Analysis of glycan polymers produced by peptidoglycan glycosyltransferases.
Bacterial cells are surrounded by a cross-linked polymer called peptidoglycan, the integrity of which is necessary for cell survival. The carbohydrate chains that form the backbone of peptidoglycan are made by peptidoglycan glycosyltransferases (PGTs), highly conserved membrane-bound enzymes that are thought to be excellent targets for the development of new antibacterials. Although structural information on these enzymes recently became available, their mechanism is not well understood because of a dearth of methods to monitor PGT activity. Here we describe a direct, sensitive, and quantitative SDS-PAGE method to analyze PGT reactions. We apply this method to characterize the substrate specificity and product length profile for two different PGT domains, PBP1A from Aquifex aeolicus and PBP1A from Escherichia coli. We show that both disaccharide and tetrasaccharide diphospholipids (Lipid II and Lipid IV) serve as substrates for these PGTs, but the product distributions differ significantly depending on which substrate is used as the starting material. Reactions using the disaccharide substrate are more processive and yield much longer glycan products than reactions using the tetrasaccharide substrate. We also show that the SDS-PAGE method can be applied to provide information on the roles of invariant residues in catalysis. A comprehensive mutational analysis shows that the biggest contributor to turnover of 14 mutated residues is an invariant glutamate located in the center of the active site cleft. The assay and results described provide new information about the process by which PGTs assemble bacterial cell walls.
Barrett
Dianah
D
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Wang
Tsung-Shing Andrew
TS
Yuan
Yanqiu
Y
Zhang
Yi
Y
Kahne
Daniel
D
Walker
Suzanne
S
D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008055:Lipids / Q000032:analysis; D008954:Models, Biological; D046928:Peptidoglycan Glycosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010458:Peptidyl Transferases; D011135:Polysaccharides, Bacterial / Q000032:analysis*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008055:Lipids; D011135:Polysaccharides, Bacterial; D010458:Peptidyl Transferases; D046928:Peptidoglycan Glycosyltransferase
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