Crystal structures of Giardia lamblia guanine phosphoribosyltransferase at1.75 angstrom

Giardia lamblia, the protozoan parasite responsible for giardiasis, require s purine salvage from its host for RNA and DNA synthesis. G, lamblia expres ses 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 immuc illinGP 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 unexpe ctedly to a GPRTase.immucillinG binary complex with an open catalytic site loop. Diffusion of ligands into preformed crystals gave a GPRTase immucilli nGP . Mg2+ . pyrophosphate complex in which the open loop is stabilized by crystal contacts. C. lamblia GPRTase exhibits substantial structural differ ences from known purine phosphoribosyltransferases at positions remote from the catalytic site, but conserves most contacts to the bound inhibitor. Th e filled catalytic site with an open catalytic loop provides insight into l igand binding. One active site Mg2+ ion is chelated to pyrophosphate, but t he other is chelated to two conserved catalytic site carboxylates, suggesti ng a role for these amino acids. This arrangement of Mg2+ and pyrophosphate has not been reported in purine phosphoribosyltransferases, ImmucillinG in the binary complex is anchored by its 9-deazaguanine group, and the iminor ibitol is disordered. No Mg2+ or pyrophosphate is detected; thus, the 5'-ph osphoryl 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) b inding the first Mg2+ to Asp125 and Glu126 carboxyl groups and binding Mg2. pyrophosphate, and (4) closing the catalytic site loop and formation of b ound (Mg2+)(2). pyrophosphate prior to catalysis. Guanine specificity is pr ovided by two peptide carbonyl oxygens hydrogen-bonded to the exocyclic ami no group and a weak interaction to O6, Transition state formation involves N7 protonation by Asp129 acting as the general acid.