Crystal Structures of Giardia lamblia Guanine Phosphoribosyltransferase at 1.75 Å^,

Abstract

Giardia lamblia, the protozoan parasite responsible for giardiasis, requires purine salvage from its host for RNA and DNA synthesis. G. lamblia expresses an unusual purine phosphoribosyltransferase with a high specificity for guanine (GPRTase). The enzyme's sequence significantly diverges from those of related enzymes in other organisms. The transition state analogue immucillinGP is a powerful inhibitor of HGXPRTase from malaria [Li, C. M., et al. (1999) Nat. Struct. Biol. 6, 582-587] and is also a 10 nM inhibitor of G. lamblia GPRTase. Cocrystallization of GPRTase with immucillinGP led unexpectedly to a GPRTase.immucillinG binary complex with an open catalytic site loop. Diffusion of ligands into preformed crystals gave a GPRTase.immucillinGP.Mg(2+).pyrophosphate complex in which the open loop is stabilized by crystal contacts. G. lamblia GPRTase exhibits substantial structural differences from known purine phosphoribosyltransferases at positions remote from the catalytic site, but conserves most contacts to the bound inhibitor. The filled catalytic site with an open catalytic loop provides insight into ligand binding. One active site Mg(2+) ion is chelated to pyrophosphate, but the other is chelated to two conserved catalytic site carboxylates, suggesting a role for these amino acids. This arrangement of Mg(2+) and pyrophosphate has not been reported in purine phosphoribosyltransferases. ImmucillinG in the binary complex is anchored by its 9-deazaguanine group, and the iminoribitol is disordered. No Mg(2+) or pyrophosphate is detected; thus, the 5'-phosphoryl group is needed to immobilize the iminoribitol prior to magnesium pyrophosphate binding. Filling the catalytic site involves (1) binding the purine ring, (2) anchoring the 5'-phosphate to fix the ribosyl group, (3) binding the first Mg(2+) to Asp125 and Glu126 carboxyl groups and binding Mg(2+).pyrophosphate, and (4) closing the catalytic site loop and formation of bound (Mg(2+))(2). pyrophosphate prior to catalysis. Guanine specificity is provided by two peptide carbonyl oxygens hydrogen-bonded to the exocyclic amino group and a weak interaction to O6. Transition state formation involves N7 protonation by Asp129 acting as the general acid.

Virtual Screening of Combinatorial Libraries across a Gene Family: in Search of Inhibitors of Giardia lamblia Guanine Phosphoribosyltransferase

“…The most apparent feature of the predicted binding modes for the two inhibitor series is that in both cases the aromatic ring positions itself in the 5Ј-phosphate binding site, (23). Electrostatic potential surface shown for GPRT (negative potential in red, positive in blue) was generated using MOLCAD (Sybyl, version 6.5; Tripos Associates).…”

“…Purine phosphoribosyltransferases (PRTs) catalyze the Mg 2ϩ -dependent synthesis of purine nucleotides via reaction of a purine base with ␣-D-5-phosphoribosyl-1-pyrophosphate (PRPP). Crystal structures of the type I PRTs share a common Rossman's fold and a hood that is composed primarily of antiparallel ␤-sheets positioned around the enzyme's active site (8,12,(20)(21)(22)(23)28). Inhibitors of PRTs that are able to block purine salvage in vivo could represent an efficient approach to antiparasite chemotherapy (31,32).…”

Biology ofGiardia lamblia

“…The amino acid sequence of the GPRTase enzyme shows less than 20% identity to the human enzyme (311). The crystallographic structure of the G. lamblia GPRTase has suggested possible reasons for the unique substrate of the G. lamblia enzyme, such as an aspartate substitution for leucine at position 181 (303).…”

A phosphoenzyme mimic, overlapping catalytic sites and reaction coordinate motion for human NAMPT

“…Additional interactions are provided by the second monomer (Y18*-␣1*, K415*/K423*-loop between ␤14* and ␤15*) and consist primarily of hydrogen bonds with the PPi moiety of the substrates. This arrangement is proposed to sequester the active site from solvent as discussed for other phosphoribosyltransferases (18)(19)(20)(21).…”