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The β-acetoacetyl-acyl carrier protein synthase FabY is a key enzyme in the initiation of fatty acid biosynthesis in Pseudomonas aeruginosa. Deletion of fabY results in an increased susceptibility of P. aeruginosa in vitro to a number of antibiotics, including vancomycin and cephalosporins. Because antibiotic susceptibility can be influenced by changes in membrane lipid composition, we determined the total fatty acid profile of the ΔfabY mutant, which suggested alterations in the lipid A region of the lipopolysaccharide. The majority of lipid A species in the ΔfabY mutant lacked a single secondary lauroyl group, resulting in hypoacylated lipid A. Adding exogenous fatty acids to the growth media restored the wild-type antibiotic susceptibility profile and the wild-type lipid A fatty acid profile. We suggest that incorporation of hypoacylated lipid A species into the outer membrane contributes to the shift in the antibiotic susceptibility profile of the ΔfabY mutant.

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.

Peptidoglycan is an essential polymer that forms a protective shell around bacterial cell membranes. Peptidoglycan biosynthesis is the target of many clinically used antibiotics, including the beta-lactams, imipenems, cephalosporins, and glycopeptides. Resistance to these and other antibiotics has prompted interest in an atomic-level understanding of the enzymes that make peptidoglycan. Representative structures have been reported for most of the enzymes in the pathway. Until now, however, there have been no structures of any peptidoglycan glycosyltransferases (also known as transglycosylases), which catalyze formation of the carbohydrate chains of peptidoglycan from disaccharide subunits on the bacterial cell surface. We report here the 2.1-A crystal structure of the peptidoglycan glycosyltransferase (PGT) domain of Aquifex aeolicus PBP1A. The structure has a different fold from all other glycosyltransferase structures reported to date, but it bears some resemblance to lambda-lysozyme, an enzyme that degrades the carbohydrate chains of peptidoglycan. An analysis of the structure, combined with biochemical information showing that these enzymes are processive, suggests a model for glycan chain polymerization.

Wall teichoic acids (WTAs) are anionic polymers that coat the cell walls of Gram-positive bacteria. Because they are essential for survival or virulence in many organisms, the enzymes involved in the biosynthesis of WTAs are attractive antibiotic targets. The first committed step in the WTA biosynthetic pathway in Bacillus subtilis is catalyzed by TagA, which transfers N-acetylmannosamine (ManNAc) to the C4 hydroxyl of a membrane-anchored N-acetylglucosaminyl diphospholipid (GlcNAc-pp-undecaprenyl, lipid I) to make ManNAc-beta-(1,4)-GlcNAc-pp-undecaprenyl (lipid II). We have previously shown that TagA utilizes an alternative substrate containing a saturated C(13)H(27) lipid chain. Here we use unnatural substrates and products to establish the lipid preferences of the enzyme and to characterize the kinetic mechanism. We report that TagA is a metal ion-independent glycosyltransferase that follows a steady-state ordered Bi-Bi mechanism in which UDP-ManNAc binds first and UDP is released last. TagA shares homology with a large family of bacterial glycosyltransferases, and the work described here should facilitate structural analysis of the enzyme in complex with its substrates.

EryCIII is a desosaminyltransferase that converts an inactive macrolide precursor to a biologically active antibiotic. It may have potential for the synthesis of unnatural macrolides with useful biological activities. However, it has been difficult to reconstitute the activity of EryCIII in vitro. We report here that purified, inactive EryCIII can be converted to an active catalyst by the addition of another protein encoded in the same gene cluster, EryCII. The EryCII-treated protein retains activity even when EryCII is removed. We also show that AknT, an activator protein from an unrelated gene cluster, is capable of activating EryCIII. Although the mechanism of activation is not yet understood, we have concluded from these experiments that these antibiotic Gtf activator proteins do not function to deliver substrates to EryCIII and do not exert their effects by forming stable complexes with the Gtf during the glycosyltransfer reaction. We report that activated EryCIII is capable of utilizing an alternative sugar donor, so these results lay the groundwork for the production of novel macrolides.