title: Closed site complexes of adenine phosphoribosyltransferase from Giardia lamblia reveal a mechanism of ribosyl migration.
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 A 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 (Glu(61)-Ser(62)) 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 Glu(100) 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 Arg(63), 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-A 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.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Wuxian
authors.initials: W
authors.lastname: Shi
authors.forename: Anne E
authors.initials: AE
authors.lastname: Sarver
authors.forename: Ching C
authors.initials: CC
authors.lastname: Wang
authors.forename: Kelly S E
authors.initials: KS
authors.lastname: Tanaka
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.forename: Vern L
authors.initials: VL
authors.lastname: Schramm
mesh_terms: D000225:Adenine / Q000378:metabolism; D000228:Adenine Phosphoribosyltransferase / Q000737:chemistry*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D016829:Giardia lamblia / Q000201:enzymology*; D006147:Guanine / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D006147:Guanine; D014364:Tryptophan; D001120:Arginine; D000228:Adenine Phosphoribosyltransferase; D008274:Magnesium; D000225:Adenine