Analysis of Glycan Polymers Produced by Peptidoglycan Glycosyltransferases

Barrett, Dianah; Wang, Tsung-Shing Andrew; Yuan, Yanqiu; Zhang, Yi; Kahne, Daniel; Walker, Suzanne

doi:10.1074/jbc.m705440200

Cited by 80 publications

(124 citation statements)

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“…The reactions were analyzed using a recently developed gel electrophoresis assay. 3a, 6 In the absence of Lipid II, the distribution of oligomers was unchanged (compare Lanes 3, 6, and 8 to Control Lane 2, Figure 3B). In the presence of Lipid II, the oligomers disappeared and a smear of radioactivity appeared higher up in the gel (lanes 4, 5, 7, and 9, Figure 3B), indicating that longer peptidoglycan products were formed.…”

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confidence: 87%

“…Nascent chains containing three or more disaccharide subunits meet this criterion. 6 To synthesize the longer Gal-blocked substrates ( Figure 3A) required to test the direction of chain elongation, we incubated compound 3 with a high concentration of E. coli PBP1A. Under these conditions, 3 reacted to give a mixture of products in which the major product, identified by its exact mass, was the octasaccharide Lipid VIII (5, Figure 1; Table S1).…”

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The Direction of Glycan Chain Elongation by Peptidoglycan Glycosyltransferases

et al. 2007

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Peptidoglycan glycosyltransferases (PGTs) are highly conserved enzymes that catalyze the polymerization of Lipid II to form the glycan strands of bacterial murein. Because they play a key role in bacterial cell wall synthesis, these enzymes are potentially important antibiotic targets; however, their mechanisms are not yet understood. One longstanding question about these enzymes is whether they elongate glycan chains by adding subunits to the anomeric (reducing) end or to the 4-hydroxyl (non-reducing) end. We have developed an approach to test the direction of chain elongation that involves the use of nascent peptidoglycan chains which are blocked at their non-reducing ends. In the presence of the PGT domains of Staphylococcus aureus PBP2, Aquifex aeolicus PBP1A, Escherichia coli PBP1A or Escherichia coli PBP1B, these blocked substrates react with Lipid II to form longer glycan chains. These results establish that PGTs elongate nascent peptidoglycan chains by the addition of disaccharide subunits to the anomeric (reducing) end of the growing polymer.Peptidoglycan glycosyltransferases (PGTs) are highly conserved bacterial enzymes that catalyze the polymerization of a disaccharide called Lipid II (Figure 1) to form the glycan strands of peptidoglycan. PGTs are regarded as desirable antibiotic targets because they are extracellular, they do not have eukaryotic counterparts, and they play an essential role in a validated therapeutic pathway. 1 Although their importance has been appreciated for decades, the mechanism of glycan chain polymerization is not yet understood. One aspect of the PGT reaction that is central to defining the mechanism is the direction of glycan chain elongation ( Figure 2A). 2 Here, we describe an approach to test the direction of glycan chain growth. We have applied this strategy to four different PGTs from both Gram-negative and Grampositive organisms, including two PGTs for which crystal structures were recently reported. 3 The results show that all these PGTs polymerize Lipid II by the addition of new disaccharide units to the anomeric (diphospholipid) end of the elongating polymer (called the "reducing end" by convention, although not a lactol).Our strategy to determine the direction of glycan polymerization, illustrated in Figure 2B, relies on the synthesis of nascent peptidoglycan chains that are blocked at their non-reducing ends ( Figure 2B). If elongation occurs by reaction at the reducing end of the growing polymer, PGTs should elongate these substrates in the presence of Lipid II ( Figure 2B NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript right). If elongation occurs in the other direction, then these blocked oligomers will not be substrates for PGTs ( Figure 2B, left).To enable our strategy for probing the direction of chain elongation, we required a facile method to selectively modify the non-reducing ends of the nascent glycan chains. We chose to utilize β-1,4-galactosyltransferase (GalT), an enzyme that transfers galactose (Gal) from UDP-Gal ...

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The Direction of Glycan Chain Elongation by Peptidoglycan Glycosyltransferases

et al. 2007

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“…However, a multiply defective mutant lacking six (Stl70, MltA, MltB, MltC, MltD, and MltE) out of the seven identified LTs exhibited no significant decrease of the 1,6-anhydro-MurNAc muropeptide content (92). Moreover, the PG material synthesized in vitro by purified glycosyltransferases remained bound to undecaprenyl pyrophosphate (8,300). Thus, neither glycosyltransferases nor most LTs appear to be directly involved in the termination step.…”

Section: 6-anhydro-n-acetylmuramic Acidmentioning

confidence: 98%

Peptidoglycan Hydrolases of Escherichia coli

Heijenoort

2011

Microbiol Mol Biol Rev

187

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SUMMARY The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway.

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“…Reactions were initiated on addition of proteins to lipid-II (4 μM) and incubated at room temperature for 15 min before heat inactivation (90°C, 10 min). Gel electrophoresis analysis of the products was carried out as described previously (22).…”

Section: Methodsmentioning

confidence: 99%

Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity

Markovski

Bohrhunter

Lupoli

et al. 2016

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

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To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a of Escherichia coli require the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b-LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed.T he peptidoglycan (PG) layer forms a protective shell that surrounds the cytoplasmic membrane of bacteria to prevent osmotic rupture and provide cells with their characteristic shape (1). This complex macromolecule is composed of glycan strands crosslinked to one another by attached peptide chains to form the exoskeletal matrix. Because of its essentiality and uniqueness to bacteria, the PG layer is an important therapeutic target. Many antibiotics in our current arsenal block PG assembly, with penicillin and related beta-lactam drugs being the most wellknown and widely used. These molecules target major PG synthase enzymes called penicillin-binding proteins (PBPs) (1).The PBPs function in the final stage of the three-part pathway for PG biogenesis (1). Precursor synthesis begins in the cytoplasm with the production of the activated sugar molecules uridine diphosphate (UDP)-N-acetylmuramic acid pentapeptide (UDP-MurNAc-pep 5 ) and UDP-N-acetylglucosamine (UDPGlcNAc). In the second, membrane-associated phase, UDPMurNAc-pep 5 is converted to the precursor lipid-I by MraY, which transfers phospho-MurNAc-pep to the lipid carrier undecaprenol-phosphate (Und-P). Lipid-II is formed by MurG via the addition of GlcNAc to lipid-I from UDP-GlcNAc. This final precursor contains the basic monomeric unit of PG, the disaccharide-pentapeptide. After its production, lipid-II must be flipped by MurJ (2, 3) ...

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Analysis of Glycan Polymers Produced by Peptidoglycan Glycosyltransferases

Cited by 80 publications

References 32 publications

The Direction of Glycan Chain Elongation by Peptidoglycan Glycosyltransferases

The Direction of Glycan Chain Elongation by Peptidoglycan Glycosyltransferases

Peptidoglycan Hydrolases of Escherichia coli

Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity

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