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Acyl carrier protein represents one of the most highly conserved proteins across all domains of life and is nature's way of transporting hydrocarbon chains in vivo. Notably, type II acyl carrier proteins serve as a crucial interaction hub in primary cellular metabolism by communicating transiently between partner enzymes of the numerous biosynthetic pathways. However, the highly transient nature of such interactions and the inherent conformational mobility of acyl carrier protein have stymied previous attempts to visualize structurally acyl carrier protein tied to an overall catalytic cycle. This is essential to understanding a fundamental aspect of cellular metabolism leading to compounds that are not only useful to the cell, but also of therapeutic value. For example, acyl carrier protein is central to the biosynthesis of the lipid A (endotoxin) component of lipopolysaccharides in Gram-negative microorganisms, which is required for their growth and survival, and is an activator of the mammalian host's immune system, thus emerging as an important therapeutic target. During lipid A synthesis (Raetz pathway), acyl carrier protein shuttles acyl intermediates linked to its prosthetic 4′-phosphopantetheine group among four acyltransferases, including LpxD. Here we report the crystal structures of three forms of Escherichia coli acyl carrier protein engaging LpxD, which represent stalled substrate and liberated products along the reaction coordinate. The structures show the intricate interactions at the interface that optimally position acyl carrier protein for acyl delivery and that directly involve the pantetheinyl group. Conformational differences among the stalled acyl carrier proteins provide the molecular basis for the association-dissociation process. An unanticipated conformational shift of 4′-phosphopantetheine groups within the LpxD catalytic chamber shows an unprecedented role of acyl carrier protein in product release. [ABSTRACT FROM AUTHOR]

Summary: PTPMT1 was the first protein tyrosine phosphatase found localized to the mitochondria, but its biological function was unknown. Herein, we demonstrate that whole body deletion of Ptpmt1 in mice leads to embryonic lethality, suggesting an indispensable role for PTPMT1 during development. Ptpmt1 deficiency in mouse embryonic fibroblasts compromises mitochondrial respiration and results in abnormal mitochondrial morphology. Lipid analysis of Ptpmt1-deficient fibroblasts reveals an accumulation of phosphatidylglycerophosphate (PGP) along with a concomitant decrease in phosphatidylglycerol. PGP is an essential intermediate in the biosynthetic pathway of cardiolipin, a mitochondrial-specific phospholipid regulating the membrane integrity and activities of the organelle. We further demonstrate that PTPMT1 specifically dephosphorylates PGP in vitro. Loss of PTPMT1 leads to dramatic diminution of cardiolipin, which can be partially reversed by the expression of catalytic active PTPMT1. Our study identifies PTPMT1 as the mammalian PGP phosphatase and points to its role as a regulator of cardiolipin biosynthesis. [Copyright &y& Elsevier]

Escherichia coli LpxB, an inverting glycosyl transferase of the GT-B superfamily and a member of CAZy database family 19, catalyzes the fifth step of lipid A biosynthesis: UDP-2,3-diacylglucosamine + 2, 3-diacylglucosamine 1-phosphate → 2′,3′-diacylglucosamine-(β, 1′-6)-2,3-diacylglucosamine 1-phosphate + UDP. LpxB is a target for the development of new antibiotics, but no member of family 19, which consists entirely of LpxB orthologues, has been characterized mechanistically or structurally. Here, we have purified E. coil and Haemophilus influenzae LpxB to near homogeneity on a 10-100mg scale using protease-cleavable His10-tagged constructs. E. coli LpxB activity is dependent upon the bulk surface concentration of its substrates in a mixed micelle assay system, suggesting that catalysis occurs at the membrane interface. E. coli LpxB (Mr ∼ 43 kDa) sediments with membranes at low salt concentrations but is largely solubilized with buffers of high ionic strength. It purifies with 1.6-3.5 mol of phospholipid/mol of LpxB polypeptide. Transmission electron microscopy reveals the accumulation of aberrant intracellular membranes when LpxB is overexpressed. Mutagenesis of LpxB identified two conserved residues, D89A and R201A, for which no residual catalytic activity was detected. Our results provide a rational starting point for structural studies. [ABSTRACT FROM AUTHOR]

Escherichia coli mutants deficie'nt in 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) biosynthesis are conditionally lethal, but their phenotypes are bypassed by certain suppressor mutations or by over- expression of MsbA, the inner membrane flippase for core-lipid A. These strains grow on broth with the tetraacylated precursor lipid IVA replacing lipopolysaccharide [Meredith, T. C., et al. (2006) ACS Chem. Biol. 1, 33-42]. Deletion of kdtA, which encodes the Kdo transferase, is possible under these conditions. We now show that lipid IVA reaches the Outer surface of the outer membrane in these strains, as judged by its accessibility to the lipase PagL. On the assumption that MsbA is optimized to transport penta- or hexaacylated lipid A, we overexpressed the lauroyl- or the myristoyltransferase of lipid A biosynthesis, encoded by IpxL and ipxM, respectively, and demonstrated that kdtA deletion mutants were also viable in this setting. Although E. coli LpxL is stimulated by the presence of the Kdo disaccharide in its acceptor substrate, LpxL does slowly acylate lipid IVA. Overexpression of LpxL from a plasmid suppressed the lethality of kdtA deletions on nutrient broth at 30 or 37 °C without the need for MsbA overproduction. These strains accumulated penta- and hexaacylated free lipid A containing a secondary laurate chain or a laurate and a myristate chain, respectively. Deletion of kdtA in strains overexpressing LpxM accumulated pentaacylated lipid A with a secondary myristate moiety. None of the strains lacking kdtA grew in the presence of bile salts at any temperature or on nutrient broth at 42 °C. Our findings show that the main function of Kdo is to provide the right substrates for the acyltransferases LpxL and LpxM, resulting in the synthesis of penta- and hexaacylated lipid A, which is optimal for the MsbA flippase. [ABSTRACT FROM AUTHOR]

LpxD catalyzes the third step of lipid A biosynthesis, the R-3-hydroxyacyl-ACP-dependent N-acylation of UDP-3-O-(acyl)-α-D-glucosamine, and is a target for new antibiotic development. Here we report the 2.6 Å crystal Structure of the Escherichia coli LpxD homotrimer (EcLpxD). As is the case in Chiamydia trochoniatis LpxD (CtLxpD), each EcLpxD chain consists of an N-terminal uridine-binding region, a left-handed parallel β-helix (LβH), and a C-terminal a-helical domain. The backbones of the LβH domains of the two enzymes are similar, as are the positions of key active site residues. The N-terminal nucleotide binding domains are oriented differently relative to the LβH regions, but are similar when overlaid on each other. The orientation of the EcLpxD tripeptide (residues 303-305), connecting the distal end of the LβH and the proximal end of the C-terminal helical domains, differs from its counterpart in CtLpxD (residues 311-312); this results in a 120° rotation of the C-terminal domain relative to the LβH region in EcLpxD versus CtLpxD. M290 of EcLpxD appears to cap the distal end of a hydrophobic cleft that binds the acyl chain of the R-3-hydroxyacyl-ACP donor substrate. Under standard assay conditions, wild-type EcLpxD prefers R,S-3-hydroxymyristoyl-ACP over R,S-3-hydroxypalmitoyl-ACP by a factor of 3, whereas the M290A mutant has the opposite selectivity. Both wild-type and M290A EcLpxD rescue the conditional lethality of E. coli RL25, a temperature-sensitive strain harboring point mutations in lpxD. Complementation with wild-type EcLpxD restores normal lipid A containing only N-linked hydroxymyristate to RL25 at 42 °C, as judged by mass spectrometry, whereas the M290A mutant generates multiple lipid A species containing one or two longer hydroxy fatty acids in place of the usual R-3-hydroxymyristate at positions 2 and 2′. [ABSTRACT FROM AUTHOR]

The core-lipid A domain of Escherichia coil lipopolysaccharide (LPS) is synthesized on the inner surface of the inner membrane (IM) and flipped to its outer surface by the ABC transporter MsbA. Recent studies with deletion mutants implicate the periplasmic protein LptA, the cytosolic protein LptB, and the IM proteins LptC, LptF, and LptG in the subsequent transport of nascent LPS to the outer membrane (OM), where the LptD/LptE complex flips LPS to the outer surface. We have isolated a temperature-sensitive mutant (MB1) harboring the S22C and QuiP substitutions in LptA. MB1 stops growing after 30 mm at 42°C. 32Pi and [35S]methionine labeling show that export of newly synthesized phospholipids and proteins is not severely impaired, but export of LPS is defective. Using the lipid A 1-phosphatase LpxE as a periplasmic IM marker and the lipid A 3-O-deacylase PagL as an OM marker, we show that core-lipid A reaches the periplasmic side of the IM at 42°C in MB1 but not the outer surface of the OM. Electron microscopy of MB1 reveals dense periplasmic material and a smooth OM at 42°C, consistent with a role for LptA in shuttling LPS across the periplasm. [ABSTRACT FROM AUTHOR]

LpxD catalyzes the third step of lipid A biosynthesis, the (R)-3-hydroxymyristoyl-acyl carrier protein (R-3-OHC14-ACP)-dependent N-acylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-α-D-glucosamine [UDP-3-O-(R-3-OHC14)-GlcN]. We have now overexpressed and purified Escherichia coli LpxD to homogeneity. Steady-state kinetics suggest a compulsory ordered mechanism in which R-3-OHC14-ACP binds prior to UDP-3-O-(R-3-OHC14)-GlcN. The product, UDP-2,3-diacylglucosamine, dissociates prior to ACP; the latter is a competitive inhibitor against R-3-OHC14-ACP and a noncompetitive inhibitor against UDP-3-O-(R-3-OHC14)-GIcN. 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-hydroxylauroyl-methylphosphopantetheine is an uncompetitive inhibitor against R-3-OHC14-ACP and a competitive inhibitor against UDP-3-O-(R-3-OHC14)-GlcN, but (R)-3-hydroxylauroyl-methylphos- phopantetheine is also a very poor substrate. A compulsory ordered mechanism is consistent with the fact that R-3-OHC14-ACP has a high binding affinity for free LpxD whereas UDP-3-O-(R-3-OHC14)-GlcN does not. Divalent cations inhibit R-3-OHC14-ACP-dependent acylation but not (R)-3-hydroxylauroyl- methyiphosphopantetheine-dependent acylation, indicating that the acidic recognition helix of R-3-OHC14- ACP contributes to binding. The F41A mutation increases the KM for UDP-3-O-(R-3-OHC14)-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 Chiamydia trachomatis LpxD. Mutagenesis implicates E. coli H239 but excludes H276 as the catalytic base, and neither residue is likely to stabilize the oxyanion intermediate. [ABSTRACT FROM AUTHOR]

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 β-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 β-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 β-turn structure of peptide 920 bound to LpxA may open previously uncharacterized approaches to the rational design of LpxA inhibitors with antibiotic activity. [ABSTRACT FROM AUTHOR]

The zinc-dependent UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) catalyzes the first committed step in the biosynthesis of lipid A, the hydrophobic anchor of lipopolysaccharide (LPS) that constitutes the outermost monolayer of Gram-negative bacteria. As LpxC is crucial for the survival of Gram-negative organisms and has no sequence homology to known mammalian deacetylases or amidases, it is an excellent target for the design of new antibiotics. The solution structure of LpxC from Aquifex aeolicus in complex with a substrate-analog inhibitor, TU-514, reveals a novel a/ß fold, a unique zinc-binding motif and a hydrophobic passage that captures the acyl chain of the inhibitor. On the basis of biochemical and structural studies, we propose a catalytic mechanism for LpxC, suggest a model for substrate binding and provide evidence that mobility and dynamics in structural motifs close to the active site have key roles in the capture of the substrate. [ABSTRACT FROM AUTHOR]

Bacterial lipopolysaccharides (LPS) typically consist of a hydrophobic domain known as lipid A (or endotoxin), a nonrepeating “core” oligosaccharide, and a distal polysaccharide (or O-antigen). Recent genomic data have facilitated study of LPS assembly in diverse Gram-negative bacteria, many of which are human or plant pathogens, and have established the importance of lateral gene transfer in generating structural diversity of O-antigens. Many enzymes of lipid A biosynthesis like LpxC have been validated as targets for development of new antibiotics. Key genes for lipid A biosynthesis have unexpectedly also been found in higher plants, indicating that eukaryotic lipid A-like molecules may exist. Most significant has been the identification of the plasma membrane protein TLR4 as the lipid A signaling receptor of animal cells. TLR4 belongs to a family of innate immunity receptors that possess a large extracellular domain of leucine-rich repeats, a single trans-membrane segment, and a smaller cytoplasmic signaling region that engages the adaptor protein MyD88. The expanding knowledge of TLR4 specificity and its downstream signaling pathways should provide new opportunities for blocking inflammation associated with infection. [ABSTRACT FROM AUTHOR]

Lipid A of Francisella tularensis subsp. novicida contains a galactosamine (GaIN) residue linked to its 1-phosphate group. As shown in the preceding paper, this GaIN unit is transferred to lipid A from the precursor undecaprenyl phosphate-β-D-GalN. A small portion of the free lipid A of Francisella novicida is further modified with a glucose residue at position-6'. We now demonstrate that the two F. novicida homologues of Escherichia coli ArnC, designated FImFL and FlmF2, are essential for lipid A modification with glucose and GaIN, respectively. Recombinant FImFI expressed in E. coli selectively condenses undecaprenyl phosphate and UDP-glucose in vitro to form undecaprenyl phosphate-glucose. Recombinant FImF2 selectively catalyzes the condensation of undecaprenyl phosphate and UDP-N- acetylgalactosamine to generate undecaprenyl phosphate-N-acetylgalactosainine. On the basis of an analysis of the lipid A composition of flmF1 and flmF2 mutants of F. novieida, we conclude that FImF1 generates the donor substrate for the modification of F. novicida free lipid A with glucose, whereas FImF2 generates the immediate precursor of the GalN donor substrate, undecaprenyl phosphate-β-D-GalN. A novel deacetylase, present in membranes of F. novicida, removes the acelyl group from undecaprenyl phosphate- N-acetylgalactosamine to yield undecaprenyl phosphate-β-D-GalN. This deacetylase may have an analogous function to the deformylase that generates undecaprenyl phosphate-4-amino-4-deoxy-α-L-arabinose from undecaprenyl phosphate-4-deoxy-4-formylamino-α-L-arabinose in polymyxin-resistant strains of E. coli and Salmonella iyphimurium. [ABSTRACT FROM AUTHOR]

Modification of lipid A with the 4-amino-4-deoxy-L-arabinose (L-Ara4N) moiety is required for resistance to polymyxin and cationic antimicrobial peptides in Escherichia coil and Salmonella typhimurium. An operon of seven genes (designated pmrHFIJKLM in S. typhimurium), which is regulated by the PmrA transcription factor and is also present in E. coil, is necessary for the maintenance of polymyxin resistance. We previously elucidated the roles of pmrHFIJK in the biosynthesis and attachment of L-Ara4N to lipid A and renamed these genes arnBCADT, respectively. We now propose functions for the last two genes of the operon, pmrL and pmrM. Chromosomal inactivation of each of these genes in an E. coli pmrAc parent switched its phenotype from polymyxin-resistant to polymyxin-sensitive. Lipid A was no longer modified with L-Ara4N, even though the levels of the lipid-linked donor of the L-Ara4N moiety, undecaprenyl phosphate-n-L-Ara4N, were not reduced in the mutants. However, the undecaprenyl phosphate-α-L-Ara4N present in the mutants was less concentrated on the periplasmic surface of the inner membrane, as judged by 4-5-fold reduced labeling with the inner membrane-impermeable amine reagent N-hydroxysulfosuccin-imidobiotin. In an arnT mutant of the same pmrAc parent, which lacks the enzyme that transfers the L-Ara4N unit to lipid A but retains the same high levels of undecaprenyl phosphate-α-L-Ara4N as the parent, N-hydroxysulfosuccinimidobiotin labeling was not reduced. These results implicate pmrL and pmrM, but not arnT, in transporting undecaprenyl phosphate-α-L-Ara4N across the inner membrane. PmrM and PmrL, now renamed ArnE and ArnF because of their involvement in L-Ara4N modification of lipid A, may be subunits of an undecaprenyl phosphate-α-L-Ara4N flippase. [ABSTRACT FROM AUTHOR]

Investigates the role of aminotransferase in uridine-diphospho-beta-(4-amino-4-deoxy-L-arabinose) biosynthesis. Analysis of the covalent structure of the aminotransferase; Evaluation of the carbon structure of the aminotransferase product; Covalent structure of the aminotransferase product.

Abstract During lipopolysaccharide biosynthesis in several pathogens, including Burkholderia and Yersinia , 3-deoxy- d - manno -oct-2-ulosonic acid (Kdo) 3-hydroxylase, otherwise referred to as KdoO, converts Kdo to d -glycero- d -talo-oct-2-ulosonic acid (Ko) in an Fe(II)/α-ketoglutarate (α-KG)/O 2 -dependent manner. This conversion renders the bacterial outer membrane more stable and resistant to stresses such as an acidic environment. KdoO is a membrane-associated, deoxy-sugar hydroxylase that does not show significant sequence identity with any known enzymes, and its structural information has not been previously reported. Here, we report the biochemical and structural characterization of KdoO, Minf_1012 (Kdo MI), from Methylacidiphilum infernorum V4. The de novo structure of Kdo MI apoprotein indicates that KdoO MI consists of 13 α helices and 11 β strands, and has the jelly roll fold containing a metal binding motif, HXDX 111 H. Structures of Kdo MI bound to Co(II), Kdo MI bound to α-KG and Fe(III), and Kdo MI bound to succinate and Fe(III), in addition to mutagenesis analysis, indicate that His146, His260, and Asp148 play critical roles in Fe(II) binding, while Arg127, Arg162, Arg174, and Trp176 stabilize α-KG. It was also observed that His225 is adjacent to the active site and plays an important role in the catalysis of KdoO MI without affecting substrate binding, possibly being involved in oxygen activation. The crystal structure of KdoO MI is the first completed structure of a deoxy-sugar hydroxylase, and the data presented here have provided mechanistic insights into deoxy-sugar hydroxylase, KdoO, and lipopolysaccharide biosynthesis. Graphical Abstract Unlabelled Image Highlights • KdoO converts Kdo to Ko during LPS biosynthesis. • Minf_1012 from Methylacidiphilum infernorum functions as KdoO MI. • The first completed structures of KdoO MI are determined at 1.45- to 1.94-Å resolution. • The structure of KdoO MI reveals a metal binding motif HXDX n > 40 H. • Cosubstrate bound KdoO MI and mutagenesis study show important residues for catalysis. [ABSTRACT FROM AUTHOR]

The lipopolysaccharide (LPS) isolated from certain important Gram-negative pathogens including a human pathogen Yersinia pestis and opportunistic pathogens Burkholderia mallei and Burkholderia pseudomallei contains d -glycero- d - talo -oct-2-ulosonic acid (Ko), an isosteric analog of 3-deoxy- d - manno -oct-2-ulosonic acid (Kdo). Kdo 3-hydroxylase (KdoO), a Fe 2+ / α -KG/O 2 dependent dioxygenase from Burkholderia ambifaria and Yersinia pestis is responsible for Ko formation with Kdo 2 -lipid A as a substrate, but in which stage KdoO functions during the LPS biosynthesis has not been established. Here we purify KdoO from B. ambifaria (BaKdoO) to homogeneity for the first time and characterize its substrates. BaKdoO utilizes Kdo 2 -lipid IV A or Kdo 2 -lipid A as a substrate, but not Kdo-lipid IV A in vivo as well as in vitro and Kdo-(Hep)kdo-lipid A in vitro . These data suggest that KdoO is an inner core assembly enzyme that functions after the Kdo-transferase KdtA but before the heptosyl-transferase WaaC enzyme during the Ko-containing LPS biosynthesis. [ABSTRACT FROM AUTHOR]

The sixth step in the lipid A biosynthetic pathway involves phosphorylation of the tetraacyldisaccharide-1-phosphate (DSMP) intermediate by the cytosol-facing inner membrane kinase LpxK, a member of the P-loop-containing nucleoside triphosphate (NTP) hydrolase superfamily. We report the kinetic characterization of LpxK from Aquifex aeolicus and the crystal structures of LpxK in complex with ATP in a precatalytic binding state, the ATP analogue AMP-PCP in the closed catalytically competent conformation, and a chloride anion revealing an inhibitory conformation of the nucleotide-binding P-loop. We demonstrate that LpxK activity in vitro requires the presence of a detergent micelle and formation of a ternary LpxK-ATP/Mg2+-DSMP complex. Using steady-state kinetics, we have identified crucial active site residues, leading to the proposal that the interaction of D99 with H261 acts to increase the pKa of the imidazole moiety, which in turn serves as the catalytic base to deprotonate the 4'-hydroxyl of the DSMP substrate. The fact that an analogous mechanism has not yet been observed for other P-loop kinases highlights LpxK as a distinct member of the P-loop kinase family, a notion that is also reflected through its localization at the membrane, lipid substrate, and overall structure. [ABSTRACT FROM AUTHOR]

Abstract: A study of the polar lipids of Clostridium novyi NT has revealed the presence of phosphatidylethanolamine (PE) and cardiolipin as major phospholipids with smaller amounts of phosphatidylglycerol (PG), lysyl-PG and alanyl-PG. Other minor phospholipids included phosphatidic acid, CDP-diacylglycerol, phosphatidylserine (PS) and phosphatidylthreonine (PT). PE, PG and amino acyl PG were present in both the diacyl and alk-1′-enyl acyl (plasmalogen) forms and cardiolipin plasmalogens were found to contain one or two alk-1′-enyl chains. In contrast, the precursor lipids phosphatidic acid, CDP-diacylglycerol and PS were present almost exclusively as diacyl phospholipids. These findings are consistent with the hypothesis that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid formation in Clostridium species. This novel pathway contrasts with the route in animals in which a saturated ether bond is formed at an early stage of plasmalogen biosynthesis and the alk-1-enyl bond is formed by an aerobic mechanism. [Copyright &y& Elsevier]

Summary: LpxC is an essential enzyme in the lipid A biosynthetic pathway in gram-negative bacteria. Several promising antimicrobial lead compounds targeting LpxC have been reported, though they typically display a large variation in potency against different gram-negative pathogens. We report that inhibitors with a diacetylene scaffold effectively overcome the resistance caused by sequence variation in the LpxC substrate-binding passage. Compound binding is captured in complex with representative LpxC orthologs, and structural analysis reveals large conformational differences that mostly reflect inherent molecular features of distinct LpxC orthologs, whereas ligand-induced structural adaptations occur at a smaller scale. These observations highlight the need for a molecular understanding of inherent structural features and conformational plasticity of LpxC enzymes for optimizing LpxC inhibitors as broad-spectrum antibiotics against gram-negative infections. [ABSTRACT FROM AUTHOR]

Abstract: Compounds inhibiting LpxC in the lipid A biosynthetic pathway are promising leads for novel antibiotics against multidrug-resistant Gram-negative pathogens. We report the syntheses and structural and biochemical characterizations of LpxC inhibitors based on a diphenyl-diacetylene (1,4-diphenyl-1,3-butadiyne) threonyl-hydroxamate scaffold. These studies provide a molecular interpretation for the differential antibiotic activities of compounds with a substituted distal phenyl ring as well as the absolute stereochemical requirement at the C2, but not C3, position of the threonyl group. [ABSTRACT FROM AUTHOR]

We report the lipidomic response of the murine macrophage RAW cell line to Kdo2-lipid A, the active component of an inflammatory lipopolysaccharide functioning as a selective TLR4 agonist and compactin, a statin inhibitor of cholesterol biosynthesis. Analyses of lipid molecular species by dynamic quantitative mass spectrometry and concomitant transcriptomic measurements define the lipidome and demonstrate immediate responses in fatty acid metabolism represented by increases in eicosanoid synthesis and delayed responses characterized by sphingolipid and sterol biosynthesis. Lipid remodeling of glycerolipids, glycerophospholipids, and prenols also take place, indicating that activation of the innate immune system by inflammatory mediators leads to alterations in a majority of mammalian lipid categories, including unanticipated effects of a statin drug. Our studies provide a systems-level view of lipid metabolism and reveal significant connections between lipid and cell signaling and biochemical pathways that contribute to innate immune responses and to pharmacological perturbations. [ABSTRACT FROM AUTHOR]

ABSTRACT: The UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine deacetylase LpxC catalyzes the committed reaction of lipid A (endotoxin) biosynthesis in Gram-negative bacteria and is a validated antibiotic target. Although several previously described compounds bind to the unique acyl chain binding passage of LpxC with high affinity, strategies to target the enzyme's UDP-binding site have not been reported. Here the identification of a series of uridine-based LpxC inhibitors is presented. The most potent examined, 1-68A, is a pH-dependent, two-step, covalent inhibitor of Escherichia coli LpxC that' competes with UDP to bind the enzyme in the first step of inhibition. Compound l-68A exhibits a K[sub1] of 54 μM and a maximal rate of inactivation (k[subinact]) of 1.7 min[sup-1] at pH 7.4. Dithiothreitol, glutathione and the C207A mutant of E. coli LpxC prevent the formation of a covalent complex by 1-68A, suggesting a role for Cys-207 in inhibition. The inhibitory activity of 1-68A and a panel of synthetic analogues identified moieties necessary for inhibition. 1-68A and a 2-dehydroxy analogue, 1-68Aa, inhibit several purified LpxC orthologues. These compounds may provide new scaffolds for extension of existing LpxC-inhibiting antibiotics to target the UDP binding pocket. [ABSTRACT FROM AUTHOR]

Escherichia coli lipid A is a hexaacylated disaccharide of glucosamine with secondary laurate and myristate chains on the distal unit. Hexaacylated lipid A is a potent agonist of human Toll-like receptor 4, whereas its tetra- and pentaacylated precursors are antagonists. The inner membrane enzyme LpxL transfers laurate from lauroyl-acyl carrier protein to the 2′-R-3-hydroxymyristate moiety of the tetraacylated lipid A precursor Kdo2-lipid IVA. LpxL has now been overexpressed, solubilized with n-dodecyl β-D-maltopyranoside (DDM), and purified to homogeneity. LpxL migration on a gel filtration column is consistent with a molecular mass of 80 kDa, suggestive of an LpxL monomer (36 kDa) embedded in a DDM micelle. Mass spectrometry showed that deformylated LpxL was the predominant species, noncovalently bound to as many as 12 DDM molecules. Purified LpxL catalyzed not only the formation in vitro of Kdo2-(lauroyl)-lipid IVA but also a slow second acylation, generating Kdo2-(dilauroyl)-lipid IVA. Consistent with the Kdo dependence of crude LpxL in membranes, Kdo2-lipid IVA is preferred 6000-fold over lipid IVA by the pure enzyme. Sequence comparisons suggest that LpxL shares distant homology with the glycerol-3-phosphate acyltransferase (GPAT) family, including a putative catalytic dyad located in a conserved H(X)4D/E motif. Mutation of H132 or E137 to alanine reduces specific activity by over 3 orders of magnitude. Like many GPATs, LpxL can also utilize acyl-CoA as an alternative acyl donor, albeit at a slower rate. Our results show that the acyltransferases that generate the secondary acyl chains of lipid A are members of the GPAT family and set the stage for structural studies. [ABSTRACT FROM AUTHOR]

The deacetylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine (UDP-3-O-acyl-GlcNAc) by LpxC is the committed reaction of lipid A biosynthesis. CHIR-090, a novel N-aroyl-ʟ-threonine hydroxamic acid, is a potent, slow, tight-binding inhibitor of the LpxC deacetylase from the hyperthermophile Aquifex aeolicus, and it has excellent antibiotic activity against Pseudomonas aeruginosa and Escherichia coli, as judged by disk diffusion assays. We now report that CHIR-090 is also a two-step slow, tight-binding inhibitor of E. coli LpxC with Ki = 4.0 nM, Ki* = 0.5 nM, k5 = 1.9 min-1, and k6 = 0.18 min-1. CHIR-090 at low nanomolar levels inhibits LpxC orthologues from diverse Gram-negative pathogens, including P. aeruginosa, Neisseria meningitidis, and Helicobacter pylori. In contrast, CHIR-090 is a relatively weak competitive and conventional inhibitor (lacking slow, tight-binding kinetics) of LpxC from Rhizobium leguminosarum (Ki = 340 nM), a Gram-negative plant endosymbiont that is resistant to this compound. The KM (4.8 μM) and the kcat (1.7 s-1) of R. leguminosarum LpxC with UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine as the substrate are similar to values reported for E. coli LpxC. R. leguminosarum LpxC therefore provides a useful control for validating LpxC as the primary target of CHIR-090 in vivo. An E. coli construct in which the chromosomal lpxC gene is replaced by R. leguminosarum lpxC is resistant to CHIR-090 up to 100 μg/mL, or 400 times above the minimal inhibitory concentration for wild-type E. coli. Given its relatively broad spectrum and potency against diverse Gram-negative pathogens, CHIR-090 is an excellent lead for the further development of new antibiotics targeting the lipid A pathway. [ABSTRACT FROM AUTHOR]

The zinc-dependent enzyme LpxC catalyzes the deacetylation of UDP-3-O-acyl-GlcNAc, the first committed step of lipid A biosynthesis. Lipid A is an essential component of the outer membranes of most Gram-negative bacteria, including Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa, making LpxC an attractive target for antibiotic design. The inhibition of LpxC by a novel N-aroyl-L-threonine hydroxamic acid (CHIR-090) from a recent patent application (International Patent WO 2004/062601 A2 to Chiron and the University of Washington) is reported here. CHIR-090 possesses remarkable antibiotic activity against both E. coli and P. aeruginosa, comparable to that of ciprofloxacin. The biological activity of CHIR-090 is explained by its inhibition of diverse LpxC orthologues at low nanomolar concentrations, including that of Aquifex aeolicus, for which structural information is available. The inhibition of A. aeolicus LpxC by CHIR-090 occurs in two steps. The first step is rapid and reversible, with a Ki of 1.0- 1.7 nM, depending upon the method of assay. The second step involves the conversion of the El complex with a half-life of about a minute to a tightly bound form. The second step is functionally irreversible but does not result in the covalent modification of the enzyme, as judged by electrospray ionization mass spectrometry. CHIR-090 is the first example of a slow, tight-binding inhibitor for LpxC and may be the prototype for a new generation of LpxC inhibitors with therapeutic applicability. [ABSTRACT FROM AUTHOR]

The first committed step of lipid A biosynthesis in Gram-negative bacteria is catalyzed by the zinc-dependent hydrolase LpxC that removes an acetate from the nitrogen at the 2"-position of UDP-3- O-acyl-N-acetylglucosamine. Recent structural characterization by both NMR and X-ray crystallography provides many important details about the active site environment of LpxC from Aqujfex aeolicus, a heat-stable orthologue that displays 32% sequence identity to LpxC from Escherichia coil. The detailed reaction mechanism and specific roles of active site residues for LpxC from A. aeolicus are further analyzed here. The pH dependencies of /CCaI/KM and kcat for the deacetylation of the substrate UDP-3-O-[(R)-3- hydroxymyristoyl)-GlcNAc are both bell-shaped. The ascending acidic limb (pKi) was fitted to 6.1 ± 0.2. for kcat and 5.7 ± 0.2 for kcat/KM. The descending basic limb (pKz) was fitted to 8.0 ± 0.2 for kcat and 8.4 ± 0.2 for kcat/KM. The pH dependence of the E73A mutant exhibits loss of the acidic limb, and the mutant retains only 0.15% activity versus the wild type. The pH dependencies of the other active site mutants H253A, K227A, H253A/K227A, and D234N remain bell-shaped, although their significantly lower activities (0.25%, 0.05%, 0.007%, and 0.57%, respectively) suggest that they contribute significantly to catalysis. Our cumulative data support a mechanism for LpxC wherein Glu73 serves as the general base for deprotonation and activation of the zinc-bound water. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide, the major constituent of the outer monolayer of the outer membrane of Gram-negative bacteria, is anchored into the membrane through the hydrophobic moiety lipid A, a hexaacylated disaccharide. The zinc-dependent metalloamidase UDP- 3.- O-acyl-N-acetylglucosamine deacetylase (LpxC) catalyzes the second and committed step in the biosynthesis of lipid A. LpxC shows no homology to mammalian metalloamidases and is essential for cell viability, making it an important target for the development of novel antibacterial compounds. Recent NMR and X-ray studies of the LpxC from Aqu-fex aeolicus have provided the first structural information about this family of proteins. Insight into the catalytic mechanism and the design of effective inhibitors could be facilitated by more detailed structural and biochemical studies that define substrate-protein interactions and the roles of specific residues in the active site. Here, we report the synthesis of the `3C-labeled substrate-analogue inhibitor TU-514, and the subsequent refinement of the solution structure of the A. aeolicus LpxC-TU-5 14 complex using residual dipolar couplings. We also reevaluate the catalytic role of an active site histidine, H253, on the basis of both its pKa as determined by NMR titration and pH-dependent kinetic analyses. These results provide a structural basis for the design of more potent LpxC inhibitors than those that are currently available. [ABSTRACT FROM AUTHOR]

Examines the genetic aspects associated with lipid biosynthesis. Methods used to catalyze the early steps of lipids; Findings of studies conducted; Methodology used in studies.