Screen for inhibitors of the coupled transglycosylase-transpeptidase of peptidoglycan biosynthesis in Escherichia coli.
Class A high-molecular-weight penicillin-binding protein 1a (PBP1a) and PBP1b of Escherichia coli have both transglycosylase (TG) and transpeptidase (TP) activity. These enzymes are difficult to assay, since their substrates are difficult to prepare. We show the activity of PBP1a or PBP1b can be measured in membranes by cloning the PBP into an E. coli ponB::Spcr strain. Using this assay, we show that PBP1a is approximately 10-fold more sensitive to penicillin than PBP1b and that the 50% inhibitory concentration (IC50) of moenomycin, a TG inhibitor, is approximately 10-fold higher in the PBP transformants than in wild-type membranes; this increase in IC50 in transformants can be used to test the specificity of test compounds for inhibition of the TG. Alternatively, the coupled TG-TP activity of PBP1b can be directly measured in a two-step microplate assay. In the first step, radiolabeled lipid II, the TG substrate, was made in membranes of the E. coli ponB::Spcr strain by incubation with the peptidoglycan sugar precursors. In the second step, the TG-TP activity was assayed by adding a source of PBP1b to the membranes. The coupled TG-TP activity converts lipid II to cross-linked peptidoglycan, which was specifically captured by wheat germ agglutinin-coated scintillation proximity beads in the presence of 0.2% Sarkosyl (B. Chandrakala et al., Antimicrob. Agents Chemother. 48:30-40, 2004). The TG-TP assay was inhibited by penicillin and moenomycin as expected. Surprisingly, tunicamycin and nisin also inhibited the assay, and paper chromatography analysis revealed that both inhibited the transglycosylase. The assay can be used to screen for novel antibacterial agents.
AstraZeneca India Pvt. Ltd., Hebbal, Bellary Road, Bangalore 560 024, India.
Vasanthi
V
Ramachandran
B
B
Chandrakala
Vidya P
VP
Kumar
Veeraraghavan
V
Usha
Suresh M
SM
Solapure
Sunita M
SM
de Sousa
D002854:Chromatography, Paper; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors*; D046915:Penicillin-Binding Proteins / Q000037:antagonists & inhibitors*; D010457:Peptidoglycan / Q000096:biosynthesis*; D046928:Peptidoglycan Glycosyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010458:Peptidyl Transferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase / Q000037:antagonists & inhibitors*
D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan; D010458:Peptidyl Transferases; D046928:Peptidoglycan Glycosyltransferase; C478681:penicillin-binding protein 1B, E coli; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase