title: Functional characterization of the glycosyltransferase domain of penicillin-binding protein 1a from Thermotoga maritima.
abstract: Class A penicillin-binding proteins (A-PBPs) are high-molecular weight membrane-bound bifunctional enzymes that catalyze the penicillin-sensitive transpeptidation and transglycosylation reaction steps involved in peptidoglycan assembling. We have over-expressed and characterized a soluble form of the glycosyltransferase domain of PBP1a (GT-PBP1a*) from the hyperthermophilic bacteria Thermotoga maritima. GT-PBP1a* efficiently catalyses peptidoglycan biosynthesis, as shown using an in vitro biosynthetized dansylated-lipid II substrate and a HPLC-coupled assay, and is specifically inhibited by moenomycin. GT-PBP1a* tends to spontaneously aggregate in detergent-free solution, a feature that supports existence of a secondary site for membrane association, distinct from the N-terminal transmembrane anchoring region. Overall, our preliminary data document the biochemical properties of GT-PBP1a* and should guide further studies aimed at deciphering the structural determinants involved into membrane binding by this class of enzymes.
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Case 932, Campus de Luminy, F13288 Marseille Cedex 09, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Offant
authors.forename: François
authors.initials: F
authors.lastname: Michoux
authors.forename: Annabelle
authors.initials: A
authors.lastname: Dermiaux
authors.forename: Jacques
authors.initials: J
authors.lastname: Biton
authors.forename: Yves
authors.initials: Y
authors.lastname: Bourne
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D005990:Glycerol / Q000737:chemistry; D016695:Glycosyltransferases / Q000737:chemistry*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D020124:Thermotoga maritima / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D003902:Detergents; D046915:Penicillin-Binding Proteins; D016695:Glycosyltransferases; D005990:Glycerol