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J. Biol. Chem., Vol. 282, Issue 44, 31964-31971, November 2, 2007

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Analysis of Glycan Polymers Produced by Peptidoglycan Glycosyltransferases^*

Dianah Barrett, Tsung-Shing Andrew Wang, Yanqiu Yuan, Yi Zhang, Daniel Kahne¹, and Suzanne Walker²

From the Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115 and Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138

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.

Received for publication, July 2, 2007 , and in revised form, August 10, 2007.

^* This work was supported by National Institutes of Health Grant GM076710 and by a UNCF ·Merck Graduate Science Research Fellowship (to D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Experimental Procedures, Table 1–3, and Fig. S1.

¹ To whom correspondence may be addressed: Dept. of Chemistry and Chemical Biology, Harvard University, 12 Oxford St., Cambridge, MA 02138. Fax: 617-496-0215; E-mail: kahne{at}chemistry.harvard.edu. Ph.: 617-496-0208. ² To whom correspondence may be addressed: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Fax: 617-736-7664; E-mail: suzanne_walker{at}hms.harvard.edu. Ph.: 617-432-5488.

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