Structural basis for the acyl chain selectivity and mechanism of UDP-
            <i>N</i>
            -acetylglucosamine acyltransferase

Williams, Allison H.; Raetz, Christian R. H.

doi:10.1073/pnas.0705833104

Cited by 71 publications

(152 citation statements)

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“…8A), the catalytic base as determined by structural studies and mutagenesis of LpxA (15)(16)(17). As shown by the 1000-fold reduction in k cat of the H239A mutant (Table 4), H239 of EcLpxD is now directly implicated as the catalytic base.…”

Section: Discussionmentioning

confidence: 92%

“…The equivalent residue (G143 in E. coli LpxA) is likewise present in all LpxA sequences, and its function as the oxyanion hole can be inferred directly from structural studies of the LpxA/3-O-(R-3-hydroxymyristoyl)-GlcNAc product complex (Fig. 8A) in which the G143 backbone NH group of LpxA is appropriately positioned (17).…”

Section: Discussionmentioning

confidence: 99%

“…The side chain of D126 serves this purpose for H125 in E. coli LpxA (15,17), whereas a backbone threonine carbonyl group plays this role in the P. aeuroginosa xenobiotic acetyltransferase (12) and an aspartate side chain is utilized in E. coli serine acetyl transferase (47). Given the conservation of D126 in most LpxA sequences, the homologous EcLpxD N240 residue seemed to be a possible candidate for interaction with EcLpxD H239.…”

Section: Discussionmentioning

confidence: 99%

“…1A) (9,41). A crystal structure of C. trachomatis LpxD has recently appeared (14), demonstrating that LpxD resembles LpxA (10,17,42,43) in its homotrimeric architecture and β-helical fold (Figs. 1B and 1C).…”

Section: Discussionmentioning

confidence: 99%

“…When H239 is mutated to alanine, the k cat of EcLpxD is reduced about 1000-fold with very little effect on the K M of either substrate (Table 4). Conserved in all LpxD sequences, H239 of EcLpxD (equivalent to H247 of CtLpxD) (14) also aligns with H125 in E. coli LpxA (15,17); the Nε2 atom of the latter is the general base in LpxA, being situated within H-bonding distance of the GlcNAc 3-OH group in the LpxA structure and functioning to activate it for attack on the thioester carbonyl moiety of acyl-ACP (16,17). Our suggestion that H239 of EcLpxD is the catalytic base is, however, inconsistent with the CtLpxD structure (Table 3) (14).…”

Section: Site Directed Mutagenesis Of Eclpxd Group I Residuesmentioning

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: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Site Directed Mutagenesis Of Eclpxd Group I Residuesmentioning

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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Characterization of left‐handed beta helix‐domains, and identification and functional annotation of proteins containing such domains

Prabha

Balaji

2020

Proteins

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Only about 0.3% of the entries in UniProt database have manually curated annotation. Annotation at the molecular level often relies on low‐throughput one‐protein‐at‐a‐time approach. Computational methods bridge this gap by assigning function based on sequence and/or fold similarity. Left‐handed beta helix (LbH) consists of three repeating six‐stranded beta‐strands forming an 18‐mer turn of the helix. Analysis of LbH‐domains showed that variations are found in the number of residues in a beta‐strand (5‐7, 6 being the most common), number of turns (4–10) of the helix, insertions of one or more loops of variable length (0‐36 residues), and the location of loop insertion. An 18‐mer HMM profile was created which identifies LbH‐domain containing proteins using sequence as the only input; the number of false positives is zero when proteins tested were those with known 3D structures. 136 474 entries of TrEMBL database were found to contain LbH‐domain. Rules developed by analyzing LbH‐domain containing acyltransferases, gamma‐class carbonic anhydrases, and nucleotidyltransferases have led to the annotation of 17 389 TrEMBL entries which currently have no functional tag.

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Structural basis for the acyl chain selectivity and mechanism of UDP- N -acetylglucosamine acyltransferase

Abstract: UDP-N-acetylglucosamine (UDP-GlcNAc

Cited by 71 publications

References 49 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?

Characterization of left‐handed beta helix‐domains, and identification and functional annotation of proteins containing such domains

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