Development of a liquid chromatography/mass spectrometry assay for the bacterial transglycosylation reaction through measurement of Lipid II.
Transglycosylation is the second to last step in the production of bacterial peptidoglycan. It is catalyzed by a transglycosylation site in class A penicillin-binding proteins (PBPs) or monofunctional glycosyl transferases. Several potential inhibitors have been suggested and need to be tested for activity. In this article, we describe the development and validation of an LC/MS assay for Lipid II, the substrate for transglycosylation. The developed assay can be used to monitor the transglycosylation activity of Staphylococcus aureus PBP2. There was no need for modification of Lipid II with a fluorescent tag that could alter affinity of inhibitors toward Lipid II. Recombinant PBP2 was produced in Escherichia coli and has been tested for activity. This LC/MS method is suitable for a transglycosylation assay for PBP2 and since it is relatively fast, it can be used to test inhibitors.
Department of Pharmaceutical and Pharmacological sciences, Pharmaceutical analysis, KU Leuven - University of Leuven, Leuven, Belgium.
Bart
B
Blanchaert
Department of Pharmaceutical and Pharmacological sciences, Therapeutic and Diagnostic Antibodies, KU Leuven - University of Leuven, Leuven, Belgium.
Tine
T
Wyseure
Department of Biochemistry of Membranes, Bijvoet Center for Biomolecular Research, University of Utrecht, Utrecht, The Netherlands.
Eefjan
E
Breukink
Department of Pharmaceutical and Pharmacological sciences, Pharmaceutical analysis, KU Leuven - University of Leuven, Leuven, Belgium.
Erwin
E
Adams
Department of Pharmaceutical and Pharmacological sciences, Therapeutic and Diagnostic Antibodies, KU Leuven - University of Leuven, Leuven, Belgium.
Paul
P
Declerck
Department of Pharmaceutical and Pharmacological sciences, Pharmaceutical analysis, KU Leuven - University of Leuven, Leuven, Belgium.
Ann
A
Van Schepdael
Bacterial transglycosylation; Lipid II; Liquid chromatography/mass spectrometry assay; Penicillin-binding protein 2