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Binding of cations in Bacillus subtilis phosphoribosyldiphosphate synthetase and their role in catalysis

¹ Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, 2100 Copenhagen, Denmark

Reprint requests to: Sine Larsen, Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark; e-mail: sine{at}ccs.ki.ku.dk; fax: 45-35-32-02-99.

The binding sites for the two cations essential for the catalytic function of 5-phospho-D-ribosyl -1-diphosphate (PRPP) synthases have been identified from the structure of the Bacillus subtilis phosphoribosyldiphosphate synthetase (PRPPsase) with bound Cd²⁺. The structure determined from X-ray diffraction data to 2.8-Å resolution reveals the same hexameric arrangement of the subunits that was observed in the complexes of the enzyme with the activator sulfate and the allosteric inhibitor ADP. Two cation binding sites were localized in each of the two domains of the subunits that compose the hexamer; each domain of the subunit has an associated cation. In addition to the bound Cd²⁺, the Cd²⁺-PRPPsase structure contains a sulfate ion in the regulatory site, a sulfate ion at the ribose-5-phosphate binding site, and an AMP moiety at the ATP binding site. Comparison of the Cd²⁺-PRPPsase to the structures of the PRPPsase complexed with sulfate and mADP reveals the structural rearrangement induced by the binding of the free cation, which is essential for the initiation of the reaction. The comparison to the cPRPP complex of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli, a type I phosphoribosyltransferase, provided information about the binding of PRPP. This strongly indicates that the binding of both substrates must lead to a stabilized conformation of the loop region, which remains unresolved in the known PRPPsase complex structures.

Keywords: Phosphoribosylpyrophosphate synthetase; allosteric enzyme; Cd2+ complex structure; nucleotide binding; phosphoribosyltransferase

Abbreviations: PRPPsase, phosphoribosyldiphosphate synthetase • PP, diphosphate • R-5-P, ribose 5-phosphate • PRPP, 5-phospho-d-ribosyl -1-diphosphate • PRTase, phosphoribosyltransferase • GPATase, glutamine PRPP amidotransferase • mATP, ,ß-methylene ATP • mADP, ,ß-methylene ADP • mAMP, -methylene AMP • cPRPP, cyclopentyl analog of 5-phosphoribosyl-1-diphosphate

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