Life with too much polyprenol: polyprenol reductase deficiency.

Congenital disorders of glycosylation (CDG) are caused by a dysfunction of glycosylation, an essential step in the manufacturing process of glycoproteins. This paper focuses on a 6-year-old patient with a new type of CDG-I caused by a defect of the steroid 5α reductase type 3 gene (SRD5A3). The clinical features were psychomotor retardation, pathological nystagmus, slight muscular hypotonia and microcephaly. SRD5A3 was recently identified encoding the polyprenol reductase, an enzyme catalyzing the final step of the biosynthesis of dolichol, which is required for the assembly of the glycans needed for N-glycosylation. Although an early homozygous stop-codon (c.57G>A [W19X]) with no functional protein was found in the patient, about 70% of transferrin (Tf) was correctly glycosylated. Quantification of dolichol and unreduced polyprenol in the patient's fibroblasts demonstrated a high polyprenol/dolichol ratio with normal amounts of dolichol, indicating that high polyprenol levels might compete with dolichol for the initiation of N-glycan assembly but without supporting normal glycosylation and that there must be an alternative pathway for dolichol biosynthesis.

Lipid A modifications in polymyxin-resistant Salmonella typhimurium: PMRA-dependent 4-amino-4-deoxy-L-arabinose, and phosphoethanolamine incorporation.

Lipid A of Salmonella typhimurium can be resolved into multiple molecular species. Many of these substances are more polar than the predominant hexa-acylated lipid A 1,4'-bisphosphate of Escherichia coli K-12. By using new isolation methods, we have purified six lipid A subtypes (St1 to St6) from wild type S. typhimurium. We demonstrate that these lipid A variants are covalently modified with one or two 4-amino-4-deoxy-l-arabinose (l-Ara4N) moieties. Each lipid A species with a defined set of polar modifications can be further derivatized with a palmitoyl moiety and/or a 2-hydroxymyristoyl residue in place of the secondary myristoyl chain at position 3'. The unexpected finding that St5 and St6 contain two l-Ara4N residues accounts for the anomalous structures of lipid A precursors seen in S. typhimurium mutants defective in 3-deoxy-d-manno-octulosonic acid biosynthesis in which only the 1-phosphate group is modified with the l-Ara4N moiety (Strain, S. M., Armitage, I. M., Anderson, L., Takayama, K., Quershi, N., and Raetz, C. R. H. (1985) J. Biol. Chem. 260, 16089-16098). Phosphoethanolamine (pEtN)-modified lipid A species are much less abundant than l-Ara4N containing forms in wild type S. typhimurium grown in broth but accumulate to high levels when l-Ara4N synthesis is blocked in pmrA(C)pmrE(-) and pmrA(C)pmrF(-) mutants. Purification and analysis of selected compounds demonstrate that one or two pEtN moieties may be present. Our findings show that S. typhimurium contains versatile enzymes capable of modifying both the 1- and 4'-phosphates of lipid A with l-Ara4N and/or pEtN groups. PmrA null mutants of S. typhimurium produce lipid A species without any pEtN or l-Ara4N substituents. However, PmrA is not needed for the incorporation of 2-hydroxymyristate or palmitate.

An inner membrane enzyme in Salmonella and Escherichia coli that transfers 4-amino-4-deoxy-L-arabinose to lipid A: induction on polymyxin-resistant mutants and role of a novel lipid-linked donor.

Attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A is required for the maintenance of polymyxin resistance in Escherichia coli and Salmonella typhimurium. The enzymes that synthesize l-Ara4N and transfer it to lipid A have not been identified. We now report an inner membrane enzyme, expressed in polymyxin-resistant mutants, that adds one or two l-Ara4N moieties to lipid A or its immediate precursors. No soluble factors are required. A gene located near minute 51 on the S. typhimurium and E. coli chromosomes (previously termed orf5, pmrK, or yfbI) encodes the l-Ara4N transferase. The enzyme, renamed ArnT, consists of 548 amino acid residues in S. typhimurium with 12 possible membrane-spanning regions. ArnT displays distant similarity to yeast protein mannosyltransferases. ArnT adds two l-Ara4N units to lipid A precursors containing a Kdo disaccharide. However, as shown by mass spectrometry and NMR spectroscopy, it transfers only a single l-Ara4N residue to the 1-phosphate moiety of lipid IV(A), a precursor lacking Kdo. Proteins with full-length sequence similarity to ArnT are present in genomes of other bacteria thought to synthesize l-Ara4N-modified lipid A, including Pseudomonas aeruginosa and Yersinia pestis. As shown in the following article (Trent, M. S., Ribeiro, A. A., Doerrler, W. T., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 43132-43144), ArnT utilizes the novel lipid undecaprenyl phosphate-alpha-l-Ara4N as its sugar donor, suggesting that l-Ara4N transfer to lipid A occurs on the periplasmic side of the inner membrane.

Accumulation of a polyisoprene-linked amino sugar in polymyxin-resistant Salmonella typhimurium and Escherichia coli: structural characterization and transfer to lipid A in the periplasm.

Polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium accumulate a novel minor lipid that can donate 4-amino-4-deoxy-l-arabinose units (l-Ara4N) to lipid A. We now report the purification of this lipid from a pss(-) pmrA(C) mutant of E. coli and assign its structure as undecaprenyl phosphate-alpha-l-Ara4N. Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chromatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid. Matrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species [M - H](-) at m/z 977.5, consistent with undecaprenyl phosphate-alpha-l-Ara4N (M(r) = 978.41). (31)P NMR spectroscopy showed a single phosphorus atom at -0.44 ppm characteristic of a phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the l-Ara4N unit. One- and two-dimensional (1)H NMR studies confirmed the presence of a polyisoprene chain and a sugar moiety with chemical shifts and coupling constants expected for an equatorially substituted arabinopyranoside. Heteronuclear multiple-quantum coherence spectroscopy analysis demonstrated that a nitrogen atom is attached to C-4 of the sugar residue. The purified donor supports in vitro conversion of lipid IV(A) to lipid II(A), which is substituted with a single l-Ara4N moiety. The identification of undecaprenyl phosphate-alpha-l-Ara4N implies that l-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and establishes a new enzymatic pathway by which Gram-negative bacteria acquire antibiotic resistance.

Regulated covalent modifications of lipid A.

Regulated covalent modifications of lipid A are implicated in virulence of pathogenic Gram-negative bacteria. The Salmonella PhoP/PhoQ-activated gene pagP is required for resistance to cationic antimicrobial peptides and for biosynthesis of hepta-acylated lipid A species containing palmitate. Interestingly, pagP encodes an unusual enzyme of lipid A biosynthesis localized in the outer membrane, whereas all previously characterized lipid A enzymes are cytoplasmic or associated with the inner membrane. PagP is not unique, however, as pagL encodes another outer membrane enzyme in Salmonella that deacylates the 3 position of lipid A.S. typhimurium also synthesizes S-2-hydroxymyristate modified lipid A in a PhoP/PhoQ-dependent manner. We postulated that 2-hydroxylation might be catalyzed by a novel dioxygenase. Using well-characterized dioxygenase sequences as probes, tBLASTn searches revealed unassigned open reading frame(s) with similarity to mammalian aspartyl beta-hydroxylases in bacteria known to make 2-hydroxyacylated lipid A. The S. typhimurium aspartyl beta-hydroxylase homologue (lpxO) was cloned and expressed in Escherichia coli K-12, which does not contain lpxO. Analysis of the resulting construct revealed that lpxO expression induces O(2)-dependent formation of 2-hydroxymyristate-modified lipid A in E. coli. LpxO may be an inner membrane enzyme that catalyzes Fe(2+)/ascorbate/alpha-ketoglutarate dependent hydroxylation of lipid A. We propose that 2-hydroxymyristate released from LPS inside infected animal cells might be converted to 2-hydroxymyristoyl coenzyme A, a potent inhibitor of protein N-myristoyl transferase.

A Chlamydia trachomatis UDP-N-acetylglucosamine acyltransferase selective for myristoyl-acyl carrier protein. Expression in Escherichia coli and formation of hybrid lipid A species.

Chlamydia trachomatis lipid A is unusual in that it is acylated with myristoyl chains at the glucosamine 3 and 3' positions. We have cloned and expressed the gene encoding UDP-N-acetylglucosamine 3-O-acyltransferase of C. trachomatis (CtlpxA), the first enzyme of lipid A biosynthesis. C. trachomatis LpxA displays approximately 20-fold selectivity for myristoyl-ACP over R/S-3-hydroxymyristoyl-ACP under standard assay conditions, consistent with the proposed structure of C. trachomatis lipid A. CtLpxA is the first reported UDP-N-acetylglucosamine acyltransferase that prefers a non-hydroxylated acyl-ACP to a hydroxyacyl-ACP. When CtlpxA was expressed in RO138, a temperature-sensitive lpxA mutant of Escherichia coli, five new hybrid lipid A species were made in vivo after 2 h at 42 degrees C, in place of Escherichia coli lipid A. These compounds were purified and analyzed by matrix-assisted laser desorption ionization/time of flight mass spectrometry. In each case, a myristoyl chain replaced one or both of the ester linked 3-hydroxymyristoyl residues of E. coli lipid A. With prolonged growth at 42 degrees C, all the ester-linked 3-hydroxymyristoyl residues were replaced with myristate chains. Re-engineering the structure of E. coli lipid A should facilitate the microbiological production of novel agonists or antagonists of the innate immunity receptor TLR-4, with possible uses as adjuvants or anti-inflammatory agents.

An Escherichia coli mutant defective in lipid export.

Escherichia coli phospholipids and lipopolysaccharide, made on the inner surface of the inner membrane, are rapidly transported to the outer membrane by mechanisms that are not well characterized. We now report a temperature-sensitive mutant (WD2) with an A270T substitution in a trans-membrane region of the ABC transporter MsbA. As shown by (32)P(i) and (14)C-acetate labeling, export of all major lipids to the outer membrane is inhibited by approximately 90% in WD2 after 30 min at 44 degrees C. Transport of newly synthesized proteins is not impaired. Electron microscopy shows reduplicated inner membranes in WD2 at 44 degrees C, consistent with a key role for MsbA in lipid trafficking.

Site-directed mutagenesis of the bacterial metalloamidase UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC). Identification of the zinc binding site.

UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second step in the biosynthesis of lipid A in Gram-negative bacteria. Compounds targeting this enzyme are proposed to chelate the single, essential zinc ion bound to LpxC and have been demonstrated to stop the growth of Escherichia coli. A comparison of LpxC sequences from diverse bacteria identified 10 conserved His, Asp, and Glu residues that might play catalytic roles. Each amino acid was altered in both E. coli and Aquifex aeolicus LpxC and the catalytic activities of the variants were determined. Three His and one Asp residues (H79, H238, D246, and H265) are essential for catalysis based on the low activities (<0.1% of wild-type LpxC) of mutants with alanine substitutions at these positions. H79 and H238 likely coordinate zinc; the Zn(2+) content of the purified variant proteins is low and the specific activity is enhanced by the addition of Zn(2+). The third side chain to coordinate zinc is likely either H265 or D246 and a fourth ligand is likely a water molecule, as indicated by the hydroxamate inhibition, suggesting a His(3)H(2)O or His(2)AspH(2)O Zn(2+)-polyhedron in LpxC. The decreased zinc inhibition of LpxC mutants at E78 suggests that this side chain may coordinate a second, inhibitory Zn(2+) ion. Given the absence of any known Zn(2+) binding motifs, the active site of LpxC may have evolved differently than other well-studied zinc metalloamidases, a feature that should aid in the design of safe antibiotics.

A PhoP/PhoQ-induced Lipase (PagL) that catalyzes 3-O-deacylation of lipid A precursors in membranes of Salmonella typhimurium.

Pathogenic bacteria modify the structure of the lipid A portion of their lipopolysaccharide in response to environmental changes. Some lipid A modifications are important for virulence and resistance to cationic antimicrobial peptides. The two-component system PhoP/PhoQ plays a central role in regulating lipid A modification. We now report the discovery of a PhoP/PhoQ-activated gene (pagL) in Salmonella typhimurium, encoding a deacylase that removes the R-3-hydroxymyristate moiety attached at position 3 of certain lipid A precursors. The deacylase gene (pagL) was identified by assaying for loss of deacylase activity in extracts of 14 random TnphoA::pag insertion mutants. The pagL gene encodes a protein of 185 amino acid residues unique to S. typhimurium and closely related organisms such as Salmonella typhi. Heterologous expression of pagL in Escherichia coli on plasmid pWLP21 results in loss of the R-3-hydroxymyristate moiety at position 3 in approximately 90% of the lipid A molecules but does not inhibit cell growth. PagL is synthesized with a 20-amino acid N-terminal signal peptide and is localized mainly in the outer membrane, as judged by assays of separated S. typhimurium membranes and by SDS-polyacrylamide gel analysis of membranes from E. coli cells that overexpress PagL. The function of PagL is unknown, given that S. typhimurium mutants lacking pagL display no obvious phenotypes, but PagL might nevertheless play a role in pathogenesis if it serves to modulate the cytokine response of an infected animal host.

Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. A novel membrane enzyme dependent upon phosphatidylethanolamine.

Certain strains of Escherichia coli and Salmonella contain lipopolysaccharide (LPS) modified with a phosphoethanolamine (pEtN) group at position 7 of the outer 3-deoxy-d-manno-octulosonic acid (Kdo) residue. Using the heptose-deficient E. coli mutant WBB06 (Brabetz, W., Muller-Loennies, S., Holst, O., and Brade, H. (1997) Eur. J. Biochem. 247, 716-724), we now demonstrate that the critical parameter determining the presence or absence of pEtN is the concentration of CaCl(2) in the medium. As judged by mass spectrometry, half the LPS in WBB06, grown on nutrient broth with 5 mm CaCl(2), is derivatized with a pEtN group, whereas LPS from WBB06 grown without supplemental CaCl(2) is not. Membranes from E. coli WBB06 or wild-type W3110 grown on 5-50 mm CaCl(2) contain a novel pEtN transferase that uses the precursor Kdo(2)-[4'-(32)P]lipid IV(A) as an acceptor. Transferase is not present in membranes of E. coli grown with 5 mm MgCl(2), BaCl(2), or ZnCl(2). Hydrolysis of the in vitro reaction product, pEtN-Kdo(2)-[4'-(32)P]lipid IV(A), at pH 4.5 shows that the pEtN substituent is located on the outer Kdo moiety. Membranes from an E. coli pss knockout mutant grown on 50 mm CaCl(2), which lack phosphatidylethanolamine, do not contain measurable transferase activity unless exogenous phosphatidylethanolamine is added back to the assay system. The induction of the pEtN transferase by 5-50 mm CaCl(2) suggests possible role(s) in establishing transformation competence or resisting environmental stress, and represents the first example of a regulated covalent modification of the inner core of E. coli LPS.

Transfer of palmitate from phospholipids to lipid A in outer membranes of gram-negative bacteria.

Regulated covalent modifications of lipid A are implicated in virulence of pathogenic Gram-negative bacteria. The Salmonella typhimurium PhoP/PhoQ-activated gene pagP is required both for biosynthesis of hepta-acylated lipid A species containing palmitate and for resistance to cationic anti-microbial peptides. Palmitoylated lipid A can also function as an endotoxin antagonist. We now show that pagP and its Escherichia coli homolog (crcA) encode an unusual enzyme of lipid A biosynthesis localized in the outer membrane. PagP transfers a palmitate residue from the sn-1 position of a phospholipid to the N-linked hydroxymyristate on the proximal unit of lipid A (or its precursors). PagP bearing a C-terminal His(6)-tag accumulated in outer membranes during overproduction, was purified with full activity and was shown by cross-linking to behave as a homodimer. PagP is the first example of an outer membrane enzyme involved in lipid A biosynthesis. Additional pagP homologs are encoded in the genomes of Yersinia and Bordetella species. PagP may provide an adaptive response toward both Mg(2+) limitation and host innate immune defenses.

Oxygen requirement for the biosynthesis of the S-2-hydroxymyristate moiety in Salmonella typhimurium lipid A. Function of LpxO, A new Fe2+/alpha-ketoglutarate-dependent dioxygenase homologue.

Lipid A molecules of certain Gram-negative bacteria, including Salmonella typhimurium and Pseudomonas aeruginosa, may contain secondary S-2-hydroxyacyl chains. S. typhimurium has recently been shown to synthesize its S-2-hydroxymyristate-modified lipid A in a PhoP/PhoQ-dependent manner, suggesting a possible role for the 2-OH group in pathogenesis. We postulated that 2-hydroxylation might be catalyzed by a novel dioxygenase. Lipid A was extracted from a PhoP-constitutive mutant of S. typhimurium grown in the presence or absence of O(2). Under anaerobic conditions, no 2-hydroxymyristate-containing lipid A was formed. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipid A from cells grown in the presence of (18)O(2) confirmed the direct incorporation of molecular oxygen into 2-hydroxyacyl-modified lipid A. Using several well characterized dioxygenase protein sequences as probes, tBLASTn searches revealed unassigned open reading frame(s) with similarity to mammalian aspartyl/asparaginyl beta-hydroxylases in bacteria known to make 2-hydroxyacylated lipid A molecules. The S. typhimurium aspartyl/asparaginyl beta-hydroxylase homologue (designated lpxO) was cloned into pBluescriptSK and expressed in Escherichia coli K-12, which does not contain lpxO. Analysis of the resulting construct revealed that lpxO expression is sufficient to induce O(2)-dependent formation of 2-hydroxymyristate-modified lipid A in E. coli. LpxO very likely is a novel Fe(2+)/alpha-ketoglutarate-dependent dioxygenase that catalyzes the hydroxylation of lipid A (or of a key precursor). The S. typhimurium lpxO gene encodes a polypeptide of 302 amino acids with predicted membrane-anchoring sequences at both ends. We hypothesize that 2-hydroxymyristate chains released from lipopolysaccharide inside infected macrophages might be converted to 2-hydroxymyristoyl coenzyme A, a well characterized, potent inhibitor of protein N-myristoyl transferase.

Purification and mass spectrometry of six lipid A species from the bacterial endosymbiont Rhizobium etli. Demonstration of a conserved distal unit and a variable proximal portion.

Lipid A of Rhizobium etli CE3 differs dramatically from that of other Gram-negative bacteria. Key features include the presence of an unusual C28 acyl chain, a galacturonic acid moiety at position 4', and an acylated aminogluconate unit in place of the proximal glucosamine. In addition, R. etli lipid A is reported to lack phosphate and acyloxyacyl residues. Most of these remarkable structural claims are consistent with our recent enzymatic studies. However, the proposed R. etli lipid A structure is inconsistent with the ability of the precursor (3-deoxy-D-manno-octulosonic acid)(2)-4'-(32)P-lipid IV(A) to accept a C28 chain in vitro (Brozek, K. A., Carlson, R. W., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 32126-32136). To re-evaluate the structure, CE3 lipid A was isolated by new chromatographic procedures. CE3 lipid A is now resolved into six related components. Aminogluconate is present in D-1, D-2, and E, whereas B and C contain the typical glucosamine disaccharide seen in lipid A of most other bacteria. All the components possess a peculiar acyloxyacyl moiety at position 2', which includes the ester-linked C28 chain. As judged by mass spectrometry, the distal glucosamine units of A through E are the same, but the proximal units are variable. As described in the accompanying article (Que, N. L. S., Ribeiro, A. A., and Raetz, C. R. H. (2000) J. Biol. Chem. 275, 28017-28027), the discovery of component B suggests a plausible enzymatic pathway for the biosynthesis of the aminogluconate residue found in species D-1, D-2, and E of R. etli lipid A. We suggest that the unusual lipid A species of R. etli might be essential during symbiosis with leguminous host plants.

Two-dimensional NMR spectroscopy and structures of six lipid A species from Rhizobium etli CE3. Detection of an acyloxyacyl residue in each component and origin of the aminogluconate moiety.

The chemical structures of six lipid A species (A, B, C, D-1, D-2, and E) purified from Rhizobium etli CE3 were investigated by one- and two-dimensional NMR spectroscopy. The R. etli lipid A subtypes each contain an unusual acyloxyacyl residue at position 2' as part of a conserved distal glucosamine moiety but differ in their proximal units. All R. etli lipid A species lack phosphate groups. However, they are derivatized with an alpha-linked galacturonic acid group at position 4', as shown by nuclear Overhauser effect spectroscopy. Component B, which had been not been reported in previous studies, features a beta, 1'-6 linked disaccharide of glucosamine acylated at positions 2, 3, 2', and 3' in a pattern that is typical of lipid A found in other Gram-negative bacteria. D-1 contains an acylated aminogluconate unit in place of the proximal glucosamine residue of B. C and E lack ester-linked beta-hydroxyacyl chains at position 3, as judged by their H-3 chemical shifts, and may be synthesized from B and D-1, respectively, by the R. etli 3-O-deacylase. D-2 is an isomer of D-1 that forms nonenzymatically by acyl chain migration. A may be an elimination product derived from D-1 during hydrolysis at 100 degrees C (pH 4.5), a step needed to release lipid A from lipopolysaccharide. Based on these findings, we propose a biosynthetic scheme for R. etli lipid A in which B is generated first by a variation of the E. coli pathway. The aminogluconate unit of D-1 could then be made from B by enzymatic oxidation of the proximal glucosamine. As predicted by our hypothesis, enzyme(s) can be demonstrated in extracts of R. etli that convert (14)C-labeled B to D-1.

A facile enzymatic synthesis of uridine diphospho-[14C]galacturonic acid.

Galacturonic acid (GalA) is a major component of plant cell-wall-derived pectins. It can be also found in the cell-surface polysaccharides of different microorganisms, including several symbiotic and pathogenic bacteria. Uridine diphosphogalacturonic acid (UDP-GalA) is a likely donor for GalA during the biosynthesis of these polysaccharides. A highly efficient, yet simple, method is presented for generating and purifying UDP-[14C]GalA. Commercially available UDP-[14C]-galactose was quantitatively oxidized (>95% conversion) to UDP-[14C]GalA in the presence of high levels of galactose oxidase and catalase, at prolonged incubation times. Following this one-step enzymatic oxidation, UDP-[14C]GalA was purified using a polyethyleneimine cellulose column with a single-step 1 M NaCl elution. The authenticity of the purified UDP-[14C]GalA was verified by its relative mobility on thin-layer chromatograms, analysis of its chemical hydrolysis products, and 1H NMR spectroscopy. Our yield of >90% is much higher than by previously described methods. The method may serve as a prototype for the preparation of other radiolabeled uronic acids and their nucleotide derivatives.

High-resolution NMR spectroscopy of lipid A molecules containing 4-amino-4-deoxy-L-arabinose and phosphoethanolamine substituents. Different attachment sites on lipid A molecules from NH4VO3-treated Escherichia coli versus kdsA mutants of Salmonella typhimurium.

When Escherichia coli are grown on LB broth containing 25 mm NH(4)VO(3), complex modifications to the lipid A anchor of lipopolysaccharide are induced. Six modified lipid As (EV1-EV6) have been purified. Many of these variants possess 4-amino-4-deoxy-l-arabinose (l-Ara4N) and/or phosphoethanolamine (pEtN) substituents. Here we use NMR spectroscopy to investigate the attachment sites of the l-Ara4N and pEtN moieties on underivatized, intact EV3 and EV6 and on precursors II(A) and III(A) from kdsA mutants of Salmonella. CDCl(3)/CD(3)OD/D(2)O (2:3:1, v/v) is shown to be a superior solvent for homo- and heteronuclear one- and two-dimensional NMR experiments. The latter were not feasible previously because available solvents caused sample decomposition. Selective inverse decoupling difference spectroscopy is used to determine the attachment sites of substituents on EV3, EV6, II(A), and III(A). l-Ara4N is attached via a phosphodiester linkage to the 4'-phosphates of EV3 and EV6 and has the beta anomeric configuration. pEtN is attached by a pyrophosphate linkage to the 1-phosphate of EV6. The l-Ara4N and pEtN substituents of lipids II(A) and III(A) are attached in the opposite manner, with l-Ara4N on the 1-phosphate of II(A) and pEtN on the 4'-phosphate of III(A). Determination of the proper attachment sites of these substituents is necessary for elucidating the enzymology of lipid A biosynthesis and for characterizing polymyxin-resistant mutants, in which l-Ara4N and pEtN substituents are greatly increased.

Antibacterial agents that target lipid A biosynthesis in gram-negative bacteria. Inhibition of diverse UDP-3-O-(r-3-hydroxymyristoyl)-n-acetylglucosamine deacetylases by substrate analogs containing zinc binding motifs.

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second step in the biosynthesis of lipid A, a unique amphiphilic molecule found in the outer membranes of virtually all Gram-negative bacteria. Since lipid A biosynthesis is required for bacterial growth, inhibitors of LpxC have potential utility as antibiotics. The enzymes of lipid A biosynthesis, including LpxC, are encoded by single copy genes in all sequenced Gram-negative genomes. We have now cloned, overexpressed, and purified LpxC from the hyperthermophile Aquifex aeolicus. This heat-stable LpxC variant (the most divergent of all known LpxCs) displays 32% identity and 51% similarity over 277 amino acid residues out of the 305 in Escherichia coli LpxC. Although A. aeolicus LpxC deacetylates the substrate UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine at a rate comparable with E. coli LpxC, a phenyloxazoline-based hydroxamate that inhibits E. coli LpxC with K(i) of approximately 50 nM (Onishi, H. R., Pelak, B. A., Gerckens, L. S., Silver, L. L., Kahan, F. M., Chen, M. H., Patchett, A. A., Galloway, S. M., Hyland, S. A., Anderson, M. S., and Raetz, C. R. H. (1996) Science 274, 980-982) does not inhibit A. aeolicus LpxC. To determine whether or not broad-spectrum deacetylase inhibitors can be found, we have designed a new class of hydroxamate-containing inhibitors of LpxC, starting with the structure of the physiological substrate. Several of these compounds inhibit both E. coli and A. aeolicus LpxC at similar concentrations. We have also identified a phosphinate-containing substrate analog that inhibits both E. coli and A. aeolicus LpxC, suggesting that the LpxC reaction proceeds by a mechanism similar to that described for other zinc metalloamidases, like carboxypeptidase A and thermolysin. The differences between the phenyloxazoline and the substrate-based LpxC inhibitors might be exploited for developing novel antibiotics targeted either against some or all Gram-negative strains. We suggest that LpxC inhibitors with antibacterial activity be termed "deacetylins."

A Haemophilus influenzae gene that encodes a membrane bound 3-deoxy-D-manno-octulosonic acid (Kdo) kinase. Possible involvement of kdo phosphorylation in bacterial virulence.

The lipopolysaccharide of Haemophilus influenzae contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) residue derivatized with either a phosphate or an ethanolamine pyrophosphate moiety at the 4-OH position. In previous studies, we identified a kinase unique to H. influenzae extracts that phosphorylates Kdo-lipid IV(A), a key precursor of lipopolysaccharide in this organism. We have now identified the gene encoding the Kdo kinase by using an expression cloning approach. A cosmid library containing random DNA fragments from H. influenzae strain Rd was constructed in Escherichia coli. Extracts of 472 colonies containing individual hybrid cosmids were assayed for Kdo kinase activity. A single hybrid cosmid directing expression of the kinase was found. The kinase gene was identified by activity assays, sub-cloning, and DNA sequencing. When the putative kinase gene was expressed in E. coli behind a T7 promoter, massive overproduction of kinase activity was achieved ( approximately 8000-fold higher than in H. influenzae membranes). The catalytic properties and the product generated by the overexpressed kinase, assayed with Kdo-lipid IV(A) as the substrate, were the same as observed with H. influenzae membranes. Unexpectedly, the kinase gene was identical to a previously characterized open reading frame (orfZ), which had been shown to be important for establishing bacteremia in an infant rat model (Hood, D. W., Deadman, M. E., Allen, T., Masoud, H., Martin, A., Brisson, J. R., Fleischmann, R., Venter, J. C., Richards, J. C., and Moxon, E. R. (1996) Mol. Microbiol. 22, 951-965). However, based solely on the genome sequence of H. influenzae Rd, no biochemical function had been assigned to the product of orfZ, which we now designate kdkA ("Kdo kinase A"). Although Kdo phosphorylation may be critical for bacterial virulence of H. influenzae, it does not appear to be required for growth.

The active site of Escherichia coli UDP-N-acetylglucosamine acyltransferase. Chemical modification and site-directed mutagenesis.

UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes the reversible transfer of an R-3-hydroxyacyl chain from R-3-hydroxyacyl-acyl carrier protein to the glucosamine 3-OH of UDP-GlcNAc in the first step of lipid A biosynthesis. Lipid A is required for the growth and virulence of most Gram-negative bacteria, making its biosynthetic enzymes intriguing targets for the development of new antibacterial agents. LpxA is a member of a large family of left-handed beta-helical proteins, many of which are acyl- or acetyltransferases. We now demonstrate that histidine-, lysine-, and arginine-specific reagents effectively inhibit LpxA of Escherichia coli, whereas serine- and cysteine-specific reagents do not. Using this information in conjunction with multiple sequence alignments, we constructed site-directed alanine substitution mutations of conserved histidine, lysine, and arginine residues. Many of these mutant LpxA enzymes show severely decreased specific activities under standard assay conditions. The decrease in activity corresponds to decreased k(cat)/K(m,UDP-GlcNAc) values for all the mutants. With the exception of H125A, in which no activity is seen under any assay condition, the decrease in k(cat)/K(m,UDP-GlcNAc) mainly reflects an increased K(m,UDP-GlcNAc). His(125) of E. coli LpxA may therefore function as a catalytic residue, possibly as a general base. LpxA does not catalyze measurable UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc hydrolysis or UDP-GlcNAc/UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc exchange, arguing against a ping-pong mechanism with an acyl-enzyme intermediate.

Lipid A modifications characteristic of Salmonella typhimurium are induced by NH4VO3 in Escherichia coli K12. Detection of 4-amino-4-deoxy-L-arabinose, phosphoethanolamine and palmitate.

Two-thirds of the lipid A in wild-type Escherichia coli K12 is a hexa-acylated disaccharide of glucosamine in which monophosphate groups are attached at positions 1 and 4'. The remaining lipid A contains a monophosphate substituent at position 4' and a pyrophosphate moiety at position 1. The biosynthesis of the 1-pyrophosphate unit is unknown. Its presence is associated with lipid A translocation to the outer membrane (Zhou, Z., White, K. A., Polissi, A., Georgopoulos, C., and Raetz, C. R. H. (1998) J. Biol. Chem. 273, 12466-12475). To determine if a phosphatase regulates the amount of the lipid A 1-pyrophosphate, we grew cells in broth containing nonspecific phosphatase inhibitors. Na2WO4 and sodium fluoride increased the relative amount of the 1-pyrophosphate slightly. Remarkably, NH4VO3-treated cells generated almost no 1-pyrophosphate, but made six major new lipid A derivatives (EV1 to EV6). Matrix-assisted laser desorption ionization/time of flight mass spectrometry of purified EV1 to EV6 indicated that these compounds were lipid A species substituted singly or in combination with palmitoyl, phosphoethanolamine, and/or aminodeoxypentose residues. The aminodeoxypentose residue was released by incubation in chloroform/methanol (4:1, v/v) at 25 degrees C, and was characterized by 1H NMR spectroscopy. The chemical shifts and vicinal coupling constants of the two anomers of the aminodeoxypentose released from EV3 closely resembled those of synthetic 4-amino-4-deoxy-L-arabinose. NH4VO3-induced lipid A modification did not require the PhoP/PhoQ two-component regulatory system, and also occurred in E. coli msbB or htrB mutants. The lipid A variants that accumulate in NH4VO3-treated E. coli K12 are the same as many of those normally found in untreated Salmonella typhimurium and Salmonella minnesota, demonstrating that E. coli K12 has latent enzyme systems for synthesizing these important derivatives.