Closed site complexes of adenine phosphoribosyltransferase from Giardia lamblia reveal a mechanism of ribosyl migration

Wuxian Shi, Anne E. Sarver, Ching C. Wang, Kelly S E Tanaka, Steven C. Almo, Vern L. Schramm

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

The adenine phosphoribosyltransferase (APRTase) from Giardia lamblia was co-crystallized with 9-deazaadenine and sulfate or with 9-deazaadenine and Mg-phosphoribosylpyrophosphate. The complexes were solved and refined to 1.85 and 1.95 Å resolution. Giardia APRTase is a symmetric homodimer with the monomers built around Rossman fold cores, an element common to all known purine phosphoribosyltransferases. The catalytic sites are capped with a small hood domain that is unique to the APRTases. These structures reveal several features relevant to the catalytic function of APRTase: 1) a non-proline cis peptide bond (Glu61-Serr62) is required to form the pyrophosphate binding site in the APRTase·9dA·MgPRPP complex but is a trans peptide bond in the absence of pyrophosphate group, as observed in the APRTase·9dA·SO4 complex; 2) a catalytic site loop is closed and fully ordered in both complexes, with Glu100 from the catalytic loop acting as the acid/base for protonation/deprotonation of N-7 of the adenine ring; 3) the pyrophosphoryl charge is neutralized by a single Mg2+ ion and Arg63, in contrast to the hypoxanthine-guanine phosphoribosyltransferases, which use two Mg2+ ions; and 4) the nearest structural neighbors to APRTases are the orotate phosphoribosyltransferases, suggesting different paths of evolution for adenine relative to other purine PRTases. An overlap comparison of AMP and 9-deazaadenine plus Mg-PRPP at the catalytic sites of APRTases indicated that reaction coordinate motion involves a 2.1-Å excursion of the ribosyl anomeric carbon, whereas the adenine ring and the 5-phosphoryl group remained fixed. G. lamblia APRTase therefore provides another example of nucleophilic displacement by electrophile migration.

Original languageEnglish (US)
Pages (from-to)39981-39988
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number42
DOIs
StatePublished - Oct 18 2002

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Adenine Phosphoribosyltransferase
Giardia lamblia
Adenine
Catalytic Domain
Orotate Phosphoribosyltransferase
Ions
Hypoxanthine Phosphoribosyltransferase
Giardia
Peptides
Deprotonation
Protonation
Adenosine Monophosphate
Sulfates
Carbon
Monomers
Binding Sites
Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Closed site complexes of adenine phosphoribosyltransferase from Giardia lamblia reveal a mechanism of ribosyl migration. / Shi, Wuxian; Sarver, Anne E.; Wang, Ching C.; Tanaka, Kelly S E; Almo, Steven C.; Schramm, Vern L.

In: Journal of Biological Chemistry, Vol. 277, No. 42, 18.10.2002, p. 39981-39988.

Research output: Contribution to journalArticle

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abstract = "The adenine phosphoribosyltransferase (APRTase) from Giardia lamblia was co-crystallized with 9-deazaadenine and sulfate or with 9-deazaadenine and Mg-phosphoribosylpyrophosphate. The complexes were solved and refined to 1.85 and 1.95 {\AA} resolution. Giardia APRTase is a symmetric homodimer with the monomers built around Rossman fold cores, an element common to all known purine phosphoribosyltransferases. The catalytic sites are capped with a small hood domain that is unique to the APRTases. These structures reveal several features relevant to the catalytic function of APRTase: 1) a non-proline cis peptide bond (Glu61-Serr62) is required to form the pyrophosphate binding site in the APRTase·9dA·MgPRPP complex but is a trans peptide bond in the absence of pyrophosphate group, as observed in the APRTase·9dA·SO4 complex; 2) a catalytic site loop is closed and fully ordered in both complexes, with Glu100 from the catalytic loop acting as the acid/base for protonation/deprotonation of N-7 of the adenine ring; 3) the pyrophosphoryl charge is neutralized by a single Mg2+ ion and Arg63, in contrast to the hypoxanthine-guanine phosphoribosyltransferases, which use two Mg2+ ions; and 4) the nearest structural neighbors to APRTases are the orotate phosphoribosyltransferases, suggesting different paths of evolution for adenine relative to other purine PRTases. An overlap comparison of AMP and 9-deazaadenine plus Mg-PRPP at the catalytic sites of APRTases indicated that reaction coordinate motion involves a 2.1-{\AA} excursion of the ribosyl anomeric carbon, whereas the adenine ring and the 5-phosphoryl group remained fixed. G. lamblia APRTase therefore provides another example of nucleophilic displacement by electrophile migration.",
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