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Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 Å resolution) and for the enzyme in complex with the reaction products, inosine 5Ј-monophosphate (IMP) and guanosine 5Ј-monophosphate (GMP) (2.8 Å resolution). Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 C␣ atoms of 1.0 Å. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6-oxopurine PRTases have a requirement for divalent metal ion, usually Mg 2+ in vivo. In the free structure, a Mg 2+ is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg 2+ complex). However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket.Keywords: Crystal structure; phosphoribosyltransferase; purine salvage; Escherichia coli; enzymology Purine nucleoside monophosphates are either synthesized de novo from simple precursors or through a salvage pathway. Most organisms, including bacteria, possess both the de novo and salvage pathways. However, some microorganisms such as the protozoan parasites do not possess the de novo pathway and therefore, depend entirely on the transport of preformed purine bases from their host cells for production of their 6-oxopurine nucleoside monophosphates (Ullman 1995). The key enzymes in the salvage pathway are the 6-oxopurine phosphoribosyltransferases (H/G/ XPRTases), which catalyze the synthesis of the purine nucleoside monophosphates inosine 5Ј-monophosphate (IMP), guanosine 5Ј-monophosphate (GMP), or xanthosine 5Ј-monophosphate (XMP) from 5-phospho-␣-D-ribosyl-1-pyrophosphate (PRib-PP) and hypoxanthine, guanine, or xanthine. A divalent cation, usually magnesium, is essential for catalysis (Musick 1981).In contrast with most eukaryotes, which have only one 6-oxopurine PRTase, enteric bacteria such as Escherichia coli possesses two, HPRT (E.C. 2.4.2.8) and XGPRT (E.C. Reprint requests to: John de Jersey, Department of Biochemistry and Molecular Biology, School of Molecular and Microbial Science, The University of Queensland, St Lucia, Qld, 4072 Australia;3,] pyra...

Xanthine phosphoribosyltransferase (XPRT; EC 2.4.2.22) from Escherichia coli is a tetrameric enzyme having 152 residues per subunit. XPRT catalyzes the transfer of the phosphoribosyl group from 5-phospho-alpha-D-ribosyl 1-pyrophosphate (PRib-PP) to the 6-oxopurine bases guanine, xanthine, and hypoxanthine to form GMP, XMP, and IMP, respectively. Crystals grown in the absence of substrate or product were used to determine the structure of XPRT at a resolution of 1.8 A, by multiple isomorphous replacement. The core structure of XPRT includes a five-stranded parallel beta-sheet surrounded by three alpha-helices, which is similar to that observed in other known phosphoribosyltransferase (PRTase) structures. The XPRT structure also has several interesting features. A glutamine residue in the purine binding site may be responsible for the altered 6-oxopurine base specificity seen in this enzyme compared to other 6-oxopurine PRTases. Also, we observe both a magnesium ion and a sulfate ion bound at the PRib-PP binding site of XPRT. The sulfate ion interacts with Arg-37 which has a cis-peptide conformation, and the magnesium ion interacts with Asp-89, a highly conserved acidic residue in the PRib-PP binding site motif. The XPRT structure also incorporates a feature which has not been observed in other PRTase structures. The C-terminal 12 residues of XPRT adopt an unusual extended conformation and make interactions with a neighboring subunit. The very last residue, Arg-152, could form part of the active site of a symmetry-related subunit in the XPRT tetramer.

A key ion-dependent folding unit within the hepatitis C IRES comprises the IIIef junction and pseudoknot. This region is also important in recruitment of the 40S ribosomal subunit. Here, circular dichroism is used to study the influence of metal ions on the structure and stability of this region. Comparison of the thermal stability of an IRES fragment encompassing subdomains IIIe/f and IV (named 3EF4) with that of a larger fragment also possessing subdomain IIId (3DEF4) indicates an additional stabilizing effect of Mg(2+) ions on the latter fragment. Magnesium and potassium ions stabilize both fragments through nonspecific counterion effects. The additional effect of magnesium on 3DEF4, observed in the absence or presence of 100 mM KCl, is attributed to a nonspecific but high-affinity site for metal ions created by a region of unusual high charge density. Subdomain IIId presumably participates in tertiary packing interactions that provide such a site. Viomycin binds to the full-length IRES and RNA fragments with K(d) values of 25-55 microM. Interestingly, viomycin binding to the two fragments is affected differently by Mg(2+); noncompetitive inhibition of binding to 3DEF4 is observed, whereas binding to 3EF4 is not impaired. Formation of a Mg(2+)-stabilized tertiary fold, involving subdomain IIId, may thereby hinder viomycin binding to 3DEF4 indirectly. Mutational and deletion studies locate viomycin binding within subdomains IIIe/f rather than within the pseudoknot. In pseudoknot mutants, Mg(2+) ions have different effects on viomycin binding and thermal stability, suggesting altered tertiary interactions involving subdomain IIId.

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