Structural analysis of adenine phosphoribosyltransferase from Saccharomyces cerevisiae

Adenine phosphoribosyltransferase (APRTase) is a widely distributed enzyme, and its deficiency in humans causes the accumulation of 2,8-dihydroxyadeni ne. It is the sole catalyst for adenine recycling in most eukaryotes. The m ost commonly expressed APRTase has subunits of approximately 187 amino acid s, but the only crystal structure is from Leishmania donovani, which expres ses a long form of the enzyme with 237 residues. Saccharomyces cerevisiae A PRTase was selected as a representative of the short APRTases, and the stru cture of the apo-enzyme and sulfate bound forms were solved to 1.5 and 1.75 Angstrom, respectively. Yeast APRTase is a dimeric molecule, and each subu nit is composed of a central five-stranded beta -sheet surrounded by five a lpha -helices, a structural theme found in all known purine phosphoribosylt ransferases. The structures reveal several important features of APRTase fu nction: (i) sulfate ions bound at the 5'-phosphate and pyrophosphate bindin g sites; (ii) a nonproline cis peptide bond (Glu67-Ser68) at the pyrophosph ate binding site in both apo-enzyme and sulfate-bound forms; and (iii) a ca talytic loop that is open and ordered in the apo-enzyme but open and disord ered in the sulfate-bound form. Alignment of conserved amino acids in short -APRTases from 33 species reveals 13 invariant and 15 highly conserved resi dues present in hinges, catalytic site loops, and the catalytic pocket, Mut agenesis of conserved residues in the catalytic loop, subunit interface, an d phosphoribosylpyrophosphate binding site indicates critical roles for the tip of the catalytic loop (Glu106) and a catalytic site residue Arg69, res pectively. Mutation of one loop residue (Tyr103Phe) increases k(cat) by 4-f old, implicating altered dynamics for the catalytic site loop.