Crystal Structure of Toxoplasma gondii Hypoxanthine-Guanine Phosphoribosyltransferase with XMP, Pyrophosphate, and Two Mg²⁺ Ions Bound:  Insights into the Catalytic Mechanism^,

Abstract

The crystal structure of the Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-xanthosine 5'-monophosphate (XMP)-pyrophosphate-Mg(2+) ternary complex has been determined at 1. 60 A resolution. This biproduct, post-transition state structure is of a T. gondii HGPRT mutant (Asp150Ala or D150A). The D150A mutant has reduced activity (k(cat) lower by 11-, 296-, and 8.6-fold for hypoxanthine, guanine, and xanthine, respectively) compared to wild-type T. gondii HGPRT. The Michaelis constants for purine bases are altered only slightly, whereas those for alpha-D-5-phosphoribosyl 1-pyrophosphate (PRPP) are lower by approximately 6.5-fold. The ternary complex crystallizes in space group C222(1) (a = 55.21 A, b = 112.25 A, and c = 144.28 A) with two subunits in the asymmetric unit; the HGPRT tetramer is completed by the application of 2-fold crystallographic symmetry. All active sites contain XMP ¿bound in a fashion similar to that of the guanosine 5'-monophosphate (GMP) and inosine 5'-monophosphate (IMP) complexes reported in the preceding article [Héroux, A., et al. (1999) Biochemistry 38, 14485-14494]¿, pyrophosphate, and two Mg(2+) ions. Each Mg(2+) ion is octahedrally coordinated by two terminal pyrophosphate oxygen atoms and several ordered water molecules. This structure shows how HGPRT uses two Mg(2+) ions to orient and activate the pyrophosphate moiety of PRPP for attack by a purine base, and why mutation in humans of the residue corresponding to Asp206, the only HGPRT amino acid that directly contacts the Mg(2+) ions, causes Lesch-Nyhan syndrome (HGPRT(Kinston), D193N). The Leu78-Lys79 peptide bond in the active site adopts the cis configuration, which it must to bind PRPP or pyrophosphate. The contribution of cis-trans isomerization of this peptide bond to the energetics of substrate binding and product release is discussed. A comprehensive description of the HGPRT reaction mechanism is also proposed.

Mechanism of 4-(β-D-Ribofuranosyl)aminobenzene 5′-Phosphate Synthase, a Key Enzyme in the Methanopterin Biosynthetic Pathway

“…If so, because the oxocarbenium would be one of the first intermediates in the reaction, PP i must become trapped in a cavity in the protein after it is cleaved from PRPP, because PP i is the last product released during the mechanism. One of the likely candidates for binding PP i is Mg 2ϩ , which is required for ␤-RFA-P synthase activity (6) as well as that of other PRPP-dependent enzymes, such as the hypoxanthine-guanine phosphoribosyltransferases (17,18). Retaining PP i throughout the mechanism could be mechanistically important, because in other PRPP-dependent enzymes, the positive charge on the ribosyl group, which becomes a ribooxocarbenium ion, is stabilized by interactions with the oxygen atoms of the released PP i (19) Another mechanistic feature of the ␤-RFA-P synthase reaction that may be shared with other PRPP-dependent enzymes is the placement of the attacking nucleophile directly above the carbenium carbon, coordinated with formation of the intermediate.…”

The competence gene, comF, from Synechocystis sp. strain PCC 6803 is involved in natural transformation, phototactic motility and piliation

“…1). PRT enzymes also require metal ions for activity (Craig & Eakin, 2000;Heroux et al, 1999). The tetracysteine motif at the N-terminus of the ComF group of proteins suggests that metal ions may be required for activity in these proteins, since this motif is very similar in organization to the zinc-binding motif of zinc-dependent enzymes (Possot & Pugsley, 1997).…”

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). Closed active site structures of HPRTs show that the side chain of Lys-68 forms multiple hydrogen bonds with residues in the opposing sub-unit in dimers of the enzyme (21,22,25).…”

“…A third coordinated water molecule forms another hydrogen bond with the N-3 atom of purine substrates. An invariant arginine at position 199 of HPRTs participates directly in binding pyrophosphate (29) and may contribute to positioning both substrates by being close enough to the carboxyl group of Asp-193 to affect its position (21)(22)(23)25). Together, these interactions help to position both substrates for in-line nucleophilic attack at the C1Ј carbon of PRPP or a nucleotide.…”