Structural analysis of adenine phosphoribosyltransferase from Saccharomyces cerevisiae

W. Shi, K. S E Tanaka, T. R. Crother, M. W. Taylor, Steven C. Almo, Vern L. Schramm

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

Adenine phosphoribosyltransferase (APRTase) is a widely distributed enzyme, and its deficiency in humans causes the accumulation of 2,8-dihydroxyadenine. It is the sole catalyst for adenine recycling in most eukaryotes. The most commonly expressed APRTase has subunits of approximately 187 amino acids, but the only crystal structure is from Leishmania donovani, which expresses a long form of the enzyme with 237 residues. Saccharomyces cerevisiae APRTase was selected as a representative of the short APRTases, and the structure of the apo-enzyme and sulfate bound forms were solved to 1.5 and 1.75 Å, respectively. Yeast APRTase is a dimeric molecule, and each subunit is composed of a central five-stranded β-sheet surrounded by five α-helices, a structural theme found in all known purine phosphoribosyltransferases. The structures reveal several important features of APRTase function: (i) sulfate ions bound at the 5′-phosphate and pyrophosphate binding sites; (ii) a nonproline cis peptide bond (Glu67-Ser68) at the pyrophosphate binding site in both apo-enzyme and sulfate-bound forms; and (iii) a catalytic loop that is open and ordered in the apo-enzyme but open and disordered in the sulfate-bound form. Alignment of conserved amino acids in short-APRTases from 33 species reveals 13 invariant and 15 highly conserved residues present in hinges, catalytic site loops, and the catalytic pocket. Mutagenesis of conserved residues in the catalytic loop, subunit interface, and phosphoribosylpyrophosphate binding site indicates critical roles for the tip of the catalytic loop (Glu106) and a catalytic site residue Arg69, respectively. Mutation of one loop residue (Tyr103Phe) increases kcat by 4-fold, implicating altered dynamics for the catalytic site loop.

Original languageEnglish (US)
Pages (from-to)10800-10809
Number of pages10
JournalBiochemistry
Volume40
Issue number36
DOIs
StatePublished - Sep 11 2001

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Adenine Phosphoribosyltransferase
Structural analysis
Yeast
Saccharomyces cerevisiae
Sulfates
Catalytic Domain
Enzymes
Binding Sites
Leishmania donovani
Amino Acids
Mutagenesis
Adenine
Hinges
Eukaryota
Recycling
Yeasts
Crystal structure
Phosphates
Ions
Peptides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural analysis of adenine phosphoribosyltransferase from Saccharomyces cerevisiae. / Shi, W.; Tanaka, K. S E; Crother, T. R.; Taylor, M. W.; Almo, Steven C.; Schramm, Vern L.

In: Biochemistry, Vol. 40, No. 36, 11.09.2001, p. 10800-10809.

Research output: Contribution to journalArticle

Shi, W. ; Tanaka, K. S E ; Crother, T. R. ; Taylor, M. W. ; Almo, Steven C. ; Schramm, Vern L. / Structural analysis of adenine phosphoribosyltransferase from Saccharomyces cerevisiae. In: Biochemistry. 2001 ; Vol. 40, No. 36. pp. 10800-10809.
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AB - Adenine phosphoribosyltransferase (APRTase) is a widely distributed enzyme, and its deficiency in humans causes the accumulation of 2,8-dihydroxyadenine. It is the sole catalyst for adenine recycling in most eukaryotes. The most commonly expressed APRTase has subunits of approximately 187 amino acids, but the only crystal structure is from Leishmania donovani, which expresses a long form of the enzyme with 237 residues. Saccharomyces cerevisiae APRTase was selected as a representative of the short APRTases, and the structure of the apo-enzyme and sulfate bound forms were solved to 1.5 and 1.75 Å, respectively. Yeast APRTase is a dimeric molecule, and each subunit is composed of a central five-stranded β-sheet surrounded by five α-helices, a structural theme found in all known purine phosphoribosyltransferases. The structures reveal several important features of APRTase function: (i) sulfate ions bound at the 5′-phosphate and pyrophosphate binding sites; (ii) a nonproline cis peptide bond (Glu67-Ser68) at the pyrophosphate binding site in both apo-enzyme and sulfate-bound forms; and (iii) a catalytic loop that is open and ordered in the apo-enzyme but open and disordered in the sulfate-bound form. Alignment of conserved amino acids in short-APRTases from 33 species reveals 13 invariant and 15 highly conserved residues present in hinges, catalytic site loops, and the catalytic pocket. Mutagenesis of conserved residues in the catalytic loop, subunit interface, and phosphoribosylpyrophosphate binding site indicates critical roles for the tip of the catalytic loop (Glu106) and a catalytic site residue Arg69, respectively. Mutation of one loop residue (Tyr103Phe) increases kcat by 4-fold, implicating altered dynamics for the catalytic site loop.

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