Purine Phosphoribosyltransferases

purine phosphoribosyltransferase phosphoribosylpyrophosphate adenine phosphoribosyltransferase hypoxanthine phosphoribosyltransferase xanthine phosphoribosyltransferase Purine phosphoribosyltransferases (PRTs)1 of microbes and mammals are enzymes that catalyze the recovery of preformed bases for use in cellular metabolism. In free living organisms, purine nucleotides can be generated via de novo synthesis, as well as by the salvage of preformed bases. In contrast, many parasitic organisms are unable to synthesize purines via de novopathways and therefore must rely on the enzymes in salvage pathways, including PRTs, for the synthesis of purine nucleotides (1.Berens R.L. Krug E.C. Marr J.J. Marr J.J. Müller M. Biochemistry and Molecular Biology of Parasites. Academic Press Ltd., London1995: 89-117Crossref Google Scholar). For this reason, enzymes in salvage pathways were proposed more than 30 years ago as potential targets of therapeutic agents for the treatment of diseases caused by parasites (2.Walsh C.J. Sherman I.W. J. Protozool. 1968; 15: 763-770Crossref PubMed Scopus (76) Google Scholar). Purine PRTs catalyze the reversible transfer of a phosphoribosyl group from phosphoribosylpyrophosphate (PRPP) to a purine base (adenine, guanine, hypoxanthine, or xanthine). For those enzymes that have been studied, the forward reaction appears to be ordered and sequential with PRPP binding first followed by the purine base (3.Yuan L. Craig III, S.P. McKerrow J.H. Wang C.C. Biochemistry. 1992; 31: 806-810Crossref PubMed Scopus (62) Google Scholar, 4.Xu Y. Eads J. Sacchettini J.C. Grubmeyer C. Biochemistry. 1997; 36: 3700-3712Crossref PubMed Scopus (109) Google Scholar, 5.Munagala N.R. Chin M.S. Wang C.C. Biochemistry. 1998; 37: 4045-4051Crossref PubMed Google Scholar). After catalysis, pyrophosphate (PPi) is released before the nucleotide. Reaction chemistry has been reported to proceed via either a dissociative (S n1) or an associative (S n2) type mechanism (Fig.1). Based on their similarity to other enzymes, PRTs have been proposed to catalyze anS n1-type reaction with the formation of an unstable ribooxocarbenium ion intermediate (6.Goitein R.K. Chelsky D. Parsons S.M. J. Biol. Chem. 1978; 253: 2963-2971Abstract Full Text PDF PubMed Google Scholar, 7.Bhatia M.B. Vinitsky A. Grubmeyer C. Biochemistry. 1990; 29: 10480-10487Crossref PubMed Scopus (63) Google Scholar), butS n2-type chemistry for purine PRTs has not been ruled out (8.Smith J.L. Nat. Struct. Biol. 1999; 6: 502-504Crossref PubMed Scopus (23) Google Scholar). Enzymes in salvage pathways that contribute to the synthesis of AMP include adenosine kinase or adenine phosphoribosyltransferase (APRT). Alternatively, adenosine deaminase and a purine nucleoside phosphorylase catalyze the conversion of adenine to hypoxanthine, which may be salvaged by hypoxanthine phosphoribosyltransferase (HPRT). The product, IMP, is a precursor for both AMP and GMP. In most bacteria and nearly all eukaryotes, HPRTs also catalyze the salvage of guanine and, in a few cases, xanthine. In human tissues, substrates available for salvage include hypoxanthine at 8.2 ± 1.3 μm, xanthine at 2.5 ± 0.6 μm, adenine at 0.3 ± 0.15 μm, and adenosine at 0.6 ± 0.2 μm (9.Hartwick R.A. Krstulovic A.M. Brown P.R. J. Chromatogr. 1979; 186: 659-676Crossref PubMed Scopus (122) Google Scholar). The human HPRT can salvage xanthine, albeit at relatively low levels (10.Krenitsky T.A. Papaioannou R. Elion G.B. J. Biol. Chem. 1969; 244: 1263-1270Abstract Full Text PDF PubMed Google Scholar), but instead this base is usually converted to uric acid for excretion as a nitrogenous waste. The identity of the 6-oxopurine substrates salvaged by HPRTs is frequently included in the names of these enzymes, but HPRTs likely are descended from a common ancestral hpt gene of prokaryotes, and substrate specificity can be dramatically altered by single amino acid substitutions (11.Lee C.C. Craig III, S.P. Eakin A.E. Biochemistry. 1998; 37: 3491-3498Crossref PubMed Scopus (19) Google Scholar, 12.Munagala N.R. Wang C.C. Biochemistry. 1998; 37: 16612-16619Crossref PubMed Scopus (26) Google Scholar). Several bacteria possess two distinct enzymes for the salvage of 6-oxopurines. In those bacteria the primary substrate for the HPRT is hypoxanthine, and guanine is utilized with reduced efficiency (11.Lee C.C. Craig III, S.P. Eakin A.E. Biochemistry. 1998; 37: 3491-3498Crossref PubMed Scopus (19) Google Scholar). The other enzyme, referred to here as xanthine phosphoribosyltransferase (XPRT), has a preference for catalyzing the salvage of guanine and xanthine. The contributions of specific enzymes to the salvage of purine bases vary significantly among different organisms. For example, inPlasmodium falciparum, etiologic agent of the most lethal form of human malaria, the activity of adenosine kinase is barely detectable, and APRT activity is 1500-fold below that of HPRT (13.Reyes P. Rathod P.K. Sanchez D.J. Mrema J.E.K. Rieckmann K.H. Heidrich H.G. Mol. Biochem. Parasitol. 1982; 5: 275-290Crossref PubMed Scopus (181) Google Scholar). In this pathogen, adenosine deaminase and purine nucleoside phosphorylase are relatively abundant, suggesting that the major route for the salvage of purine bases leading to both AMP and GMP is through hypoxanthine, which can be converted to IMP by the HPRT-catalyzed reaction. For this review, residue numbers refer to the positions of amino acids in the human HPRT, the bacterial XPRT, or the leishmanial APRT, as these enzymes were the first of their class for which crystal structures were reported (14.Eads J.C. Scapin G. Xu Y. Grubmeyer C. Sacchettini J.C. Cell. 1994; 78: 325-334Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 15.Vos S. de Jersey J. Martin J.L. Biochemistry. 1997; 36: 4125-4134Crossref PubMed Scopus (71) Google Scholar, 16.Phillips C.L. Ullman B. Brennan R.G. Hill C.P. EMBO J. 1999; 18: 3533-3545Crossref PubMed Scopus (55) Google Scholar). Fig. 2illustrates ribbon diagrams of monomers from these representative structures, although the enzymes may be functional as dimers or tetramers. All three of these enzymes possess a core domain composed of a 4- or 5-stranded parallel β sheet flanked by 3–4 α helices. The C-terminal ends of the β sheets of the core domains of purine PRTs form the floor of the active sites of these enzymes. A poorly conserved hood domain contributes residues that complete the active site and participate in binding purine substrates. For HPRTs and XPRTs the amino acids that flank the active site are contributed largely by 4 active site loops (loops I–IV). Residues in the hood domains of HPRTs, XPRTs, and APRTs come from non-homologous regions of the protein. However, all three enzymes possess an aromatic residue that forms π-π stacking interactions with purine substrates (Fig.3).Figure 3Conserved residues in the active sites of purine PRTs. The enzyme from Trypanosoma cruzi is shown (numbered according to residues in the human HPRT) in ternary complex with a purine analog and PRPP (25.Focia P.J. Craig III, S.P. Eakin A.E. Biochemistry. 1998; 37: 17120-17127Crossref PubMed Scopus (80) Google Scholar). The bacterial XPRT is with guanine and a PRPP analog (19.Vos S. Parry R.J. Burns M.R. de Jersey J. Martin J.L. J. Mol. Biol. 1998; 282: 875-889Crossref PubMed Scopus (54) Google Scholar), and APRT has bound AMP (16.Phillips C.L. Ullman B. Brennan R.G. Hill C.P. EMBO J. 1999; 18: 3533-3545Crossref PubMed Scopus (55) Google Scholar). The main chain atoms of invariant glycines and side chains of other invariant amino acids are shown with thick bonds. Substrates, substrate analogs, and conserved aromatic side chains that form π-π stacking interactions with purine rings are illustrated with relatively thin bonds.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Data reported to GenBankTM indicate that the HPRTs of distantly related organisms share extensive primary sequence homology. For example, there is 41% identity for amino acids in the human and a bacterial HPRT (11.Lee C.C. Craig III, S.P. Eakin A.E. Biochemistry. 1998; 37: 3491-3498Crossref PubMed Scopus (19) Google Scholar). However, among well over 20 HPRT sequences reported there are only 9 invariant amino acids and all but the HPRT of Giardia lamblia also are invariant at Glu-133 and Asp-134 (Table I). In general, conserved residues of HPRTs and bacterial XPRTs differ at positions homologous with human Leu-67 and Glu-133 (Ser-36 and Asp-88 in the XPRT of Escherichia coli). Solutions for the crystal structures of HPRTs reveal that the 11 conserved residues immediately flank or are very near the active site of HPRTs (Fig. 3).Table IThe highly conserved residues of HPRTs, XPRTs, and APRTsHuman HPRTBacterial XPRTLeishmanial APRTPhe-42Asp-44Leu-67Ser-36Arg-82Gly-69Gly-38Gly-83Ser-103Ser-62Arg-102Tyr-104Tyr-63Lys-106Glu-133Asp-88Asp-146Asp-134Asp-89Asp-147Asp-137Asp-92Ala-150Lys-165Lys-115Thr-151Gly-189Gly-153Asp-193Asp-140Arg-199Residues that may have analogous functions in all purine PRTs are printed in bold type. Although located at opposite ends of the protein sequences, Asp-193 of HPRTs, Asp-140 of bacterial XPRTs, and Asp-44 of APRTs are located at analogous positions in enzyme crystal structures and thus could possibly have similar functional roles (see text). Open table in a new tab Residues that may have analogous functions in all purine PRTs are printed in bold type. Although located at opposite ends of the protein sequences, Asp-193 of HPRTs, Asp-140 of bacterial XPRTs, and Asp-44 of APRTs are located at analogous positions in enzyme crystal structures and thus could possibly have similar functional roles (see text). Among the amino acid sequences deduced from 39 APRT genes reported to GenBankTM, 11 residues are invariant. As shown in Fig. 3, these residues either flank the active site or are located within active site loop II, which is predicted to participate in forming the active site of APRTs during (16.Phillips C.L. Ullman B. Brennan R.G. Hill C.P. EMBO J. 1999; 18: 3533-3545Crossref PubMed Scopus (55) Google Scholar). the crystal structures of all purine PRTs are with the amino acid sequences reported to GenBankTM, there are only residues with human and that are invariant. A the human HPRT, in a the enzyme, in 1992; PubMed Scopus Google Scholar). The invariant may be for the formation of a and an in active site loop of purine PRTs S. de Jersey J. Martin J.L. Biochemistry. 1997; 36: 4125-4134Crossref PubMed Scopus (71) Google Scholar, 16.Phillips C.L. Ullman B. Brennan R.G. Hill C.P. EMBO J. 1999; 18: 3533-3545Crossref PubMed Scopus (55) Google Scholar, D. Ullman B. Brennan R.G. Nat. Struct. Biol. PubMed Scopus Google Scholar, S. Parry R.J. Burns M.R. de Jersey J. Martin J.L. J. Mol. Biol. 1998; 282: 875-889Crossref PubMed Scopus (54) Google Scholar, P.J. Craig III, S.P. R. R.J. Eakin A.E. Biochemistry. 1998; 37: PubMed Scopus Google Scholar, Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar, S.M. Grubmeyer C. Biochemistry. 1999; PubMed Scopus Google Scholar, A. L. R.L. Biochemistry. 1999; PubMed Scopus Google Scholar). In nearly all purine PRTs, there is a or residue located immediately from the invariant have been for the functional of and the in active site loop of HPRTs P.J. Craig III, S.P. R. R.J. Eakin A.E. Biochemistry. 1998; 37: PubMed Scopus Google Scholar, A. L. R.L. Biochemistry. 1999; PubMed Scopus Google Scholar, Xu Y. J. R. P.J. Eakin A.E. Sacchettini J.C. Craig III, S.P. 1999; PubMed Scopus (63) Google Scholar). The of the the and to the to with pyrophosphate atoms and a are in the active sites of HPRTs Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar, S.M. Grubmeyer C. Biochemistry. 1999; PubMed Scopus Google Scholar, A. L. R.L. Biochemistry. 1999; PubMed Scopus Google P.J. Craig III, S.P. Eakin A.E. Biochemistry. 1998; 37: 17120-17127Crossref PubMed Scopus (80) Google Scholar). active site structures of HPRTs that the side chain of forms with residues in the in dimers of the enzyme Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar, S.M. Grubmeyer C. Biochemistry. 1999; PubMed Scopus Google Scholar, P.J. Craig III, S.P. Eakin A.E. Biochemistry. 1998; 37: 17120-17127Crossref PubMed Scopus (80) Google Scholar). in interactions before and of the active as the enzyme the of the forward reaction with the or of main chain atoms of loop residues and in the active site (25.Focia P.J. Craig III, S.P. Eakin A.E. Biochemistry. 1998; 37: 17120-17127Crossref PubMed Scopus (80) Google Scholar). interactions with active site are by new with main chain atoms of residues in active site loop II, which over the active site during the in interactions with substrates during of the active site may the of pyrophosphate from the active site of to in the human HPRT in the of in the binding of a that is with loop in binding interactions with PRPP and pyrophosphate Xu Y. J. R. P.J. Eakin A.E. Sacchettini J.C. Craig III, S.P. 1999; PubMed Scopus (63) Google Scholar). in the HPRT of the residue at proposed to the binding of PRPP and to this enzyme Chin M.S. P.J. Wang C.C. R.J. Biochemistry. PubMed Scopus Google Scholar). All purine PRTs possess a of residues HPRT or leishmanial APRT within a conserved PRPP binding that 11 amino acids and residues of active site loop Among purine PRTs, the forms with the and of of substrates (16.Phillips C.L. Ullman B. Brennan R.G. Hill C.P. EMBO J. 1999; 18: 3533-3545Crossref PubMed Scopus (55) Google Scholar, D. Ullman B. Brennan R.G. Nat. Struct. Biol. PubMed Scopus Google Scholar, S. Parry R.J. Burns M.R. de Jersey J. Martin J.L. J. Mol. Biol. 1998; 282: 875-889Crossref PubMed Scopus (54) Google Scholar, Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar, S.M. Grubmeyer C. Biochemistry. 1999; PubMed Scopus Google Scholar, P.J. Craig III, S.P. Eakin A.E. Biochemistry. 1998; 37: 17120-17127Crossref PubMed Scopus (80) Google Scholar, A. Biochemistry. 1999; PubMed Scopus Google Scholar). Glu-133 of HPRTs forms a with a is in the active Among HPRTs, the at proposed to a in by with the of 6-oxopurine substrates Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar, S.M. Grubmeyer C. Biochemistry. 1999; PubMed Scopus Google Scholar, Y. Grubmeyer C. Biochemistry. 1998; 37: PubMed Google Scholar). of the human HPRT that this likely functions as a base by of a in forward by HPRTs Y. Grubmeyer C. Biochemistry. 1998; 37: PubMed Google Scholar). this and the purine of a analog may contribute to the very binding of this to HPRTs Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar, S.M. Grubmeyer C. Biochemistry. 1999; PubMed Scopus Google Scholar). The residue at the analogous in the crystal of the leishmanial APRT is a conserved suggesting that either the mechanism HPRTs and APRTs or that the base of APRT is contributed by an amino acid that to participate in the formation of the active site during the In HPRTs, active site loop has been shown to over the active site both substrate are bound and during the of the reaction Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar, S.M. Grubmeyer C. Biochemistry. 1999; PubMed Scopus Google Scholar, P.J. Craig III, S.P. Eakin A.E. Biochemistry. 1998; 37: 17120-17127Crossref PubMed Scopus (80) Google Scholar). the crystal of the leishmanial APRT with bound AMP that residues within active site loop of this enzyme may contribute to forming the active site during the (16.Phillips C.L. Ullman B. Brennan R.G. Hill C.P. EMBO J. 1999; 18: 3533-3545Crossref PubMed Scopus (55) Google Scholar). a base be contributed by a residue of active site loop of at of HPRTs has been shown to participate in binding pyrophosphate and purines via the formation of a protein with a ion Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar, S.M. Grubmeyer C. Biochemistry. 1999; PubMed Scopus Google Scholar, A. L. R.L. Biochemistry. 1999; PubMed Scopus Google P.J. Craig III, S.P. Eakin A.E. Biochemistry. 1998; 37: 17120-17127Crossref PubMed Scopus (80) Google (Fig. Asp-193 also forms with two by the and the forms interactions with two of PRPP or A forms with the of purine substrates. invariant at of HPRTs in binding pyrophosphate III, S.P. P.J. R.J. 1997; PubMed Scopus Google and may contribute to both substrates by to the group of Asp-193 to Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar, S.M. Grubmeyer C. Biochemistry. 1999; PubMed Scopus Google Scholar, A. L. R.L. 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In this there is a gene an as well as an The two enzymes to be the of a of the hpt with both genes in The leishmanial XPRT is in preference for catalyzing the salvage of xanthine over hypoxanthine and enzyme from all other 6-oxopurine PRTs in than at a homologous with Asp-193 in the human be predicted to the of the ion that in purine substrates in the active the in the L. XPRT is for the altered substrate specificity of this As in and are invariant in HPRTs and are located in loop II, which over the active site during the (Fig. A has been shown to the human HPRT, in 1992; PubMed Scopus Google Scholar). The main chain of forms a with a pyrophosphate of the side chain with a (Fig. forms an with a conserved as well as to a of either PRPP or a analog in the active site Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar, S.M. Grubmeyer C. Biochemistry. 1999; PubMed Scopus Google Scholar, P.J. Craig III, S.P. Eakin A.E. Biochemistry. 1998; 37: 17120-17127Crossref PubMed Scopus (80) Google Scholar). interactions the base of the loop and over the active the a in of the human HPRT 1992; PubMed Scopus Google Scholar). of these in the human HPRT R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar), with indicate that loop also contributes to the binding of purine substrates. this is through interactions of loop residues with conserved aromatic residues with human that purine substrates. of the HPRT from L. that of the residue homologous with the at of the human HPRT the for the forward reaction by the enzyme A. Ullman B. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The main chain of forms a with a pyrophosphate (Fig. and the side chain forms a with a of either PRPP or a analog in the active site Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar, S.M. Grubmeyer C. 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In the only available for an APRT, specificity for appears to be by the formation of a with a main chain of an residue at The of two in the active sites of HPRTs has for the chemistry of the reaction by this The potential of the may contribute to of as a group in HPRT-catalyzed The forms interactions with active site residues (Fig. However, 4 atoms to either PRPP or and the of substrates are within the of this Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar, S.M. Grubmeyer C. Biochemistry. 1999; PubMed Scopus Google Scholar, P.J. Craig III, S.P. Eakin A.E. Biochemistry. 1998; 37: 17120-17127Crossref PubMed Scopus (80) Google Scholar). In the of the HPRT, with PRPP and a purine analog as the of the of with the of PRPP is to the (25.Focia P.J. Craig III, S.P. Eakin A.E. Biochemistry. 1998; 37: 17120-17127Crossref PubMed Scopus (80) Google Scholar). the potential of with formation of the to as the reaction the may contribute to the for (6.Goitein R.K. Chelsky D. Parsons S.M. J. Biol. Chem. 1978; 253: 2963-2971Abstract Full Text PDF PubMed Google Scholar, 7.Bhatia M.B. Vinitsky A. Grubmeyer C. Biochemistry. 1990; 29: 10480-10487Crossref PubMed Scopus (63) Google that HPRTs catalyze anS n1-type reaction (Fig. an unstable ribooxocarbenium intermediate to be from The of residues from active site loop of the HPRT not the enzyme from catalyzing either forward or albeit at reduced C.C. of of Scholar). that from is not for the of a highly unstable intermediate in the reaction. crystal structures of the active sites of the and HPRTs reveal that the only residue near to of a intermediate is an invariant which is located the of bound substrates Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar, S.M. Grubmeyer C. Biochemistry. 1999; PubMed Scopus Google Scholar, P.J. Craig III, S.P. Eakin A.E. 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The in the for the formation of a on the analog of the at In the the potential predicted for a ribooxocarbenium intermediate n1-type structures of the human and HPRTs were with bound Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar, S.M. Grubmeyer C. Biochemistry. 1999; PubMed Scopus Google Scholar). these structures reveal interactions enzyme residues and the In the that is by interactions the of the substrate analog Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar). could be by interactions with the pyrophosphate to the for G. Xu Y. Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar). the the of the purine and the invariant at Grubmeyer C. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar, S.M. Grubmeyer C. Biochemistry. 1999; PubMed Scopus Google as well as altered of the of the may for the binding of the be in their form as of the of parasites in have to to the an potential of is that that may all PRTs that have a similar enzyme and may be more to for binding to the enzymes of a of and may be to in to HPRT the crystal structures of enzymes have been For example, the to a μm of the HPRT from N.R. S. R.J. Wang C.C. Biochemistry. 1998; 37: PubMed Scopus Google Scholar). of this were shown to of μm N.R. S. R.J. Wang C.C. Biochemistry. 1998; 37: PubMed Scopus Google and A.M. N.R. de P.R. Wang C.C. Biochemistry. PubMed Scopus Google the HPRT from with human and HPRTs indicate that the the enzyme of the of the of by these by the of purines in the that the HPRT the most likely of the N.R. S. R.J. Wang C.C. Biochemistry. 1998; 37: PubMed Scopus Google Scholar, A.M. N.R. de P.R. Wang C.C. Biochemistry. PubMed Scopus Google Scholar). are the first in which an HPRT has been shown to be in the of a Purine PRTs have been during the of the A of crystal structures have been and are available from of amino acid substitutions that for this class of enzymes. and enzyme structures have been in the and of of HPRTs, that the of HPRT activity may be within the few for the of for the treatment of diseases caused by for and and for for amino acid sequences reported to and and for this