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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.
J Mol Biol. 2003 Jul 25; 330(5):1077-86.JM

Abstract

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.

Authors+Show Affiliations

Laboratoire d'Enzymologie et Biochimie Structurales UPR 9063 CNRS, Bât. 34, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

12860129

Citation

Larivière, Laurent, et al. "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." Journal of Molecular Biology, vol. 330, no. 5, 2003, pp. 1077-86.
Larivière L, Gueguen-Chaignon V, Moréra S. 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. J Mol Biol. 2003;330(5):1077-86.
Larivière, L., Gueguen-Chaignon, V., & Moréra, S. (2003). 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. Journal of Molecular Biology, 330(5), 1077-86.
Larivière L, Gueguen-Chaignon V, Moréra S. 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. J Mol Biol. 2003 Jul 25;330(5):1077-86. PubMed PMID: 12860129.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Larivière,Laurent, AU - Gueguen-Chaignon,Virginie, AU - Moréra,Solange, PY - 2003/7/16/pubmed PY - 2003/8/26/medline PY - 2003/7/16/entrez SP - 1077 EP - 86 JF - Journal of molecular biology JO - J Mol Biol VL - 330 IS - 5 N2 - 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. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/12860129/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_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022283603006351 DB - PRIME DP - Unbound Medicine ER -