Synthesis of Heptaprenyl−Lipid IV to Analyze Peptidoglycan Glycosyltransferases

Zhang, Yi; Fechter, Eric J.; Wang, Tsung-Shing Andrew; Barrett, Dianah; Walker, Suzanne; Kahne, Daniel

doi:10.1021/ja069060g

Cited by 72 publications

(82 citation statements)

References 26 publications

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“…Under steady-state conditions, a ladder of products is observed with no accumulation of short products. Because we have previously established that elongated glycan strands such as Lipid IV are poor substrates for PGTs compared with Lipid II (26), and thus would not be expected to rebind and react faster than Lipid II, this distribution is consistent with a processive mechanism in which elongation occurs without release of the growing polymer chain rather than a distributive mechanism in which product release occurs before the next reaction cycle. The topology of the active site cleft, combined with the location of the two carboxylates, allows us to propose a model in which the elongating glycan chain (the glycosyl donor) is bound so that the reacting end of the molecule is anchored near E83/D84 behind the flap that folds over the cleft, whereas the lipid chain extends down past the hydrophobic patch and into the membrane (Fig.…”

Section: Resultssupporting

confidence: 63%

Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis

Yuan

Barrett²,

Zhang³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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138

122

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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 ␤-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-Å 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 -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.antibiotic resistance ͉ penicillin-binding protein ͉ cell wall ͉ transglycosylase T he major component of the bacterial cell wall is a cross-linked glycopeptide polymer called peptidoglycan. This polymer surrounds the cytoplasmic membrane of bacteria and functions as an exoskeleton, maintaining cell shape and stabilizing the membrane against fluctuations in osmotic pressure. Peptidoglycan is synthesized in an intracellular phase in which UDP-Nacetylglucosamine is converted to a diphospholipid-linked disaccharide-pentapeptide known as Lipid II, and in an extracellular phase in which the disaccharide (NAG-NAM) subunits of translocated Lipid II are coupled by peptidoglycan glycosyltransferases (PGTs; also known as transglycosylases) to form linear carbohydrate chains ( Fig. 1), which are cross-linked through the attached peptide moieties by transpeptidases (1). A functioning peptidoglycan pathway is required for bacterial cell growth and division, and compounds that inhibit peptidoglycan biosynthesis have antibiotic activity (2). For example, the ␤-lactams, which have been used for decades to treat bacterial infections, irreversibly inhibit the transpeptidases. The emergence of resistance to ␤-lactams and other clinically used antibiotics has prompted intense interest in understanding peptidoglycan biosynthesis in detail. Major advances have been made in recent years with respect to the characterization of key biosynthetic enzymes (3-5). Several structures have been reported for enzymes that catalyze transpeptidation (6, 7), but no PGT structures have been described.PGTs are defined by the presence of five conserved sequence motifs ( Fig. 2A) and exist in two forms: (i) as N-terminal glycosyltransferase domains in bifunctional proteins that also contain a C-terminal transpeptidase domain [called class A penicillin-bindi...

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Section: Resultssupporting

confidence: 63%

Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis

Yuan

Barrett²,

Zhang³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

138

122

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“…Cloning, Expression, and Purification of PGT ConstructsThe cloning, expression, and purification of the PGT domain of A. aeolicus ⌬PBP1A(Asn 29 -Lys 243 ) and full-length E. coli PBP1A(Met 1 -Phe 850 ) have been described previously (15,21). The isolated PGT domain of E. coli PBP1A was obtained as follows.…”

Section: Methodsmentioning

confidence: 99%

“…Alternatively, an acetyl group can be incorporated on the lysines of the substrate peptide chains, with no effect on substrate utility, by treatment with 14 C-acetic anhydride to give (1c) or 14 C-acetic anhydride to give radiolabeled (2b) as described in (21).…”

Section: Establishing a Gel Electrophoresis Assay For The Separation Ofmentioning

confidence: 99%

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Analysis of Glycan Polymers Produced by Peptidoglycan Glycosyltransferases

Barrett

Wang

Yuan

et al. 2007

Journal of Biological Chemistry

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121

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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.

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“…Despite these advantages, only a very limited number of examples of partially protected glycosyl donors have been reported in the literature. [16][17][18] In order to avoid unwanted polymerisation reactions, the use of inverse glycosylation conditions is usually employed with these donors, maintaining a higher concentration of the acceptor relative to the donor during the glycosylation.…”

Section: Introductionmentioning

confidence: 99%

An unusual glycosylation product from a partially protected fucosyl donor under silver triflate activation conditions

Daly

Scanlan

2013

Org. Biomol. Chem.

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Partially protected glycosyl donors are extremely useful reagents for oligosaccharide synthesis allowing more facile deprotection and enhanced activity due to lower steric restraints. A partially protected fucosyl donor containing tert-butyldimethylsilyl (TBDMS) protecting groups was activated under bromine-silver triflate conditions in the presence of primary alcohols and found to give difucoside products exclusively, in good yield with excellent diastereoselectivity. The dimerisation reaction appears to require a conformational relaxation of steric crowding, induced upon activation of the glycosyl donor. The scope and limitations of this unusual glycosylation methodology are reported.

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Synthesis of Heptaprenyl−Lipid IV to Analyze Peptidoglycan Glycosyltransferases

Cited by 72 publications

References 26 publications

Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis

Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis

Analysis of Glycan Polymers Produced by Peptidoglycan Glycosyltransferases

An unusual glycosylation product from a partially protected fucosyl donor under silver triflate activation conditions

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