Optimization of conditions for the glycosyltransferase activity of penicillin-binding protein 1a from Thermotoga maritima.
Cell wall biosynthesis is a key target for antibacterial drugs. The major constituent of the bacterial wall, peptidoglycan, is a netlike polymer responsible for the size and shape of the cell and for resisting osmotic pressure. It consists of glycan chains of repeating disaccharide units cross-linked through short peptide chains. Peptidoglycan assembly is catalyzed by the periplasmic domain of bifunctional class A penicillin-binding proteins. Cross-linking of the peptide chains is catalyzed by their transpeptidase module, which can be inhibited by the most widely used antibiotics, the β-lactams. In contrast, no drug in clinical use inhibits the polymerization of the glycan chains, catalyzed by their glycosyltransferase module, although it is an obvious target. We report here the purification of the ectodomain of the class A penicillin-binding protein 1a from Thermotoga maritima (Tm-1a*), expressed recombinantly in Escherichia coli. A detergent screen showed that detergents with shorter aliphatic chains were better solubilizers. Cyclohexyl-hexyl-β-D-maltoside-purified Tm-1a* was found to be monomeric and to have improved thermal stability. A miniaturized, multiwell continuous fluorescence assay of the glycosyltransferase activity was used to screen for optimal reaction conditions. Tm-1a* was active as a glycosyltransferase, catalyzing the formation of glycan chains up to 16 disaccharide units long. Our results emphasize the importance of the detergent in preparing a stable monomeric ectodomain of a class A penicillin-binding protein. Our assay could be used to screen collections of compounds for inhibitors of peptidoglycan glycosyltransferases that could serve as the basis for the development of novel antibiotics.
Offant
Julien
J
CEA, Institut de Biologie Structurale, Grenoble, France.
Terrak
Mohammed
M
Derouaux
Adeline
A
Breukink
Eefjan
E
Nguyen-Distèche
Martine
M
Zapun
André
A
Vernet
Thierry
T
D003001:Cloning, Molecular / Q000379:methods; D003902:Detergents; D016503:Drug Delivery Systems; D057075:Enzyme Assays / Q000379:methods*; D016695:Glycosyltransferases / Q000378:metabolism*; D046915:Penicillin-Binding Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D020124:Thermotoga maritima / Q000201:enzymology*
D003902:Detergents; D046915:Penicillin-Binding Proteins; D016695:Glycosyltransferases