD (or NADP) as a cofactor [12
]. In the first step D-G3H is covalently attached to the active site cysteine residue via nucleophilic attack on the carbonyl group of D-G3H, resulting in the formation of a thiohemiacetal intermediate. This is followed by a hydride transfer from the thiohemiacetal to NAD, leading to the formation of a thioacyl enzyme. Finally, the resulting thioester is phosphorylated through the nucleophilic attack of an inorganic phosphate ion (Pi) on the carbonyl carbon atom of the thioacyl group, which leads to the formation of 1,3-diphosphoglycerate.
The three-dimensional structures of GAPDH from a number of mammalian, bacterial and parasitic species have been determined [13
]. GAPDHs exist as a homotetrameric protein. Based on the location of sulfate ions originating from the crystallization medium, two anion binding sites were initially identified in each GAPDH subunit [19
]. These sites are labeled 'Ps', corresponding to the binding site for the C-3 phosphate of D-G3H, and 'Pi', corresponding to the binding site for the inorganic phosphate. The 'Ps' site is conserved in various GAPDH structures. However, two possible 'Pi' sites have been proposed [17
]. The original Pi site was identified in crystals grown from ammonium sulfate solutions and was based on the position of the sulfate ion [19
]. The second 'Pi' site was identified by Kim et al [17
] in crystals grown from a phosphate-buffered medium. This latter site, referred to as the 'new Pi' site, is 2.9 Å from the original 'Pi' site.
Skarżyñski et al [19
] proposed that the 'Pi' site is the location of the inorganic phosphate in the phosphorylation step. According to their 'flip-flop' hypothesis, the C-3 phosphate of the substrate binds first to the 'Pi' site in the acylation step and then flips from the 'Pi' to the 'Ps' site during the phosphorylation step. This mechanism is supported by kinetic studies [22
] as well as structural studies of the E. coli
GAPDH in which the substrate was covalently linked to the enzyme in a hemiacetal form [16
] and there was no cofactor. The crystal structure of Trypanosoma cruzi
GAPDH covalently bound to a substrate analogue also provided strong support for this model [14
]. In the E. coli
and T. cruzi
enzyme structures, the 'Pi' site was localized to the 'new Pi' site.
However, in the Bacillus stearothermophilus
GAPDH (BsGAPDH) ternary complex with NAD and D-G3H, the C-3 phosphate group of the non-covalently bound substrate was located in the 'Ps' site, and Didierjean et al [15
] proposed that this structure represented the productive Michaelis complex. The substrate was found in the same conformation in two ternary complexes that were prepared by using different active site mutant (Cys→Ala and Cys→Ser) forms of BsGAPDH. In a recently reported crystal structure of the thioacyl intermediate of wild type BsGAPDH the C-3 phosphate is found (with partial occupancy) in the 'new Pi' site [23
] in each subunit.
We have determined the crystal structure of C. parvum
GAPDH (CpGAPDH) in a substrate free state (apo), in the NAD-bound state (holo) and in a ternary complex with NAD and D-G3H. At no stage during purification and crystallization was the protein exposed to sulfate or phosphate ions. As a result there is no sulfate or phosphate ion in any of the CpGAPDH structures. In order to form the ternary complex with the physiological substrate, the nucleophilic cysteine residue of the active site (C153), was substituted with serine following the strategy employed by Didierjean et al [15
] to form the ternary complex of BsGAPDH. However, in contrast to their results, in three subunits of the CpGAPDH tetramer the C-3 phosphate is located at the 'new Pi' site and not in the 'Ps' site. In the remaining subunit, an unexpected new binding site for this phosphate is revealed. According to the accepted mechanism of enzymatic action the conformation of the substrate in two subunits (C and D) appears to be unproductive.