Enzymatic synthesis of lipid A molecules with four amide-linked acyl chains. LpxA acyltransferases selective for an analog of UDP-N-acetylglucosamine in which an amine replaces the 3"-hydroxyl group

LpxA of Escherichia coli catalyzes the acylation of the glucosamine 3-OH group of UDP-GlcNAc, using R-3-hydroxymyristoyl-acyl carrier protein (ACP) as the donor substrate. We now demonstrate that LpxA in cell extracts of Mesorhizobium loti and Leptospira interrogans, which synthesize lipid A molecules containing 2,3-diamino-2,3-dideoxy-d-glucopyranose (GlcN3N) units in place of glucosamine, do not acylate UDP-GlcNAc. Instead, these LpxA acyltransferases require a UDP-Glc-NAc derivative (designated UDP 2-acetamido-3-amino-2,3-dideoxy-alpha-d-glucopyranose or UDP-GlcNAc3N), characterized in the preceding paper, in which an amine replaces the glucosamine 3-OH group. L. interrogans LpxA furthermore displays absolute selectivity for 3-hydroxylauroyl-ACP as the donor, whereas M. loti LpxA functions almost equally well with 10-, 12-, and 14-carbon 3-hydroxyacyl-ACPs. The substrate selectivity of L. interrogans LpxA is consistent with the structure of L. interrogans lipid A. The mechanism of L. interrogans LpxA appears to be similar to that of E. coli LpxA, given that the essential His(125) residue of E. coli LpxA is conserved and is also required for acyltransferase activity in L. interrogans. Acidithiobacillus ferrooxidans (an organism that makes lipid A molecules containing both GlcN and GlcN3N) has an ortholog of LpxA that is selective for UDP-GlcNAc3N, but the enzyme also catalyzes the acylation of UDP-GlcNAc at a slow rate. E. coli LpxA acylates UDP-GlcNAc and UDP-GlcNAc3N at comparable rates in vitro. However, UDP-GlcNAc3N is not synthesized in vivo, because E. coli lacks gnnA and gnnB. When the latter are supplied together with A. ferrooxidans lpxA, E. coli incorporates a significant amount of GlcN3N into its lipid A.     

Relaxed acyl chain specificity of Bordetella UDP-N-acetylglucosamine acyltransferases

Lipid A (endotoxin) is a major structural component of Gram-negative outer membranes. It also serves as the hydrophobic anchor of lipopolysaccharide and is a potent activator of the innate immune response. Lipid A molecules from the genus Bordetella are reported to exhibit unusual structural asymmetry with respect to the acyl chains at the 3- and 3'-positions. These acyl chains are attached by UDP-N-acetylglucosamine acyltransferase (LpxA). To determine the origin of the acyl variability, the single lpxA ortholog present in each of the genomes of Bordetella bronchiseptica (lpxA(Br)), Bordetella parapertussis (lpxA(Pa)), and Bordetella pertussis (lpxA(Pe)) was cloned and expressed in Escherichia coli. In contrast to all LpxA proteins studied to date, LpxA(Br) and LpxA(Pe) display relaxed acyl chain length specificity in vitro, utilizing C(10)OH-ACP, C(12)OH-ACP, and C(14)OH-ACP at similar rates. Furthermore, hybrid lipid A molecules synthesized at 42 degrees C by an E. coli lpxA mutant complemented with lpxA(Pe) contain C(10)OH, C(12)OH, and C(14)OH at both the 3- and 3'-positions, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In contrast, LpxA from B. parapertussis did not display relaxed specificity but was selective for C(10)OH-ACP. This study provides an enzymatic explanation for some of the unusual acyl chain variations found in Bordetella lipid A.     

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.     

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

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 the lpxA gene of Pseudomonas aeruginosa from a library of Pseudomonas strain PAO1 expressed in Escherichia 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 of P. 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 predicted Pseudomonas 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 complemented E. coli RO138, a temperature-sensitive mutant harboring lpxA2. 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′.     

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.     

A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis

Using localized mutagenesis of whole cells, we have isolated a temperature-sensitive UDP-N-acetylglucosamine acyltransferase mutant of Escherichia coli that loses all detectable acyltransferase activity and quickly dies after a shift from 30 to 42 degrees C. Acyltransferase activity and temperature resistance are restored by transforming the mutant with a hybrid plasmid containing the E. coli gene for UDP-GlcNAc acyltransferase (lpxA). In addition, a new assay has been developed for quantitating the amount of lipid A (the active component of endotoxin) in E. coli and related Gram-negative strains. Cells are labeled with 32Pi and extracted with chloroform/methanol/water (1:2:0.8, v/v) to remove glycerophospholipids. The residue is then hydrolyzed with 0.2 M HCl to liberate the "monophosphoryl" lipid A degradation products (Qureshi, N., Cotter, R. J. and Takayama, K. (1986) J. Microbiol. Methods 5, 65-77), each of which bears a single phosphate residue at position 4'. The amount of lipid A is normalized to the total amount of labeled glycerophospholipid present in the cells. The steady state ratio of lipid A to glycerophospholipid in wild-type cells is approximately 0.12. The lipid A content of the acyltransferase mutant is reduced 2-3-fold, and the rate of lipid A synthesis is reduced 10-fold when compared to wild-type after 60 min at 42 degrees C. These results provide physiological evidence that UDP-N-acetylglucosamine acyltransferase is the major committed step for lipid A biosynthesis in E. coli and that lipid A is an essential molecule.     

Biosynthesis of lipid A precursors in Escherichia coli. A cytoplasmic acyltransferase that converts UDP-N-acetylglucosamine to UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine

Preliminary studies from our laboratory have suggested the existence of a novel set of fatty acyltransferases in extracts of Escherichia coli that attach two R-3-hydroxymyristoyl moieties to UDP-GlcNAc (Anderson, M.S., Bulawa, C.E., and Raetz, C.R.H. (1985) J. Biol. Chem. 260, 15536-15541). The resulting "glucosamine-derived" phospholipids appear to be crucial precursors for the biosynthesis of the lipid A component of lipopolysaccharide. We now describe an assay and a 1000-fold purification of the first enzyme in this pathway, which catalyzes the reaction: UDP-GlcNAc + R-3-hydroxymyristoyl-acyl carrier protein----UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc + acyl carrier protein. The covalent structure of the monoacylated UDP-GlcNAc product was established by fast atom bombardment mass spectrometry and 1H-NMR spectroscopy. The UDP-GlcNAc acyltransferase has a strict requirement for R-3-hydroxymyristoyl-acyl carrier protein, since R-3-hydroxymyristoyl coenzyme A and myristoyl-acyl carrier protein are not substrates. Of various NDP-GlcNAc preparations examined, only the uridine and thymidine derivatives were utilized to a significant extent. When the product of the reaction (UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc) was isolated and reincubated with crude E. coli extracts, it was rapidly converted to more hydrophobic products in the presence of R-3-hydroxymyristoyl-acyl carrier protein. We propose that the addition of an R-3-hydroxymyristoyl residue to the 3 position of the GlcNAc moiety of UDP-GlcNAc is the first committed step in lipid A biosynthesis and that UDP-GlcNAc is situated at a biosynthetic branchpoint in E. coli leading either to lipid A or to peptidoglycan.     

Nucleotide substrate recognition by UDP-N-acetylglucosamine acyltransferase (LpxA) in the first step of lipid A biosynthesis

Lipid A is an integral component of the lipopolysaccharide (LPS) that forms the selective and protective outer monolayer of Gram-negative bacteria, and is essential for bacterial growth and viability. UDP-N-acetylglucosamine acyltransferase (LpxA) initiates lipid A biosynthesis by catalyzing the transfer of R-3-hydroxymyristic acid from acyl carrier protein to the 3'-hydroxyl group of UDP-GlcNAc. The enzyme is a homotrimer, and previous studies suggested that the active site lies within a positively charged cleft formed at the subunit-subunit interface. The crystal structure of Escherichia coli LpxA in complex with UDP-GlcNAc reveals details of the substrate-binding site, with prominent hydrophilic interactions between highly conserved clusters of residues (Asn198, Glu200, Arg204 and Arg205) with UDP, and (Asp74, His125, His144 and Gln161) with the GlcNAc moiety. These interactions serve to bind and orient the substrate for catalysis. The crystallographic model supports previous results, which suggest that acylation occurs via nucleophilic attack of deprotonated UDP-GlcNAc on the acyl donor in a general base-catalyzed mechanism involving a catalytic dyad of His125 and Asp126. His125, the general base, interacts with the 3'-hydroxyl group of UDP-GlcNAc to generate the nucleophile. The Asp126 side-chain accepts a hydrogen bond from His125 and helps orient the general base to participate in catalysis. Comparisons with an LpxA:peptide inhibitor complex indicate that the peptide competes with both nucleotide and acyl carrier protein substrates.     

Acyl chain specificity of the acyltransferases LpxA and LpxD and substrate availability contribute to lipid A fatty acid heterogeneity in Porphyromonas gingivalis

Porphyromonas gingivalis lipid A is heterogeneous with regard to the number, type, and placement of fatty acids. Analysis of lipid A by matrix-assisted laser desorption ionization-time of flight mass spectrometry reveals clusters of peaks differing by 14 mass units indicative of an altered distribution of the fatty acids generating different lipid A structures. To examine whether the transfer of hydroxy fatty acids with different chain lengths could account for the clustering of lipid A structures, P. gingivalis lpxA (lpxA(Pg)) and lpxD(Pg) were cloned and expressed in Escherichia coli strains in which the homologous gene was mutated. Lipid A from strains expressing either of the P. gingivalis transferases was found to contain 16-carbon hydroxy fatty acids in addition to the normal E. coli 14-carbon hydroxy fatty acids, demonstrating that these acyltransferases display a relaxed acyl chain length specificity. Both LpxA and LpxD, from either E. coli or P. gingivalis, were also able to incorporate odd-chain fatty acids into lipid A when grown in the presence of 1% propionic acid. This indicates that E. coli lipid A acyltransferases do not have an absolute specificity for 14-carbon hydroxy fatty acids but can transfer fatty acids differing by one carbon unit if the fatty acid substrates are available. We conclude that the relaxed specificity of the P. gingivalis lipid A acyltransferases and the substrate availability account for the lipid A structural clusters that differ by 14 mass units observed in P. gingivalis lipopolysaccharide preparations.     

Molecular cloning of the genes for lipid A disaccharide synthase and UDP-N-acetylglucosamine acyltransferase in Escherichia coli.

Several enzymes have been discovered recently in crude extracts of Escherichia coli that appear to be involved in the biosynthesis of the lipid A component of lipopolysaccharide. Two of these are lipid A disaccharide synthase and UDP-N-acetylglucosamine acyltransferase. Lipid A disaccharide synthase activity is barely detectable in cells harboring a lesion in the lpxB (pgsB) gene. We subcloned the lpxB gene from plasmid pLC26-43 of the Clarke and Carbon collection (L. Clarke and J. Carbon, Cell 9:91-99, 1976) and localized it to a 1.7-kilobase-pair fragment of DNA counterclockwise of dnaE on the E. coli chromosome. Furthermore, we discovered a new gene (lpxA) located adjacent to and counterclockwise of lpxB that encodes or controls UDP-N-acetylglucosamine acyltransferase. Our data prove that lpxB and lpxA are transcribed in the clockwise direction and suggest that they may be cotranscribed.     

Insertional inactivation of the lpxA gene involved in the biosynthesis of lipid A in Neisseria meningitidis resulted in lpxA/lpxA::aph-3' heterodiploids

The lpxA gene is known to be involved in the biosynthesis of lipid A in Gram-negative bacteria and thought to be an essential gene. However, viable meningococcal lpxA mutants devoid of detectable endotoxin (lipooligosaccharide) have been reported. We characterised such mutants in strains of Neisseria meningitidis belonging to serogroups B and C using molecular and biochemical analysis. While lpxA mutants with no detectable or a low level of lipooligosaccharide could be obtained in N. meningitidis, the simple insertional inactivation of lpxA was not possible. In all mutants, we obtained lpxA/lpxA::aph-3' heterodiploids harbouring one copy of the wild-type lpxA gene and one copy of the inactivated lpxA gene by insertion of the kanamycin resistance cassette, aph-3'. The absence of lipooligosaccharide in these mutants may result from a negative transdominance effect of a truncated LpxA protein on the wild-type LpxA protein.     

Dual targeting antibacterial peptide inhibitor of early lipid a biosynthesis

UDP-3-O-(R-3-hydroxyacyl)GlcN N-acyltransferase (LpxD) has been shown to be essential to survival of lipid A producing Gram-negative bacteria. In this study, LpxD-binding peptides 12 amino acids in length were identified from a phage-bound random peptide library screen. Three peptides displayed antibacterial activity when expressed intracellularly, one of which (RJPXD33) represented 15% of the total hits. RJPXD33 binds to E. coli LpxD with a K(d) of 6 μM and is competitive with R-3-hydroxymyristoyl-ACP binding. RJPXD33 can be C-terminally fused in vivo with thioredoxin or N-terminally modified in vitro with β-alanyl-fluorescein and maintain LpxD binding. The latter was used to develop an LpxD fluorescent binding assay used to evaluate unlabeled ligands and is amenable to small molecule library screening. Furthermore, RJPXD33 also binds to and inhibits E. coli UDP-N-acetylglucosamine acyltransferase (LpxA) with a K(d) of 20 μM, unearthing the possibility for the development of small molecule, dual-binding LpxA/LpxD inhibitors as novel antimicrobials.     

Oxidation and transamination of the 3"-position of UDP-N-acetylglucosamine by enzymes from Acidithiobacillus ferrooxidans. Role in the formation of lipid a molecules with four amide-linked acyl chains

Lipid A, a major component of the outer membranes of Escherichia coli and other Gram-negative bacteria, is usually constructed around a beta-1',6-linked glucosamine disaccharide backbone. However, in organisms like Acidithiobacillus ferrooxidans, Leptospira interrogans, Mesorhizobium loti, and Legionella pneumophila, one or both glucosamine residues are replaced with the sugar 2,3-diamino-2,3-dideoxy-d-glucopyranose. We now report the identification of two proteins, designated GnnA and GnnB, involved in the formation of the 2,3-diamino-2,3-dideoxy-d-glucopyranose moiety. The genes encoding these proteins were recognized because of their location between lpxA and lpxB in A. ferrooxidans. Based upon their sequences, the 313-residue GnnA protein was proposed to catalyze the NAD(+)-dependent oxidation of the glucosamine 3-OH of UDP-GlcNAc, and the 369-residue GnnB protein was proposed to catalyze the subsequent transamination to form UDP 2-acetamido-3-amino-2,3-dideoxy-alpha-d-glucopyranose (UDP-GlcNAc3N). Both gnnA and gnnB were cloned and expressed in E. coli using pET23c+. In the presence of l-glutamate and NAD(+), both proteins were required for the conversion of [alpha-(32)P]UDP-GlcNAc to a novel, less negatively charged sugar nucleotide shown to be [alpha-(32)P]UDP-GlcNAc3N. The latter contained a free amine, as judged by modification with acetic anhydride. Using recombinant GnnA and GnnB, approximately 0.4 mg of the presumptive UDP-GlcNAc3N was synthesized. The product was purified and subjected to NMR analysis to confirm the replacement of the GlcNAc 3-OH group with an equatorial NH(2). As shown in the accompanying papers, UDP-GlcNAc3N is selectively acylated by LpxAs of A. ferrooxidans, L. interrogans, and M. loti. UDP-GlcNAc3N may be useful as a substrate analog for diverse enzymes that utilize UDP-GlcNAc.     

Rapid analysis of large protein-protein complexes using NMR-derived orientational constraints: the 95 kDa complex of LpxA with acyl carrier protein

Characterization of protein-protein interactions that are critical to the specific function of many biological systems has become a primary goal of structural biology research. Analysis of these interactions by structural techniques is, however, challenging due to inherent limitations of the techniques and because many of the interactions are transient, and suitable complexes are difficult to isolate. In particular, structural studies of large protein complexes by traditional solution NMR methods are difficult due to a priori requirement of extensive assignments and a large number of intermolecular restraints for the complex. An approach overcoming some of these challenges by utilizing orientational restraints from residual dipolar couplings collected on solution NMR samples is presented. The approach exploits existing structures of individual components, including the symmetry properties of some of these structures, to assemble rapidly models for relatively large protein-protein complexes. An application is illustrated with a 95 kDa homotrimeric complex of the acyltransferase protein, LpxA (UDP-N-acetylglucosamine acyltransferase), and acyl carrier protein. LpxA catalyzes the first step in the biosynthesis of the lipid A component of lipopolysaccharide in Gram-negative bacteria. The structural model generated for this complex can be useful in the design of new anti-bacterial agents that inhibit the biosynthesis of lipid A.     

Structural Basis for the Recognition of Peptide RJPXD33 by Acyltransferases in Lipid A Biosynthesis

UDP-N-acetylglucosamine acyltransferase (LpxA) and UDP-3-O-(acyl)-glucosamine acyltransferase (LpxD) constitute the essential, early acyltransferases of lipid A biosynthesis. Recently, an antimicrobial peptide inhibitor, RJPXD33, was identified with dual affinity for LpxA and LpxD. To gain a fundamental understanding of the molecular basis of inhibitor binding, we determined the crystal structure of LpxA from Escherichia coli in complex with RJPXD33 at 1.9 Å resolutions. Our results suggest that the peptide binds in a unique modality that mimics (R)-β-hydroxyacyl pantetheine binding to LpxA and displays how the peptide binds exclusive of the native substrate, acyl-acyl carrier protein. Acyltransferase binding studies with photo-labile RJPXD33 probes and truncations of RJPXD33 validated the structure and provided fundamental insights for future design of small molecule inhibitors. Overlay of the LpxA-RJPXD33 structure with E. coli LpxD identified a complementary peptide binding pocket within LpxD and serves as a model for further biochemical characterization of RJPXD33 binding to LpxD.     

Outer membrane composition of a lipopolysaccharide-deficient Neisseria meningitidis mutant.

In the pathogen Neisseria meningitidis, a completely lipopolysaccharide (LPS)-deficient but viable mutant can be obtained by insertional inactivation of the lpxA gene, encoding UDP-GlcNAc acyltransferase required for the first step of lipid A biosynthesis. To study how outer membrane structure and biogenesis are affected by the absence of this normally major component, inner and outer membranes were separated and their composition analysed. The expression and assembly of integral outer membrane proteins appeared largely unaffected. However, the expression of iron limitation-inducible, cell surface-exposed lipoproteins was greatly reduced. Major changes were seen in the phospholipid composition, with a shift towards phosphatidylethanolamine and phosphatidylglycerol species containing mostly shorter chain, saturated fatty acids, one of which was unique to the LPS-deficient outer membrane. The presence of the capsular polysaccharide turned out to be essential for viability without LPS, as demonstrated by using a strain in which LPS biosynthesis could be switched on or off through a tac promoter-controlled lpxA gene. Taken together, these results can help to explain why meningococci have the unique ability to survive without LPS.     

Defective in cuticular ridges (DCR) of Arabidopsis thaliana, a gene associated with surface cutin formation, encodes a soluble diacylglycerol acyltransferase

A key step in the triacylglycerol (TAG) biosynthetic pathway is the final acylation of diacylglycerol (DAG) by DAG acyltransferase. In silico analysis has revealed that the DCR (defective in cuticular ridges) (At5g23940) gene has a typical HX(4)D acyltransferase motif at the N-terminal end and a lipid binding motif VX(2)GF at the middle of the sequence. To understand the biochemical function, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to acylate DAG specifically in an acyl-CoA-dependent manner. Overexpression of At5g23940 in a Saccharomyces cerevisiae quadruple mutant deficient in DAG acyltransferases resulted in TAG accumulation. At5g23940 rescued the growth of this quadruple mutant in the oleate-containing medium, whereas empty vector control did not. Lipid particles were localized in the cytosol of At5g23940-transformed quadruple mutant cells, as observed by oil red O staining. There was an incorporation of 16-hydroxyhexadecanoic acid into TAG in At5g23940-transformed cells of quadruple mutant. Here we report a soluble acyl-CoA-dependent DAG acyltransferase from Arabidopsis thaliana. Taken together, these data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid.     

A gene (plsD) from Clostridium butyricum that functionally substitutes for the sn-glycerol-3-phosphate acyltransferase gene (plsB) of Escherichia coli.

The sn-glycerol-3-phosphate acyltransferase (plsB) of Escherichia coli is a key regulatory enzyme that catalyzes the first committed step in phospholipid biosynthesis. We report the initial characterization of a novel gene (termed plsD) from Clostridium butyricum, cloned based on its ability to complement the sn-glycerol-3-phosphate auxotrophic phenotype of a plsB mutant strain of E. coli. Unlike the 83-kDa PlsB acyltransferase from E. coli, the predicted plsD open reading frame encoded a protein of 26.5 kDa. Two regions of strong homology to other lipid acyltransferases, including PlsB and PlsC analogs from mammals, plants, yeast, and bacteria, were identified. PlsD was most closely related to the 1-acyl-sn-glycerol-3-phosphate acyltransferase (plsC) gene family but did not complement the growth of plsC(Ts) mutants. An in vivo metabolic labeling experiment using a plsB plsX plsC(Ts) strain of E. coli confirmed that the plsD expression restored the ability of the cells to synthesize 1-acyl-glycerol-3-phosphate. However, glycerol-3-phosphate acyltransferase activity was not detected in vitro in assays using either acyl-acyl carrier protein or acyl coenzyme A as the substrate.     

Nucleotide sequence of the Escherichia coli gene for lipid A disaccharide synthase.

The lpxB gene of Escherichia coli, believed to be the structural gene for lipid A disaccharide synthase, is located in the min 4 region of the chromosome. It is adjacent to and clockwise of the lpxA gene, which is thought to encode UDP-N-acetylglucosamine acyltransferase. Preliminary evidence suggests that lpxA and lpxB are cotranscribed in the clockwise direction and thus constitute part of a previously unknown operon (D. N. Crowell, M. S. Anderson, and C. R. H. Raetz, J. Bacteriol. 168:152-159, 1986). We now report the complete nucleotide sequence of a 1,522-base-pair PvuII-HincII fragment known to carry the lpxB gene. This sequence contained an open reading frame of 1,149 base pairs, in agreement with the predicted size, location, and orientation of lpxB. There was a second open reading frame 5' to, and in the same orientation as, lpxB that corresponded to lpxA. The ochre codon terminating lpxA was shown to overlap the methionine codon identified as the initiation codon for lpxB, suggesting that these genes are cotranscribed and translationally coupled. A third open reading frame was also shown to begin at the 3' end of lpxB with analogous overlap between the opal codon terminating lpxB and the methionine codon that putatively initiates translation downstream of lpxB in the clockwise direction. These results argue that at least three genes constitute a translationally coupled operon in the min 4 region of the E. coli chromosome. The accompanying paper by Tomasiewicz and McHenry (J. Bacteriol. 169:5735-5744, 1987) presents 4.35 kilobases of DNA sequence, beginning at the 3' end of lpxB, and argues that dnaE and several other open reading frames may be members of this operon.     

Expression of a Porphyromonas gingivalis lipid A palmitylacyltransferase in Escherichia coli yields a chimeric lipid A with altered ability to stimulate interleukin-8 secretion

In Escherichia coli the gene htrB codes for an acyltransferase that catalyses the incorporation of laurate into lipopolysaccharide (LPS) as a lipid A substituent. We describe the cloning, expression and characterization of a Porphyromonas gingivalis htrB homologue. When the htrB homologue was expressed in wild-type E. coli or a mutant strain deficient in htrB, a chimeric LPS with altered lipid A structure was produced. Compared with wild-type E. coli lipid A, the new lipid A species contained a palmitate (C16) in the position normally occupied by laurate (C12) suggesting that the cloned gene performs the same function as E. coli htrB but preferentially transfers the longer-chain palmitic acid that is known to be present in P. gingivalis LPS. LPS was purified from wild-type E. coli, the E. coli htrB mutant strain and the htrB mutant strain expressing the P. gingivalis acyltransferase. LPS from the palmitate bearing chimeric LPS as well as the htrB mutant exhibited a reduced ability to activate human embryonic kidney 293 (HEK293) cells transfected with TLR4/MD2. LPS from the htrB mutant also had a greatly reduced ability to stimulate interleukin-8 (IL-8) secretion in both endothelial cells and monocytes. In contrast, the activity of LPS from the htrB mutant bacteria expressing the P. gingivalis gene displayed wild-type activity to stimulate IL-8 production from endothelial cells but a reduced ability to stimulate IL-8 secretion from monocytes. The intermediate activation observed in monocytes for the chimeric LPS was similar to the pattern seen in HEK293 cells expressing TLR4/MD2 and CD14. Thus, the presence of a longer-chain fatty acid on E. coli lipid A altered the activity of the LPS in monocytes but not endothelial cell assays and the difference in recognition does not appear to be related to differences in Toll-like receptor utilization.