Assay for identification of inhibitors for bacterial MraY translocase or MurG transferase.
Bacterial peptidoglycan synthesis is a well-characterized system for targeting new antimicrobial drugs. Formation of the peptidoglycan precursors Lipid I and Lipid II is catalyzed by the gene products of mraY and murG, which are involved in the first and second steps of the lipid cycle reactions, respectively. Here we describe the development of an assay specific for identifying inhibitors of MraY or MurG, based on the detection of radiolabeled [(14)C]GlcNAc incorporated into Lipid II. Assay specificity is achieved with the biotin tagging of the Lipid I precursor UDP-MurNAc-pentapeptide. This allows for the separation and identification of lipid products produced by the enzymatic activity of the MraY and MurG proteins, and thus identification of specific inhibitors.
Branstrom
A A
AA
Department of Biological Research, IRL, Inc., Cranbury, New Jersey, 08512, USA. art@irl.incara.com
Midha
S
S
Longley
C B
CB
Han
K
K
Baizman
E R
ER
Axelrod
H R
HR
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019921:Biotinylation; D002473:Cell Wall / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000032:analysis*; D004926:Escherichia coli; D050356:Lipid Metabolism; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010457:Peptidoglycan / Q000378:metabolism; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives / Q000302:isolation & purification / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D010457:Peptidoglycan; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C002225:UDP-N-acetylmuramic acid pentapeptide; D014166:Transferases; D017351:N-Acetylglucosaminyltransferases; C064057:UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide)pyrophosphoryl-undecaprenol N-acetylglucosamine transferase; D017855:Transferases (Other Substituted Phosphate Groups); C079803:mraY protein, Bacteria