scholarly journals Crystal structure and acyl chain selectivity of Francisella novicida LpxA, the first enzyme in lipid A biosynthesis

2010 ◽  
Vol 24 (S1) ◽  
Author(s):  
Sang Hoon Joo ◽  
Christian R. H. Raetz
Keyword(s):  
Crystal Structure ◽  
Lipid A ◽  
Acyl Chain ◽  
Francisella Novicida ◽  
Lipid A Biosynthesis

scholarly journals Crystal structure of LpxK: The tetraacyldisaccharide 4′‐kinase of lipid A biosynthesis

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
Ryan Paul Emptage ◽  
Kelly D. Daughtry ◽  
Charles W. Pemble ◽  
Christian R. H. Raetz
Keyword(s):  
Crystal Structure ◽  
Lipid A ◽  
Lipid A Biosynthesis

scholarly journals Substrate specificity of the pyrophosphohydrolase LpxH determines the asymmetry of Bordetella pertussis lipid A

2019 ◽  
Vol 294 (20) ◽  
pp. 7982-7989 ◽  
Author(s):  
Jesús Arenas ◽  
Elder Pupo ◽  
Eline de Jonge ◽  
Jesús Pérez-Ortega ◽  
Joerg Schaarschmidt ◽  
...  
Keyword(s):  
Chain Length ◽  
Bordetella Pertussis ◽  
Lipid A ◽  
Whooping Cough ◽  
Acyl Chain ◽  
Biosynthesis Pathway ◽  
Hydrophobic Moiety ◽  
Chain Length Specificity ◽  
Lipid A Biosynthesis ◽  
The Asymmetry

Lipopolysaccharides are anchored to the outer membrane of Gram-negative bacteria by a hydrophobic moiety known as lipid A, which potently activates the host innate immune response. Lipid A of Bordetella pertussis, the causative agent of whooping cough, displays unusual structural asymmetry with respect to the length of the acyl chains at the 3 and 3′ positions, which are 3OH-C10 and 3OH-C14 chains, respectively. Both chains are attached by the acyltransferase LpxA, the first enzyme in the lipid A biosynthesis pathway, which, in B. pertussis, has limited chain length specificity. However, this only partially explains the strict asymmetry of lipid A. In attempts to modulate the endotoxicity of B. pertussis lipid A, here we expressed the gene encoding LpxA from Neisseria meningitidis, which specifically attaches 3OH-C12 chains, in B. pertussis. This expression was lethal, suggesting that one of the downstream enzymes in the lipid A biosynthesis pathway in B. pertussis cannot handle precursors with a 3OH-C12 chain. We considered that the UDP-diacylglucosamine pyrophosphohydrolase LpxH could be responsible for this defect as well as for the asymmetry of B. pertussis lipid A. Expression of meningococcal LpxH in B. pertussis indeed resulted in new symmetric lipid A species with 3OH-C10 or 3OH-C14 chains at both the 3 and 3′ positions, as revealed by MS analysis. Furthermore, co-expression of meningococcal lpxH and lpxA resulted in viable cells that incorporated 3OH-C12 chains in B. pertussis lipid A. We conclude that the asymmetry of B. pertussis lipid A is determined by the acyl chain length specificity of LpxH.

scholarly journals Expression Cloning of a Pseudomonas Gene Encoding a Hydroxydecanoyl-Acyl Carrier Protein-Dependent UDP-GlcNAc Acyltransferase

1998 ◽  
Vol 180 (2) ◽  
pp. 330-337 ◽  
Author(s):  
Garry D. Dotson ◽  
Igor A. Kaltashov ◽  
Robert J. Cotter ◽  
Christian R. H. Raetz
Keyword(s):  
Lipid A ◽  
Acyl Carrier Protein ◽  
Acyl Chain ◽  
Expression Cloning ◽  
Temperature Sensitive ◽  
Carrier Protein ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli ◽  
Lipid A Biosynthesis

UDP-N-acetylglucosamine-3-O-acyltransferase (UDP-GlcNAc acyltransferase) catalyzes the first step of lipid A biosynthesis (M. S. Anderson and C. R. H. Raetz, J. Biol. Chem. 262:5159–5169, 1987). We here report the isolation of thelpxA gene of Pseudomonas aeruginosa from a library of Pseudomonas strain PAO1 expressed inEscherichia coli LE392 (J. Lightfoot and J. S. Lam, J. Bacteriol. 173:5624–5630, 1991). Pseudomonas lpxA encodes a 10-carbon-specific UDP-GlcNAc acyltransferase, whereas the E. coli transferase is selective for a 14-carbon acyl chain. Recombinant cosmid 1137 enabled production of a 3-hydroxydecanoyl-specific UDP-GlcNAc acyltransferase in E. coli. It was identified by assaying lysozyme-EDTA lysates of individual members of the library with 3-hydroxydecanoyl-acyl carrier protein (ACP) as the substrate. Cosmid 1137 contained a 20-kb insert ofP. aeruginosa DNA. The lpxA gene region was localized to a 1.3-kb SalI-PstI fragment. Sequencing revealed that it contains one complete open reading frame (777 bp) encoding a new lpxA homolog. The predictedPseudomonas LpxA is 258 amino acids long and contains 21 complete hexapeptide repeating units, spaced in approximately the same manner as the 24 repeats of E. coli LpxA. The P. aeruginosa UDP-GlcNAc acyltransferase is 54% identical and 67% similar to the E. coli enzyme. A plasmid (pGD3) containing the 1.3-kb SalI-PstI fragment complementedE. coli RO138, a temperature-sensitive mutant harboringlpxA2. LpxA assays of extracts of this construct indicated that it is >1,000-fold more selective for 3-hydroxydecanoyl-ACP than for 3-hydroxymyristoyl-ACP. Mass spectrometry of lipid A isolated from this strain by hydrolysis at pH 4.5 revealed [M-H]−1,684.5 (versus 1,796.5 for wild-type lipid A), consistent with 3-hydroxydecanoate rather than 3-hydroxymyristate at positions 3 and 3′.

scholarly journals Structural basis of the UDP-diacylglucosamine pyrophosphohydrolase LpxH inhibition by sulfonyl piperazine antibiotics

2020 ◽  
Vol 117 (8) ◽  
pp. 4109-4116 ◽  
Author(s):  
Jae Cho ◽  
Minhee Lee ◽  
C. Skyler Cochrane ◽  
Caroline G. Webster ◽  
Benjamin A. Fenton ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Lipid A ◽  
Acyl Chain ◽  
Structural Basis ◽  
Enzymatic Assays ◽  
Substituted Piperazine ◽  
Outer Membrane Permeability ◽  
Ligand Conformation ◽  
Further Development

The UDP-2,3-diacylglucosamine pyrophosphate hydrolase LpxH is an essential lipid A biosynthetic enzyme that is conserved in the majority of gram-negative bacteria. It has emerged as an attractive novel antibiotic target due to the recent discovery of an LpxH-targeting sulfonyl piperazine compound (referred to as AZ1) by AstraZeneca. However, the molecular details of AZ1 inhibition have remained unresolved, stymieing further development of this class of antibiotics. Here we report the crystal structure of Klebsiella pneumoniae LpxH in complex with AZ1. We show that AZ1 fits snugly into the L-shaped acyl chain-binding chamber of LpxH with its indoline ring situating adjacent to the active site, its sulfonyl group adopting a sharp kink, and its N-CF3–phenyl substituted piperazine group reaching out to the far side of the LpxH acyl chain-binding chamber. Intriguingly, despite the observation of a single AZ1 conformation in the crystal structure, our solution NMR investigation has revealed the presence of a second ligand conformation invisible in the crystalline state. Together, these distinct ligand conformations delineate a cryptic inhibitor envelope that expands the observed footprint of AZ1 in the LpxH-bound crystal structure and enables the design of AZ1 analogs with enhanced potency in enzymatic assays. These designed compounds display striking improvement in antibiotic activity over AZ1 against wild-type K. pneumoniae, and coadministration with outer membrane permeability enhancers profoundly sensitizes Escherichia coli to designed LpxH inhibitors. Remarkably, none of the sulfonyl piperazine compounds occupies the active site of LpxH, foretelling a straightforward path for rapid optimization of this class of antibiotics.

scholarly journals Analyses of Lipid A Diversity in Gram-Negative Intestinal Bacteria Using Liquid Chromatography–Quadrupole Time-of-Flight Mass Spectrometry

Metabolites ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 197
Author(s):  
Nobuyuki Okahashi ◽  
Masahiro Ueda ◽  
Fumio Matsuda ◽  
Makoto Arita
Keyword(s):  
Mass Spectrometry ◽  
Immune Response ◽  
Liquid Chromatography ◽  
Mammalian Cells ◽  
Lipid A ◽  
Acyl Chain ◽  
Time Of Flight ◽  
Intestinal Bacteria ◽  
Gram Negative ◽  
Flight Mass Spectrometry

Lipid A is a characteristic molecule of Gram-negative bacteria that elicits an immune response in mammalian cells. The presence of structurally diverse lipid A types in the human gut bacteria has been suggested before, and this appears associated with the immune response. However, lipid A structures and their quantitative heterogeneity have not been well characterized. In this study, a method of analysis for lipid A using liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) was developed and applied to the analyses of Escherichia coli and Bacteroidetes strains. In general, phosphate compounds adsorb on stainless-steel piping and cause peak tailing, but the use of an ammonia-containing alkaline solvent produced sharp lipid A peaks with high sensitivity. The method was applied to E. coli strains, and revealed the accumulation of lipid A with abnormal acyl side chains in knockout strains as well as known diphosphoryl hexa-acylated lipid A in a wild-type strain. The analysis of nine representative strains of Bacteroidetes showed the presence of monophosphoryl penta-acylated lipid A characterized by a highly heterogeneous main acyl chain length. Comparison of the structures and amounts of lipid A among the strains suggested a relationship between lipid A profiles and the phylogenetic classification of the strains.

scholarly journals Identification of two late acyltransferase genes responsible for lipid A biosynthesis in Moraxella catarrhalis

FEBS Journal ◽  
2008 ◽  
Vol 275 (20) ◽  
pp. 5201-5214 ◽  
Author(s):  
Song Gao ◽  
Daxin Peng ◽  
Wenhong Zhang ◽  
Artur Muszyński ◽  
Russell W. Carlson ◽  
...  
Keyword(s):  
Moraxella Catarrhalis ◽  
Lipid A ◽  
Lipid A Biosynthesis
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