The 1.9 Å crystal structure of <i>Escherichia coli</i> MurG, a membrane‐associated glycosyltransferase involved in peptidoglycan biosynthesis

Ha, Sha; Walker, Deborah; Shi, Yigong; Walker, Suzanne

doi:10.1110/ps.9.6.1045

Cited by 246 publications

(267 citation statements)

References 26 publications

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“…The structure of the UDPGlcNAc:MurG complex (Fig. 1) shows that the UDP-GlcNAc moiety mostly contacts the C-terminal domain, which was previously proposed to be the donor-binding site based on the presence of a sequence motif that is found in most members of the GT-B superfamily (8,18). This sequence motif consists of a pattern of prolines and glycines on which are grafted other characteristic residues.…”

Section: Resultsmentioning

confidence: 86%

“…The x-ray structure of the MurG:UDP-GlcNAc complex was solved by molecular replacement by using the free enzyme as a search model (Table 1) (8). As in the free enzyme, there are two protein molecules in the asymmetric unit.…”

Section: Resultsmentioning

confidence: 99%

“…Protein expressed from pET21b and containing the C-terminal tag LEHHHHHH (designated C1 MurG) was purified as described previously (8) and concentrated to 10 mg͞ml in a storage buffer consisting of 20 mM Tris⅐HCl (pH 7.9), 150 mM NaCl, and 50 mM EDTA. The protein concentrate was stored at Ϫ70°C in 50-or 100-l aliquots and thawed on use.…”

Section: Methodsmentioning

confidence: 99%

“…These enzymes have low sequence homology, and because there was no structural information on them until recently, they have been classified into dozens of different families (1) (http:͞͞afmb.cnrs-mrs.fr͞ϳcazy͞CA ZY͞in-dex.html). In the past few years, 11 different UDP͞TDP-GTase structures from 10 different families have been reported (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), but all belong to only two different structural superfamilies, GT-A and GT-B (16). Eight of the eleven crystal structures obtained to date belong to the GT-A superfamily, which includes most of the GTases found in the Golgi apparatus and the endoplasmic reticulum.…”

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confidence: 99%

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Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases

Chen

et al. 2003

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

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MurG is an essential glycosyltransferase that forms the glycosidic linkage between N-acetyl muramyl pentapeptide and N-acetyl glucosamine in the biosynthesis of the bacterial cell wall. This enzyme is a member of a major superfamily of NDP-glycosyltransferases for which no x-ray structures containing intact substrates have been reported. Here we present the 2.5-Å crystal structure of Escherichia coli MurG in complex with its donor substrate, UDPGlcNAc. Combined with genomic analysis of other superfamily members and site-specific mutagenesis of E. coli MurG, this structure sheds light on the molecular basis for both donor and acceptor selectivity for the superfamily. This structural analysis suggests that it will be possible to evolve new glycosyltransferases from prototypical superfamily members by varying two key loops while maintaining the overall architecture of the family and preserving key residues.

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Section: Resultsmentioning

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases

Chen

et al. 2003

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

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236

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“…A genetic approach indicated that the N terminus of nisin is involved in the interaction with Lipid II (6), and more recently we could map the binding interface toward specific residues in the N terminus using 15 N-labeled nisin (7). It is not clear yet what events lead to pore formation after the initial interaction.…”

mentioning

confidence: 99%

Lipid II Is an Intrinsic Component of the Pore Induced by Nisin in Bacterial Membranes

Breukink

Heusden

Vollmerhaus

et al. 2003

Journal of Biological Chemistry

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307

311

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The peptidoglycan layers surrounding bacterial membranes are essential for bacterial cell survival and provide an important target for antibiotics. Many antibiotics have mechanisms of action that involve binding to Lipid II, the prenyl chain-linked donor of the peptidoglycan building blocks. One of these antibiotics, the pore-forming peptide nisin uses Lipid II as a receptor molecule to increase its antimicrobial efficacy dramatically. Nisin is the first example of a targeted membranepermeabilizing peptide antibiotic. However, it was not known whether Lipid II functions only as a receptor to recruit nisin to bacterial membranes, thus increasing its specificity for bacterial cells, or whether it also plays a role in pore formation. We have developed a new method to produce large amounts of Lipid II and variants thereof so that we can address the role of the lipidlinked disaccharide in the activity of nisin. We show here that Lipid II is not only the receptor for nisin but an intrinsic component of the pore formed by nisin, and we present a new model for the pore complex that includes Lipid II.The cell wall is an essential structure of a bacterium, providing its shape and protecting it from bursting because of the high osmotic pressures of the cytoplasm. This wall is a threedimensional network built of identical subunits consisting of two amino sugars, N-acetylglucosamine (GlcNAc) and N-acetylmu-is attached to the carboxyl group of MurNAc. These subunits are assembled in the cytosol of bacteria using UDP-activated precursors on a special lipid carrier, undecaprenyl phosphate (for a review see Ref. 1). The integral membrane protein MraY and the peripherally membraneassociated MurG that synthesize the precursors Lipid I and II, respectively, are the key enzymes in the last two cytoplasmic steps in the formation of the subunits (Fig. 1). Subsequently, Lipid II is transported across the plasma membrane via an as of yet unknown mechanism. Thereafter, the subunits are polymerized and inserted into the pre-existing cell wall by means of the penicillin-binding proteins (for review see Ref.2). Numerous antibiotics target the cell wall synthesis, including a diverse group of antibiotics that bind to Lipid II. Perhaps the best known of these antibiotics is vancomycin, the antibiotic of last resort to treat MRSA infections (3). However, there are many others, including the polypeptide nisin, that kill cells by permeabilizing bacterial membranes. Efficient membrane permeabilization by nisin requires an interaction with Lipid II (4, 5). This designates nisin as the first example of a targeted poreforming peptide antibiotic.Two recent studies have shed light on the structural requirements within nisin for the interaction with Lipid II. A genetic approach indicated that the N terminus of nisin is involved in the interaction with Lipid II (6), and more recently we could map the binding interface toward specific residues in the N terminus using 15 N-labeled nisin (7). It is not clear yet what events lead to pore formation after the...

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Kohlenhydrate in der Antibiotikaforschung: ein neuer Ansatz zur Resistenzbekämpfung

Ritter

Wong

2001

Angew. Chem.

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The 1.9 Å crystal structure of Escherichia coli MurG, a membrane‐associated glycosyltransferase involved in peptidoglycan biosynthesis

Cited by 246 publications

References 26 publications

Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases

Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases

Lipid II Is an Intrinsic Component of the Pore Induced by Nisin in Bacterial Membranes

Kohlenhydrate in der Antibiotikaforschung: ein neuer Ansatz zur Resistenzbekämpfung

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