A 1.4 Å Crystal Structure for the Hypoxanthine Phosphoribosyltransferase of Trypanosoma cruzi^,

Abstract

The hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi, etiologic agent of Chagas' disease, was cocrystallized with the inosine analogue Formycin B (FmB) and the structure determined to 1.4 A resolution. This is the highest resolution structure yet reported for a phosphoribosyltransferase (PRT), and the asymmetric unit of the crystal contains a dimer of closely associated, nearly identical subunits. A conserved nonproline cis peptide in one active-site loop exposes the main-chain nitrogen to the enzyme active site, while the adjacent lysine side chain interacts with the other subunit of the dimer, thereby providing a possible mechanism for communication between the subunits and their active sites. The three-dimensional coordinates for the invariant Ser103-Tyr104 dipeptide are reported here for the first time. These are the only highly conserved residues in a second active-site loop, termed the long flexible loop, which is predicted to close over the active site of HPRTs to protect a labile transition state [Eads et al. (1994) Cell 78, 325-334]. This structure represents a major step forward in efforts to design/discover potent selective inhibitors of the HPRT of T. cruzi.

Structural and functional analysis of mutations at the human hypoxanthine phosphoribosyl transferase (HPRT1) locus

“…Indeed, the high polarity and charges in the interfaces explains the experimentally-observed dependency of the dimer-tetramer transition on the ionic strength of the solvent [Johnson et al, 1979;Strauss et al, 1978]. There are a few HPRT structures solved for other species, and the two structures with bound transition-state-like ligands were all tetrameric [Shi et al, 1999a, b], while the product/substrate binding structures can be either dimer [Balendiran et al, 1999;Focia et al, 1998aFocia et al, , 1998bSomoza et al, 1996] or tetramer [Guddat et al, 2002;Heroux et al, 1999a, b]. It is tempting to conclude that HPRT goes through a dimer-tetramer-dimer cycle during the catalysis.…”

Interactions at the Dimer Interface Influence the Relative Efficiencies for Purine Nucleotide Synthesis and Pyrophosphorolysis in a Phosphoribosyltransferase

“…Furthermore, the cis-peptide linkage enables the Lys68 side-chain to interact with residues in the opposing subunit of the dimer. 13,14 These interactions can be seen in the structures of the HPRTs from humans, 18 Plasmodium falciparum (etiologic agent of human malaria), 19 Toxoplasma gondii (etiologic agent of toxoplasmosis), 16 and T. cruzi. 14 In all of these cases, the side-chain of the active-site loop I lysine residue forms a hydrogen bond (in at least one of the subunits of the dimer) with the main-chain carbonyl group of a residue (analogous with the human Val96) in the opposing dimer subunit.…”

Purine Phosphoribosyltransferases

“…There have been several suggestions for the functional role of the cispeptide and the lysine (Lys-68) in active site loop I of HPRTs (20,23,24). The presence of the cis-peptide enables the carbonyl oxygen and amide nitrogen, adjacent to the peptide bond, to interact with pyrophosphate atoms and a metal-associated water molecule when they are present in the active sites of HPRTs (21)(22)(23)25).…”