title: Structural evidence of a passive base-flipping mechanism for AGT, an unusual GT-B glycosyltransferase.
abstract: The Escherichia coli T4 bacteriophage uses two glycosyltransferases to glucosylate and thus protect its DNA: the retaining alpha-glucosyltransferase (AGT) and the inverting beta-glucosyltransferase (BGT). They glucosylate 5-hydroxymethyl cytosine (5-HMC) bases of duplex DNA using UDP-glucose as the sugar donor to form an alpha-glucosidic linkage and a beta-glucosidic linkage, respectively. Five structures of AGT have been determined: a binary complex with the UDP product and four ternary complexes with UDP or UDP-glucose and oligonucleotides containing an A:G, HMU:G (hydroxymethyl uracyl) or AP:G (apurinic/apyrimidinic) mismatch at the target base-pair. AGT adopts the GT-B fold, one of the two folds known for GTs. However, while the sugar donor binding mode is classical for a GT-B enzyme, the sugar acceptor binding mode is unexpected and breaks the established consensus: AGT is the first GT-B enzyme that predominantly binds both the sugar donor and acceptor to the C-terminal domain. Its active site pocket is highly similar to four retaining GT-B glycosyltransferases (trehalose-6-phosphate synthase, glycogen synthase, glycogen and maltodextrin phosphorylases) strongly suggesting a common evolutionary origin and catalytic mechanism for these enzymes. Structure-guided mutagenesis and kinetic analysis do not permit identification of a nucleophile residue responsible for a glycosyl-enzyme intermediate for the classical double displacement mechanism. Interestingly, the DNA structures reveal partially flipped-out bases. They provide evidence for a passive role of AGT in the base-flipping mechanism and for its specific recognition of the acceptor base.
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales, UPR 9063 CNRS, Bât.34, 91198-Gif-sur-Yvette, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Larivière
authors.forename: Nicole
authors.initials: N
authors.lastname: Sommer
authors.forename: Solange
authors.initials: S
authors.lastname: Moréra
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000821:virology; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D014532:Uridine Diphosphate Glucose / Q000737:chemistry / Q000378:metabolism
chemical_list: D016695:Glycosyltransferases; D014532:Uridine Diphosphate Glucose