Structure and reactivity of LpxD, the
            <i>N</i>
            -acyltransferase of lipid A biosynthesis

Buetow, Lori; Smith, Terry; Dawson, Alice; Fyffe, S.A.; Hunter, William N.

doi:10.1073/pnas.0606356104

Cited by 70 publications

(174 citation statements)

References 44 publications

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“…Unless otherwise noted, the 10 μl assay mixture contained 40 mM Hepes, pH 7.5, 0.02 mg/ml BSA, 0.1 nM pure wild-type E. coli LpxD (or up to 10 nM mutant LpxD), [α-32 P]-UDP-3-O-(R-3-OHC 14 )-GlcN (4 μM, 0.005-0.04 μCi/μl) and 6 μM R-3-OHC 14 -ACP. The concentration of UDP-3-O-(R-3-OHC 14 )-GlcN was determined using the UDP extinction coefficient of 9900 M −1 cm −1 (24). The components were equilibrated at 30 °C, and the reaction was started by addition of enzyme.…”

Section: The Lpxd Assaymentioning

confidence: 99%

“…G-50 spin columns were purchased from GE Healthcare Piscataway, NJ, and C 18 SepPak columns were purchased from Waters, Milford, MA. Substrates and reagents, such as R-3-OHC 14 -ACP, R/S-3-hydroxylauroyl-ACP (9), [α-32 P]-UDP-GlcNAc (9), and [α-32 P]-UDP-3-O-(R-3-OHC 14 )-GlcN (9,23), were prepared by minor modifications of published methods (see Supporting Information).…”

Section: Reagents and Materialsmentioning

confidence: 99%

“…After removal of the solvent, the plates were exposed overnight to a Molecular Dynamics PhosphorImager Screen. Percent of the [α-32 P]-UDP-3-O-(R-3-OHC 14 )-GlcN converted to [α-32 P]-UDP-2,3-diacylglucosamine was quantified with ImageQuant Software.…”

Section: The Lpxd Assaymentioning

confidence: 99%

“…We have purified untagged EcLpxD and an active N-terminally His 6 -tagged LpxD variant to near homogeneity. Based on the steady state kinetics and the effects of several inhibitors, we suggest a compulsory ordered mechanism in which R-3-OHC 14 -ACP binds prior to UDP-3-O-(R-3-OHC 14 )-GlcN, and UDP-2,3-diacylglucosamine dissociates prior to ACP (Scheme 1). Direct binding studies with the physiological LpxD substrates, R-3-OHC 14 -ACP and UDP-3-O-(R-3-OHC 14 )-GlcN (Fig.…”

mentioning

confidence: 99%

“…1A) (3). LpxD catalyzes the third reaction in this scheme, the R-3-hydroxymyristoyl-acyl carrier protein (R-3-OHC 14 (Fig. 1A).…”

mentioning

confidence: 99%

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Steady-State Kinetics and Mechanism of LpxD, the N-Acyltransferase of Lipid A Biosynthesis

2008

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LpxD catalyzes the third step of lipid A biosynthesis, the R-3-hydroxymyristoyl-acyl carrier protein (R-3-OHC 14 -ACP)-dependent N-acylation of UDP-3-O-(R-3-hydroxymyristoyl)-α-D-glucosamine [UDP-3-O-(R-3-OHC 14 )-GlcN]. We have now over-expressed and purified E. coli LpxD to homogeneity. Steady state kinetics suggest a compulsory ordered mechanism in which R-3-OHC 14 -ACP binds prior to UDP-3-O-(R-3-OHC 14 )-GlcN. The product, UDP-2,3-diacylglucosamine, dissociates prior to ACP; the latter is a competitive inhibitor against R-3-OHC 14 -ACP and a noncompetitive inhibitor against UDP-3-O-(R-3-OHC 14 )-GlcN. UDP-2-N-(R-3-hydroxymyristoyl)-α-D-glucosamine, obtained by mild base hydrolysis of UDP-2,3-diacylglucosamine, is a noncompetitive inhibitor against both substrates. Synthetic R-3-hydroxylauroylmethylphosphopantetheine is an uncompetitive inhibitor against R-3-OHC 14 -ACP and a competitive inhibitor against UDP-3-O-(R-3-OHC 14 )-GlcN, but R-3-hydroxylauroyl-methylphosphopantetheine is also a very poor substrate. A compulsory ordered mechanism is consistent with the fact that R-3-OHC 14 -ACP has a high binding affinity for free LpxD, whereas UDP-3-O-(R-3-OHC 14 )-GlcN does not. Divalent cations inhibit R-3-OHC 14 -ACP-dependent acylation but not R-3-hydroxylauroylmethylphosphopantetheine-dependent acylation, indicating that the acidic recognition helix of R-3-OHC 14 -ACP contributes to binding. The F41A mutation increases the K M for UDP-3-O-(R-3-OHC 14 )-GlcN 30-fold, consistent with aromatic stacking of the corresponding F43 side chain against the uracil moiety of bound UDP-GlcNAc in the x-ray structure of Chlamydia trachomatis LpxD. Mutagenesis implicates E. coli H239 but excludes H276 as the catalytic base, and neither residue is likely to stabilize the oxyanion intermediate.Lipid A is the hydrophobic moiety of lipopolysaccharide (LPS) 1 , which constitutes the outer leaflet of the outer membrane of most Gram-negative bacteria (1-3). The lipid A moiety of LPS is usually required for bacterial growth (3,4) and is a potent activator of the mammalian innate immune system via the TLR4/MD-2 complex (5,6). Over-production of cytokines due to excessive stimulation of TLR4/MD-2 may occur during severe Gram-negative infections and may contribute to the life-threatening complications of septic shock (7,8).The Kdo 2 -lipid A substructure of Escherichia coli LPS is synthesized by a conserved system of nine constitutive enzymes (Fig. 1A) (3). LpxD catalyzes the third reaction in this scheme, the R-3-hydroxymyristoyl-acyl carrier protein (R-3-OHC 14 (Fig. 1A). Although essential for growth and an excellent target for the design of new antibiotics (9), LpxD is one of the least characterized enzymes in the pathway. Kelly and Raetz identified *Author to whom correspondence should be addressed: C. R. H. Raetz at (919) 684-3384; Fax (919) 684-8885; raetz@biochem.duke.edu. 1 The abbreviations are: ACP, acyl carrier protein; Bis-Tris, 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol; CMC, critical micelle concentration;...

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Section: The Lpxd Assaymentioning

confidence: 99%

Section: Reagents and Materialsmentioning

confidence: 99%

Section: The Lpxd Assaymentioning

confidence: 99%

mentioning

confidence: 99%

“…1A) (3). LpxD catalyzes the third reaction in this scheme, the R-3-hydroxymyristoyl-acyl carrier protein (R-3-OHC 14 (Fig. 1A).…”

mentioning

confidence: 99%

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Steady-State Kinetics and Mechanism of LpxD, the N-Acyltransferase of Lipid A Biosynthesis

2008

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The Inhibition of Lipid A Biosynthesis—The Antidote Against Superbugs?

González‐Bello

2018

Advanced Therapeutics

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After many years of success in the battle against infectious diseases, ground is being lost in this fight with the worldwide increasing appearance of "superbugs," which are resistant to most antibiotics in clinical use. The impact of superbugs on the older population, healthcare-associated patients or patients with a compromised immune system is highly worrisome since no treatment options are available in some cases, especially for Gram-negative pathogens. Efforts are currently devoted to develop novel chemical entities with new mechanisms of action that can inactivate unexplored or underexplored bacterial objectives and to better understand bacterial behavior. The present article highlights the therapeutic potential of the enzymes involved in the biosynthesis of lipid A, which is the lipidic component of lipopolysaccharide-a lipid-anchored complex carbohydrate and a well-designed natural barrier to protect Gram-negative bacteria from external agents, such as antibiotics. An overview of the state-of-the-art inhibitors currently available along with the biochemical and structural knowledge of the enzyme/ligand complexes available is provided. This insight will contribute to the rational design of the next generation of inhibitors or the development of new ones for those promising targets for which inhibitors have not yet been developed.

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Crystal Structures of Acyl Carrier Protein in Complex with Two Catalytic Partners Show a Dynamic Role in Cellular Metabolism

Newmister

Sherman

2014

ChemBioChem

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Structure and reactivity of LpxD, the N -acyltransferase of lipid A biosynthesis

Abstract: Chlamydia trachomatis ͉ enzyme structure ͉ fatty acid binding ͉ enzyme mechanism

Cited by 70 publications

References 44 publications

Steady-State Kinetics and Mechanism of LpxD, the N-Acyltransferase of Lipid A Biosynthesis

Steady-State Kinetics and Mechanism of LpxD, the N-Acyltransferase of Lipid A Biosynthesis

The Inhibition of Lipid A Biosynthesis—The Antidote Against Superbugs?

Crystal Structures of Acyl Carrier Protein in Complex with Two Catalytic Partners Show a Dynamic Role in Cellular Metabolism

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