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.