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 5′‐monophosphate (IMP) and guanosine 5′‐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 Cα 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‐ P Rib‐ 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.

Structures of free, substrate-bound and product-bound forms of Escherichia coli xanthine-guanine phosphoribosyltransferase (XGPRT) have been determined by X-ray crystallography. These are compared with the previously determined structure of magnesium and sulphate-bound XPRT. The structure of free XGPRT at 2.25 Å resolution confirms the flexibility of residues in and around a mobile loop identified in other PRTases and shows that the cis-peptide conformation of Arg37 at the active site is maintained in the absence of bound ligands. The structures of XGPRT complexed with the purine base substrates guanine or xanthine in combination with cPRib-PP, an analog of the second substrate PRib-PP, have been solved to 2.0 Å resolution. In these two structures the disordered phosphate-binding loop of uncomplexed XGPRT becomes ordered through interactions with the 5′-phosphate group of cPRib-PP. The cyclopentane ring of cPRib-PP has the C3 exo pucker conformation, stabilised by the cPRib-PP-bound Mg2+. The purine base specificity of XGPRT appears to be due to water-mediated interactions between the 2-exocyclic groups of guanine or xanthine and side-chains of Glu136 and Asp140, as well as the main-chain oxygen atom of Ile135. Asp92, together with Lys115, could help stabilise the N7-protonated tautomer of the incoming base and could act as a general base to remove the proton from N7 when the nucleotide product is formed. The 2.6 Å resolution structure of XGPRT complexed with product GMP is similar to the substrate-bound complexes. However, the ribose ring of GMP is rotated by ∼24° compared with the equivalent ring in cPRib-PP. This rotation results in the loss of all interactions between the ribosyl group and the enzyme in the product complex.

Phosphoribosyltransferases (PRTases) catalyse the transfer of the > phosphoribosyl group from 5-phospho-a-l) ribosyl- 1-pyrophosphate (PRib PP) to a nitrogenous base. Catalysis requires the presence of a divalent metal ion which may conceivably function by forming a complex with PRib-PP (to form the true substrate) and/or by binding directly to the enzyme. Kxperi ments using stable recombinant mutant forms of human hypoxanthine-guanine PRI'ase and K. coli xanthine PRTase have been performed which comment on these two possibilities. These include: (ij nrnr spectra support the proposi lion that Mg2+ binds to both the 5-phosphate and 1-pyrophosphate groups of PRib-PP under physiological conditions, with a greater affinity for the pvrophosphate group; (ii) various divalent metal ion.s ran substitute for Mg24 in catalysis by the human enzyme in the order Mn2f >Mg2+ >C'o2+>/n';+ at p H 7.4: (iii) the crystal structure of xanthine PRTase has been found to contain bound Mg2 + ; (iv) human hypoxanthine-guanine PRTase requires the presence of Mg2+ to maintain maximal activity: removal of the metal ion by dialysis results in loss of activity. These results indicate that Mg2+ hinds To the en zynies under certain conditions in the absence of PRib PP. as well as binding to PRib PP.

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.

Xanthine phosphoribosyltransferase (XPRT; EC 2.4.2.22) from Escherichia coli is a purine salvage enzyme which synthesizes the nucleotides GMP, XMP, and IMP. A mutant C59A, which is more stable than wild-type XPRT while retaining high activity, has been prepared and crystallized to give three different crystal forms (A, B, and C). Form A crystals are orthorhombic (P21212), with unit cell dimensions a = 59.2 A, b = 92.9 A, c = 53.2 A. Form B crystals are monoclinic (C2) with unit cell dimensions a = 84.4 A, b = 70.8 A, c = 54.1 A, and beta = 113.4 degrees, and form C crystals are tetragonal (P41212 or P43212) with unit cell dimensions a,b = 94 A, c = 167.5 A. Wild-type XPRT and a selenomethionine derivative of C59A XPRT have also been crystallized in the orthorhombic form. The selenomethionine derivative was prepared by expressing XPRT in the usual E. coli strain without the need for a methionine auxotroph. Cells were grown in a methionine-deficient medium supplemented with selenomethionine which gave >95% incorporation. Both the wild-type and selenomethionine C59A XPRT crystals are isomorphous with C59A form A crystals.