Crystal structures of Giardia lamblia guanine phosphoribosyltransferase at 1.75 Å

Wuxian Shi, Narsimha R. Munagala, Ching C. Wang, Caroline M. Li, Peter C. Tyler, Richard H. Furneaux, Charles Grubmeyer, Vern L. Schramm, Steven C. Almo

Research output: Contribution to journalArticle

37 Citations (Scopus)

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·Mg2+·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 Mg2+ 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 Mg2+ 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. Mg2+ 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 Mg2+ to Asp125 and Glu126 carboxyl groups and binding Mg2+·pyrophosphate, and (4)closing the catalytic site loop and formation of bound (Mg2+)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.

Original languageEnglish (US)
Pages (from-to)6781-6790
Number of pages10
JournalBiochemistry
Volume39
Issue number23
DOIs
StatePublished - Jun 13 2000

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Hypoxanthine Phosphoribosyltransferase
Giardia lamblia
Catalytic Domain
Crystal structure
Guanine
Ligands
Salvaging
Crystals
Protonation
Enzymes
Giardiasis
Catalysis
Hydrogen
Phosphates
diphosphoric acid
RNA
Ions
Oxygen
Malaria
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Shi, W., Munagala, N. R., Wang, C. C., Li, C. M., Tyler, P. C., Furneaux, R. H., ... Almo, S. C. (2000). Crystal structures of Giardia lamblia guanine phosphoribosyltransferase at 1.75 Å. Biochemistry, 39(23), 6781-6790. https://doi.org/10.1021/bi000128t

Crystal structures of Giardia lamblia guanine phosphoribosyltransferase at 1.75 Å. / Shi, Wuxian; Munagala, Narsimha R.; Wang, Ching C.; Li, Caroline M.; Tyler, Peter C.; Furneaux, Richard H.; Grubmeyer, Charles; Schramm, Vern L.; Almo, Steven C.

In: Biochemistry, Vol. 39, No. 23, 13.06.2000, p. 6781-6790.

Research output: Contribution to journalArticle

Shi, W, Munagala, NR, Wang, CC, Li, CM, Tyler, PC, Furneaux, RH, Grubmeyer, C, Schramm, VL & Almo, SC 2000, 'Crystal structures of Giardia lamblia guanine phosphoribosyltransferase at 1.75 Å', Biochemistry, vol. 39, no. 23, pp. 6781-6790. https://doi.org/10.1021/bi000128t
Shi W, Munagala NR, Wang CC, Li CM, Tyler PC, Furneaux RH et al. Crystal structures of Giardia lamblia guanine phosphoribosyltransferase at 1.75 Å. Biochemistry. 2000 Jun 13;39(23):6781-6790. https://doi.org/10.1021/bi000128t
Shi, Wuxian ; Munagala, Narsimha R. ; Wang, Ching C. ; Li, Caroline M. ; Tyler, Peter C. ; Furneaux, Richard H. ; Grubmeyer, Charles ; Schramm, Vern L. ; Almo, Steven C. / Crystal structures of Giardia lamblia guanine phosphoribosyltransferase at 1.75 Å. In: Biochemistry. 2000 ; Vol. 39, No. 23. pp. 6781-6790.
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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·Mg2+·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 Mg2+ 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 Mg2+ 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. Mg2+ 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 Mg2+ to Asp125 and Glu126 carboxyl groups and binding Mg2+·pyrophosphate, and (4)closing the catalytic site loop and formation of bound (Mg2+)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.",
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AU - Shi, Wuxian

AU - Munagala, Narsimha R.

AU - Wang, Ching C.

AU - Li, Caroline M.

AU - Tyler, Peter C.

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AU - Grubmeyer, Charles

AU - Schramm, Vern L.

AU - Almo, Steven C.

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N2 - 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·Mg2+·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 Mg2+ 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 Mg2+ 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. Mg2+ 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 Mg2+ to Asp125 and Glu126 carboxyl groups and binding Mg2+·pyrophosphate, and (4)closing the catalytic site loop and formation of bound (Mg2+)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.

AB - 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·Mg2+·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 Mg2+ 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 Mg2+ 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. Mg2+ 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 Mg2+ to Asp125 and Glu126 carboxyl groups and binding Mg2+·pyrophosphate, and (4)closing the catalytic site loop and formation of bound (Mg2+)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.

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