Publications

• Crystal structures of the UDP-diacylglucosamine pyrophosphohydrase LpxH from Pseudomonas aeruginosa.

  Okada C., Wakabayashi H., Kobayashi M., Shinoda A., Tanaka I., Yao M.

  Lipid A (also known as endotoxin) is the hydrophobic portion of lipopolysaccharides. It is an essential membrane component required for the viability of gram-negative bacteria. The enzymes involved in its biosynthesis are attractive targets for the development of novel antibiotics. LpxH catalyzes ... >> More

  Lipid A (also known as endotoxin) is the hydrophobic portion of lipopolysaccharides. It is an essential membrane component required for the viability of gram-negative bacteria. The enzymes involved in its biosynthesis are attractive targets for the development of novel antibiotics. LpxH catalyzes the fourth step of the lipid A biosynthesis pathway and cleaves the pyrophosphate bond of UDP-2,3-diacylglucosamine to yield 2,3-diacylglucosamine 1-phosphate (lipid X) and UMP. Here we present the structures of LpxH from Pseudomonas aeruginosa (PaLpxH). PaLpxH consists of two domains: a catalytic domain that is homologous to the metallophosphoesterases and a helical insertion domain. Lipid X was captured in the crevice between these two domains, with its phosphate group facing the dinuclear metal (Mn(2+)) center and two acyl chains buried in the hydrophobic cavity. The structures reveal that a large conformational change occurs at the lipid X binding site surface upon the binding/release of the product molecule. Based on these observations, we propose a novel model for lipid X embedding, which involves the scissor-like movement of helix α6, resulting in the release of lipid X into the lipid bilayer. << Less

  Sci. Rep. 6:32822-32822(2016) [PubMed] [EuropePMC]

• The Escherichia coli gene encoding the UDP-2,3-diacylglucosamine pyrophosphatase of lipid A biosynthesis.

  Babinski K.J., Ribeiro A.A., Raetz C.R.H.

  UDP-2,3-diacylglucosamine hydrolase is believed to catalyze the fourth step of lipid A biosynthesis in Escherichia coli. This reaction involves pyrophosphate bond hydrolysis of the precursor UDP-2,3-diacylglucosamine to yield 2,3-diacylglucosamine 1-phosphate and UMP. To identify the gene encoding ... >> More

  UDP-2,3-diacylglucosamine hydrolase is believed to catalyze the fourth step of lipid A biosynthesis in Escherichia coli. This reaction involves pyrophosphate bond hydrolysis of the precursor UDP-2,3-diacylglucosamine to yield 2,3-diacylglucosamine 1-phosphate and UMP. To identify the gene encoding this hydrolase, E. coli lysates generated with individual lambda clones of the ordered Kohara library were assayed for overexpression of the enzyme. The sequence of lambda clone 157[6E7], promoting overproduction of hydrolase activity, was examined for genes encoding hypothetical proteins of unknown function. The amino acid sequence of one such open reading frame, ybbF, is 50.5% identical to a Haemophilus influenzae hypothetical protein and is also conserved in most other Gram-negative organisms, but is absent in Gram-positives. Cell extracts prepared from cells overexpressing ybbF behind the T7lac promoter have approximately 540 times more hydrolase activity than cells with vector alone. YbbF was purified to approximately 60% homogeneity, and its catalytic properties were examined. Enzymatic activity is maximal at pH 8 and is inhibited by 0.01% (or more) Triton X-100. The apparent K(m) for UDP-2,3-diacylglucosamine is 62 microm. YbbF requires a diacylated substrate and does not cleave CDP-diacylglycerol. (31)P NMR studies of the UMP product generated from UDP-2,3-diacylglucosamine in the presence of 40% H(2)180 show that the enzyme attacks the alpha-phosphate group of the UDP moiety. Because ybbF encodes the specific UDP-2,3-diacylglucosamine hydrolase involved in lipid A biosynthesis, it is now designated lpxH. << Less

  J. Biol. Chem. 277:25937-25946(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Accumulation of the lipid A precursor UDP-2,3-diacylglucosamine in an Escherichia coli mutant lacking the lpxH gene.

  Babinski K.J., Kanjilal S.J., Raetz C.R.H.

  The lpxH gene encodes the UDP-2,3-diacylglucosamine-specific pyrophosphatase that catalyzes the fourth step of lipid A biosynthesis in Escherichia coli. To confirm the function of lpxH, we constructed KB21/pKJB5. This strain contains a kanamycin insertion element in the chromosomal copy of lpxH, c ... >> More

  The lpxH gene encodes the UDP-2,3-diacylglucosamine-specific pyrophosphatase that catalyzes the fourth step of lipid A biosynthesis in Escherichia coli. To confirm the function of lpxH, we constructed KB21/pKJB5. This strain contains a kanamycin insertion element in the chromosomal copy of lpxH, complemented by plasmid pKJB5, which is temperature-sensitive for replication and harbors lpxH(+). KB21/pKJB5 grows at 30 degrees C but loses viability at 44 degrees C, demonstrating that lpxH is essential. CDP-diglyceride hydrolase (Cdh) catalyzes the same reaction as LpxH in vitro but is non-essential and cannot compensate for the absence of LpxH. The presence of Cdh in cell extracts interferes with the LpxH assay. We therefore constructed KB25/pKJB5, which contains both an in-frame deletion of cdh and a kanamycin insertion mutation in lpxH, covered by pKJB5. When KB25/pKJB5 cells are grown at 44 degrees C, viability is lost, and all in vitro LpxH activity is eliminated. A lipid migrating with synthetic UDP-2,3-diacylglucosamine accumulates in KB25/pKJB5 following loss of the covering plasmid at 44 degrees C. This material was converted to the expected products, 2,3-diacylglucosamine 1-phosphate and UMP, by LpxH. Pseudomonas aeruginosa contains two proteins with sequence similarity to E. coli LpxH. The more homologous protein catalyzes UDP-2,3-diacylglucosamine hydrolysis in vitro. The corresponding gene complements KB25/pKJB5 at 44 degrees C, but the less homologous gene does not. The accumulation of UDP-2,3-diacylglucosamine in our lpxH mutant is consistent with the observation that the lipid A disaccharide synthase LpxB, the next enzyme in the pathway, cannot condense two UDP-2,3-diacylglucosamine molecules, but instead utilizes UDP-2,3-diacylglucosamine as its donor and 2,3-diacylglucosamine 1-phosphate as its acceptor. << Less

  J. Biol. Chem. 277:25947-25956(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structure of the essential Haemophilus influenzae UDP-diacylglucosamine pyrophosphohydrolase LpxH in lipid A biosynthesis.

  Cho J., Lee C.J., Zhao J., Young H.E., Zhou P.

  In most Gram-negative pathogens, the hydrolysis of UDP-2,3-diacylglucosamine to generate lipid X in lipid A biosynthesis is catalysed by the membrane-associated enzyme LpxH. We report the crystal structure of LpxH in complex with its product, lipid X, unveiling a unique insertion lid above the con ... >> More

  In most Gram-negative pathogens, the hydrolysis of UDP-2,3-diacylglucosamine to generate lipid X in lipid A biosynthesis is catalysed by the membrane-associated enzyme LpxH. We report the crystal structure of LpxH in complex with its product, lipid X, unveiling a unique insertion lid above the conserved architecture of calcineurin-like phosphoesterases. This structure reveals elaborate interactions surrounding lipid X and provides molecular insights into the substrate selectivity, catalysis and inhibition of LpxH. << Less

  Nat. Microbiol. 1:16154-16154(2016) [PubMed] [EuropePMC]

• Substrate specificity of the pyrophosphohydrolase LpxH determines the asymmetry of <i>Bordetella pertussis</i> lipid A.

  Arenas J., Pupo E., de Jonge E., Perez-Ortega J., Schaarschmidt J., van der Ley P., Tommassen J.

  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 <i>Bordetella pertussis</i>, the causative agent of whooping cough, displays unusual structural asymmetry ... >> More

  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 <i>Bordetella pertussis</i>, 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 <i>B. pertussis</i>, has limited chain length specificity. However, this only partially explains the strict asymmetry of lipid A. In attempts to modulate the endotoxicity of <i>B. pertussis</i> lipid A, here we expressed the gene encoding LpxA from <i>Neisseria meningitidis</i>, which specifically attaches 3OH-C12 chains, in <i>B. pertussis</i> This expression was lethal, suggesting that one of the downstream enzymes in the lipid A biosynthesis pathway in <i>B. pertussis</i> 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 <i>B. pertussis</i> lipid A. Expression of meningococcal LpxH in <i>B. pertussis</i> 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 <i>lpxH</i> and <i>lpxA</i> resulted in viable cells that incorporated 3OH-C12 chains in <i>B. pertussis</i> lipid A. We conclude that the asymmetry of <i>B. pertussis</i> lipid A is determined by the acyl chain length specificity of LpxH. << Less

  J Biol Chem 294:7982-7989(2019) [PubMed] [EuropePMC]