Premium
Development of a liquid chromatography/mass spectrometry assay for the bacterial transglycosylation reaction through measurement of Lipid II
Author(s) -
Blanchaert Bart,
Wyseure Tine,
Breukink Eefjan,
Adams Erwin,
Declerck Paul,
Schepdael Ann
Publication year - 2015
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201500201
Subject(s) - chemistry , recombinant dna , lipid ii , chromatography , substrate (aquarium) , escherichia coli , liquid chromatography–mass spectrometry , penicillin binding proteins , peptidoglycan , mass spectrometry , biochemistry , glycosyl , enzyme , affinity chromatography , staphylococcus aureus , bacteria , biology , ecology , genetics , gene
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.