Acyl-acyl carrier protein specificity of UDP-GlcNAc acyltransferases from gram-negative bacteria: relationship to lipid A structure

Williamson, Joanne M.; Anderson, Matt S.; Raetz, C. R. H.

doi:10.1128/jb.173.11.3591-3596.1991

Cited by 65 publications

(64 citation statements)

References 28 publications

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“…The corresponding coenzyme A thioesters are not substrates (8,25). E. coli LpxA is highly selective for R-3-hydroxymyristoyl-ACP (8,25), whereas the Pseudomonas aeruginosa enzyme strongly prefers R-3-hydroxydecanoyl-ACP (35,36). The G173M substitution converts the E. coli enzyme from a 14-to a 10-carbonspecific acyltransferase, whereas the converse M169G substitution in P. aeruginosa LpxA does the opposite (10).…”

Section: Discussionmentioning

confidence: 99%

“…The acyl-ACP donor selectivity of LpxA has previously been studied in several systems (8,17,24,(35)(36)(37). In general, LpxA orthologs show strong preferences for acyl chain length and the presence of the R-3-hydroxyl group (8,17,24,(35)(36)(37).…”

Section: Discussionmentioning

confidence: 99%

“…In general, LpxA orthologs show strong preferences for acyl chain length and the presence of the R-3-hydroxyl group (8,17,24,(35)(36)(37). The corresponding coenzyme A thioesters are not substrates (8,25).…”

Section: Discussionmentioning

confidence: 99%

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Enzymatic Synthesis of Lipid A Molecules with Four Amide-linked Acyl Chains

Sweet

Williams

Karbarz

et al. 2004

Journal of Biological Chemistry

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LpxA of Escherichia coli catalyzes the acylation of the glucosamine 3-OH group of UDP-GlcNAc, using R-3-hydroxymyristoyl-acyl carrier protein (ACP) as the donor substrate. We now demonstrate that LpxA in cell extracts of Mesorhizobium loti and Leptospira interrogans, The mechanism of L. interrogans LpxA appears to be similar to that of E. coli LpxA, given that the essential His 125 residue of E. coli LpxA is conserved and is also required for acyltransferase activity in L. interrogans. Acidithiobacillus ferrooxidans (an organism that makes lipid A molecules containing both GlcN and GlcN3N) has an ortholog of LpxA that is selective for UDP-GlcNAc3N, but the enzyme also catalyzes the acylation of UDPGlcNAc at a slow rate. E. coli LpxA acylates UDP-GlcNAc and UDP-GlcNAc3N at comparable rates in vitro. However, UDP-GlcNAc3N is not synthesized in vivo, because E. coli lacks gnnA and gnnB. When the latter are supplied together with A. ferrooxidans lpxA, E. coli incorporates a significant amount of GlcN3N into its lipid A.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Enzymatic Synthesis of Lipid A Molecules with Four Amide-linked Acyl Chains

Sweet

Williams

Karbarz

et al. 2004

Journal of Biological Chemistry

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“…1) and is essential for growth in almost all Gram-negative pathogens (2). E. coli LpxA, which contains a glycine residue at position 173, is highly selective for a 14-carbon R-3-hydroxyacyl chain activated on ACP, whereas P. aeruginosa LpxA, which contains a methionine residue at the equivalent position, is selective for 10 carbons (8,36). Indeed, the G173M mutation converts E. coli LpxA into a C10 enzyme, whereas the reciprocal M169G substitution converts P. aeruginosa LpxA to a C14 acyltransferase (8).…”

Section: Discussionmentioning

confidence: 99%

Structure of UDP-N-acetylglucosamine acyltransferase with a bound antibacterial pentadecapeptide

Williams

Immormino

Gewirth

et al. 2006

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

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UDP-GlcNAc acyltransferase (LpxA) catalyzes the first step of lipid A biosynthesis, the transfer of the R-3-hydroxyacyl chain from R-3-hydroxyacyl acyl carrier protein (ACP) to the glucosamine 3-OH group of UDP-GlcNAc. LpxA is essential for the growth of Escherichia coli and related Gram-negative bacteria. The crystal structure of the E. coli LpxA homotrimer, determined previously at 2.6 Å in the absence of substrates or inhibitors, revealed that LpxA contains an unusual, left-handed parallel ␤-helix fold. We now present the crystal structure at 1.8 Å resolution of E. coli LpxA in a complex with a pentadecapeptide, peptide 920. Three peptides, each of which adopts a ␤-hairpin conformation, are bound per LpxA trimer. The peptides are located at the interfaces of adjacent subunits in the vicinity of the three active sites. Each peptide interacts with residues from both adjacent subunits. Peptide 920 is a potent inhibitor of E. coli LpxA (Ki ‫؍‬ 50 nM). It is competitive with respect to acyl-ACP but not UDP-GlcNAc. The compact ␤-turn structure of peptide 920 bound to LpxA may open previously uncharacterized approaches to the rational design of LpxA inhibitors with antibiotic activity.antibiotic ͉ crystal structure ͉ Escherichia coli ͉ inhibitor ͉ outer membrane L ipopolysaccharide constitutes the outer monolayer of the outer membrane in Gram-negative bacteria (1-3). Lipopolysaccharide maintains the integrity of the outer membrane, which functions as a barrier to molecules Ͼ500 Da (4). Lipid A (endotoxin) is the hydrophobic moiety that anchors lipopolysaccharide into the outer membrane (1, 2). It is a potent activator of the human innate immune system via Toll-like receptor 4 (TLR4) (5-7). The minimal lipopolysaccharide required for growth usually consists of two 3-deoxy-D-manno-octulosonic acid (Kdo) residues attached to lipid A ( Fig. 1; ref. 2). Because lipid A biosynthetic enzymes have no mammalian counterparts, they are attractive targets for the design of new antibiotics (10, 11).Kdo 2 -lipid A is synthesized by a conserved pathway consisting of nine enzymes (1, 2). LpxA catalyzes the first step (Fig. 1), the thermodynamically unfavorable 3-O-acylation of UDP-GlcNAc (K eq ϭ 0.01) (12-14). The crystal structure of Escherichia coli LpxA, previously determined at 2.6 Å (15), revealed that the enzyme is a homotrimer. It adopts a distinctive, left-handed parallel ␤-helix fold ( Fig. 2 A and B), which is specified by the presence of 24 complete and six partial hexad repeats (15). Three contiguous hexad repeats (18-aa residues) fold into one coil of the ␤-helix (Fig. 2 A and B). Many other bacterial acyl and acetyl transferases later were shown to possess this fold (17)(18)(19)(20)(21)(22).Structures of LpxA have not been solved in the presence of substrates or inhibitors. Mutagenesis suggests that the active site is located in a large cleft between adjacent subunits (8, 23). This region contains several conserved basic residues (K76, H122, H125, H144, H160, and R204) (23). H125 (Fig. 2 C and D) may be the catalytic ...

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“…R-3-hydroxydecanoyl-ACP is used at Ϸ0.1% the rate of R-3-hydroxymyristoyl-ACP (18,19). Pseudomonas aeruginosa LpxA is maximally active with R-3-hydroxydecanoyl-ACP, which it prefers over R-3-hydroxymyristoyl-ACP by a factor of 1,000 (18)(19)(20). The in vitro fatty acyl chain length selectivity of these LpxA orthologues is consistent with the structures of the lipid A molecules isolated from E. coli and P. aeruginosa (2).…”

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confidence: 99%