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

• Published in: The Journal of biological chemistry Vol. 277; no. 42; pp. 39981 - 39988
• Main Authors: Shi, Wuxian, Sarver, Anne E, Wang, Ching C, Tanaka, Kelly S E, Almo, Steven C, Schramm, Vern L
• Format: Journal Article
• Language: English
• Published: United States 18.10.2002
• Subjects: Adenine - metabolism; Adenine Phosphoribosyltransferase - chemistry; Adenosine Monophosphate - metabolism; Amino Acid Sequence; Animals; Arginine - chemistry; Binding Sites; Catalysis; Catalytic Domain; Cloning, Molecular; Giardia lamblia - enzymology; Guanine - metabolism; Hydrogen Bonding; Kinetics; Ligands; Magnesium - metabolism; Models, Chemical; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Substrate Specificity; Tryptophan - chemistry
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M205596200

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.