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Crystal structures of free, IMP-, and GMP-bound Escherichia coli hypoxanthine phosphoribosyltransferase

¹ Department of Biochemistry and Molecular Biology, School of Molecular and Microbial Science, The University of Queensland, St. Lucia, Qld, 4072 Australia
² Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Qld, 4072 Australia

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; e-mail: J.dejersey{at}mailbox.uq.edu.au; fax: 61-7-3365-4699.

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 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 Mg²⁺ in vivo. In the free structure, a Mg²⁺ 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-Mg²⁺ 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

Abbreviations: HPP, 7-hydroxy [4,3,-d] pyrazolo pyrimidine • PRib-PP, 5-phospho--D-ribosyl-1-pyrophosphate • cPRPP, (+)-(1S)-1-pyrophosphoryl-(2R,3R)-2,3-dihydroxy-(4S)-4-(phosphoryloxy-methyl)cyclopentane • DTT, dithiothreitol • PPi, pyrophosphate • ImmucillinHP, (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribotol 5-phosphate • ImmucillinGP, (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-D-ribotol 5-phosphate • H/G/XPRTase, hypoxanthine/guanine/xanthine phosphoribosyltransferase. The nomenclature adopted to describe the 6-oxopurine PRTases from different sources is based on their ability to use the three naturally occurring purine bases, hypoxanthine (H), guanine (G), or xanthine (X)

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