Crystal structures of the T4 phage beta-glucosyltransferase and the D100A mutant in complex with UDP-glucose: glucose binding and identification of the catalytic base for a direct displacement mechanism.
T4 phage beta-glucosyltransferase (BGT) is an inverting glycosyltransferase (GT) that transfers glucose from uridine diphospho-glucose (UDP-glucose) to an acceptor modified DNA. BGT belongs to the GT-B structural superfamily, represented, so far, by five different inverting or retaining GT families. Here, we report three high-resolution X-ray structures of BGT and a point mutant solved in the presence of UDP-glucose. The two co-crystal structures of the D100A mutant show that, unlike the wild-type enzyme, this mutation prevents glucose hydrolysis. This strongly indicates that Asp100 is the catalytic base. We obtained the wild-type BGT-UDP-glucose complex by soaking substrate-free BGT crystals. Comparison with a previous structure of BGT solved in the presence of the donor product UDP and an acceptor analogue provides the first model of an inverting GT-B enzyme in which both the donor and acceptor substrates are bound to the active site. The structural analyses support the in-line displacement reaction mechanism previously proposed, locate residues involved in donor substrate specificity and identify the catalytic base.
Larivière
Laurent
L
Laboratoire d'Enzymologie et Biochimie Structurales UPR 9063 CNRS, Bât. 34, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
Gueguen-Chaignon
Virginie
V
Moréra
Solange
S
D017122:Bacteriophage T4 / Q000201:enzymology*; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005947:Glucose / Q000737:chemistry; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics*; D007477:Ions; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014530:Uridine Diphosphate / Q000737:chemistry*; D014532:Uridine Diphosphate Glucose / Q000737:chemistry
D007477:Ions; D008670:Metals; D008345:Manganese; D014530:Uridine Diphosphate; D005964:Glucosyltransferases; D005947:Glucose; D014532:Uridine Diphosphate Glucose