Biomedical subjects
C R Raetz
Publications and source records attributed to C R Raetz.
Chinese hamster ovary cell mutants defective in peroxisome biogenesis. Comparison to Zellweger syndrome.
We have previously reported the isolation of Chinese hamster ovary (CHO) cell mutants that are defective in the biosynthesis of plasmalogens, deficient in at least two peroxisomal enzymes (dihydroxyacetonephosphate (DHAP) acyltransferase and alkyl-DHAP synthase), and in which catalase is not found within peroxisomes (Zoeller, R. A., and Raetz, C. R. H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5170). We now provide further evidence that three such strains are more generally defective in peroxisome biogenesis. Electron microscopic cytochemistry revealed that the mutants did not contain recognizable peroxisomes. However, immunofluorescence microscopy using an antibody directed against peroxisomal integral membrane proteins revealed the presence of peroxisomal membrane ghosts resembling those seen in cells of patients suffering from one of the human peroxisomal disorders, Zellweger syndrome. Immunoblot analyses, using antibodies specific for peroxisomal matrix proteins, demonstrated deficiencies of peroxisomal proteins in the mutant CHO cells that were similar to those in Zellweger syndrome. Fusion of a CHO mutant with fibroblasts obtained from Zellweger patients resulted in restoration of peroxisomal dihydroxyacetonephosphate acyltransferase and peroxisomal acyl-coenzyme A oxidation activities. The hybrid cells also regained the ability to synthesize plasmenylethanolamine. Moreover, normal peroxisomes were seen by immunofluorescence in the hybrid cells. These results indicate that the hybrid cells have recovered the ability to assemble peroxisomes and that, although the mutant CHO cells are biochemically and morphologically very similar to cells from patients with Zellweger syndrome, the genetic lesions are distinct. Our somatic cell mutants should be useful in identifying factors and genes involved in peroxisome biogenesis and may aid the genetic categorization of the various peroxisomal disorders.
Biosynthesis of lipopolysaccharide in Escherichia coli. Cytoplasmic enzymes that attach 3-deoxy-D-manno-octulosonic acid to lipid A.
Previous studies in our laboratory led to the elucidation of the covalent structure of a tetraacyldisaccharide 1,4'-bisphosphate precursor of lipid A (designated lipid IVA), that accumulates at 42 degrees C in temperature-sensitive mutants defective in 3-deoxy-D-manno-octulosonic acid (KDO) biosynthesis (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). Using [4'-32P]lipid IVA as the probe, we now demonstrate the existence of cytoplasmic KDO-transferases in Escherichia coli capable of attaching 2 KDO residues, derived from CMP-KDO, to lipid IVA. A partial purification has been developed to obtain a cytoplasmic subfraction that adds these 2 KDO residues with a 90% yield. The product is shown to have the stoichiometry of (KDO)2-IVA by fast atom bombardment mass spectrometry and NMR spectroscopy. The partially purified enzyme can utilize alternative lipid-disaccharide cosubstrates bearing five or six fatty acyl chains, but it has an absolute requirement for a monophosphate residue at position 4' of the lipid acceptor. When reincubated with a crude cytoplasmic fraction, a nucleoside triphosphate and Mg2+, (KDO)2-IVA is rapidly metabolized to more polar substances, the identity of which is unknown. The KDO-transferase(s) described in the present study should be very useful for the semisynthetic preparation of complex lipopolysaccharide substructures and analogs.
Cytoplasmic requirement for peroxisome biogenesis in Chinese hamster ovary cells.
Hybrids constructed by fusion of wild-type Chinese hamster ovary cells (CHO-K1) to peroxisome-deficient CHO mutants (ZR-78.1) contain normal peroxisomes, demonstrating that the mutation(s) are recessive. "Nuclear hybrids" prepared by fusion of CHO-K1 karyoplasts to mutant ZR-78.1 occasionally fail to regain intact peroxisomes (approximately 1/300 cells). These peroxisome-deficient nuclear hybrids closely resemble the original mutant cells by biochemical criteria, but their modal chromosome number is 36-38, the same as that of CHO hybrids generated from intact cells. When the peroxisome-deficient nuclear hybrids are fused to wild-type cytoplasts, a fraction of the fusion products (at least 70%) continue to propagate normal peroxisomes indefinitely. Peroxisome biogenesis cannot be reinitiated in cells of mutant ZR-78.1 by fusion to wild-type cytoplasts. Our results suggest that a wild-type nucleus by itself is necessary but not sufficient for restoration of normal peroxisome biogenesis and that a cytoplasmic component of wild-type cells, possibly a normal peroxisome, is also required.
Purification and properties of lipid A disaccharide synthase of Escherichia coli.
Lipid A disaccharide synthase of Escherichia coli catalyzes the reaction 2,3-diacyl-GlcN-1-P + UDP-2,3-diacyl-GlcN----2',3'-diacyl-GlcN (beta,1'----6)2,3-diacyl-GlcN-1-P + UDP (Ray, B. L., Painter, G., and Raetz, C. R. H. (1984) J. Biol. Chem. 259, 4852-4859). Using a strain that overproduces the enzyme about 200-fold we have devised a simple purification to near homogeneity, utilizing two types of dye-ligand resins and heparin-agarose. The overall purification starting with membrane-free extracts was 54-fold (16,000-fold relative to wild-type extracts) with a 31% yield. The subunit molecular mass determined by sodium dodecyl sulfate gel electrophoresis is approximately 42,000 daltons, and the native enzyme appears to be a dimer. The amino-terminal sequence is (X)-(Thr)-Glu-Gln-(X)-Pro-Leu-Thr-Ie-Ala..., consistent with the results predicted from the DNA sequence, Met-Thr-Glu-Gln-Arg-Pro-Leu-Thr-Ile-Ala.... The purified enzyme displays a strong kinetic preference for sugar substrates bearing two fatty acyl moieties, but it is, nevertheless, very useful for the semisynthetic preparation of many lipid A analogs. Gel filtration studies demonstrate that the natural substrates (2,3-diacyl-GlcN-1-P and UDP-2,3-diacyl-GlcN) form micelles (n approximately equal to 300), rather than bilayers, under conditions used to assay the enzyme. Unlike most enzymes of glycerophospholipid synthesis, the lipid A disaccharide synthase does not require the presence of a detergent for catalytic activity. At 1 mM UDP-2,3-diacyl-GlcN the Vmax and Km values for 2,3-diacyl-GlcN-1-P are 14,028 +/- 513 nmol/min/mg and 0.27 +/- 0.02 mM. When 2,3-diacyl-GlcN-1-P is maintained at 1 mM, they are 12,368 +/- 472 nmol/min/mg and 0.11 +/- 0.01 mM for UDP-2,3-diacyl-GlcN.
Lipid A binding sites in membranes of macrophage tumor cells.
Lipopolysaccharide affects a variety of eukaryotic cells and mammalian organisms. These actions are involved in the pathogenesis of Gram-negative septicemia. Many of the actions of lipopolysaccharide are believed to be caused by its active moiety, lipid A. Our laboratory has previously identified a bioactive lipid A precursor, termed lipid IVA (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16888), which can be labeled with 32P of high specific activity and purified. In this work we have used the labeled probe, 4'-32P-lipid IVA, to develop a novel assay for the specific binding of lipid IVA to whole cells. We have also demonstrated its use in a ligand blotting assay of immobilized cellular proteins. Using the whole cell assay, we show that 4'-32P-lipid IVA specifically binds to RAW 264.7 macrophage-like cultured cells. The binding is saturable, is inhibited with excess unlabeled lipid IVA, and is proteinase K-sensitive. It displays cellular and pharmacological specificity. Using the ligand blotting assay, we show that several RAW 264.7 cell proteins can bind 4'-32P-lipid IVA. The two principal binding proteins have Mr values of 31 and 95 kDa, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fractionation studies indicate that the 31-kDa protein is enriched in the nuclear fraction and may be a histone, whereas the 95-kDa protein is enriched in the membrane fraction. The binding assays that we have developed should lead to a clearer understanding of lipid A/animal cell interactions.
A convenient synthesis of 4-amino-4-deoxy-L-arabinose and its reduction product, 1,4-dideoxy-1,4-imino-L-arabinitol.
Methyl beta-D-xylopyranoside in a mixture of N,N-dimethylformamide and 2-methoxypropene containing a little hydrogen chloride gave preponderantly the 2,3-O-isopropylidene derivative, which was readily converted into its 4-trifluoromethanesulfonate. The facile displacement of the triflate group gave a 4-azido-4-deoxy-alpha-L-arabinopyranoside derivative, and this, on mild acid treatment, was hydrolyzed to the 2,3-diol, or under more vigorous conditions to 4-azido-4-deoxy-L-arabinose. Methyl 2,3-di-O-acetyl-4-azido-4-deoxy-alpha-L-arabinopyranoside, from the diol, appears (1H-n.m.r. data) to exist as an equilibrating mixture of the 4C1 and 1C4 conformers in chloroform solution. The reduction of the azido sugar by hydrogen over Pd/C in .6M HCl yielded 4-amino-4-deoxy-L-arabinopyranose as its hydrochloride; in 0.1M HCl, further reactions occurred to give 1,4-dideoxy-1,4-imino-L-arabinitol as the final product. The aminodeoxypentose from lipid A precursor IIA, isolated from a Salmonella mutant by Raetz et al. in 1985, was shown to be identical with the synthetic aminoarabinose by t.l.c., 1H-n.m.r. spectroscopy, and g.l.c. of the acetylated reduction products.
A possible role for plasmalogens in protecting animal cells against photosensitized killing.
Chinese hamster ovary (CHO) cells incorporate 12-(1'-pyrene) dodecanoic acid (P12) into membrane lipids. Exposure of P12-labeled cells to long wavelength ultraviolet light causes cell killing, presumably because excitation of the pyrene moiety (a photosensitizer) leads to the generation of reactive oxygen species. Cytotoxicity is dependent upon the concentration of P12 used to label the cells, and time of UV exposure, and the presence of oxygen during irradiation. CHO mutant cells deficient in plasmalogen biosynthesis and peroxisome assembly (Zoeller, R.A., and Raetz, C.R.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5170-5174) are several orders of magnitude more sensitive to P12/UV treatment than wild-type cells, permitting direct selection of one wild-type cell in 1 X 10(4) mutant cells. A major factor responsible for the P12/UV hypersensitivity of these mutants appears to be the absence of plasmalogens. Supplementation of the mutants with 1-O-hexadecyl-sn-glycerol restores plasmalogen levels and nearly normal resistance to P12/UV treatment, whereas the biogenesis of peroxisomes is not restored. The P12/UV hypersensitivity of the plasmalogen-deficient mutants, together with the selective, P12/UV-induced decomposition of plasmalogens in wild-type cells, documented in the accompanying manuscript, suggest that the vinyl ether linkage of plasmalogens plays a direct role in protecting animal cell membranes against certain oxidative stresses.
Disappearance of plasmalogens from membranes of animal cells subjected to photosensitized oxidation.
Chinese hamster ovary cells (CHO-K1) photosensitized with 12-(1'-pyrene)dodecanoic acid (P12) are killed when exposed to long wavelength ultraviolet (UV) light (greater than 300 nm). Mutants deficient in plasmalogen biosynthesis are hypersensitive to this treatment. We now demonstrate that plasmenylethanolamine is rapidly and preferentially destroyed when CHO-K1 cells, photosensitized either with P12 or merocyanine 540, are irradiated with light of the appropriate wavelength. Using [2-14C]ethanolamine, [1-14C]hexadecanol, or [U-14C]hexadecanol to follow the turnover of plasmenylethanolamine, we show that 2-monoacylglycerophosphoethanolamine, formic acid, and pentadecanal are formed during P12/UV treatment of CHO-K1 cells, but not of mutant cells deficient in plasmalogen synthesis. The decomposition of plasmenylethanolamine is O2-dependent, is enhanced in D2O, and is reduced in the presence of sodium azide. The process may be explained, in part, by the cycloaddition of singlet oxygen to the vinyl ether linkage of plasmenylethanolamine, generating a dioxetane intermediate that would be expected to decompose under physiological conditions to the observed products. An additional possibility is the formation of an allylic hydroperoxide at the 1'-carbon of the alkyl moiety by an "ene" reaction of singlet oxygen, or by radical-mediated oxidation, followed by metabolism or chemical decomposition of the hydroperoxide. Given the P12/UV hypersensitivity of plasmalogen-deficient mutants, we suggest that plasmalogens might protect animal cell membranes from singlet oxygen and/or radical-initiated oxidation by functioning as scavengers and decomposing to products that can be reutilized.
Induction of kappa light chain synthesis in 70Z/3 B lymphoma cells by chemically defined lipid A precursors.
The murine B cell lymphoma 70Z/3 is a tumor line that resembles an early B cell. It responds to lipopolysaccharide by synthesizing kappa light chains, resulting in the appearance of surface IgM. We now demonstrate that this effect is triggered by the lipid A domain of lipopolysaccharide since a defined tetraacyl disaccharide 1,4'-bisphosphate precursor of mature lipid A, designated lipid IVA (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088), is fully active in stimulating kappa chain mRNA synthesis. In contrast, the monosaccharide lipid A precursor, lipid X, shows only slight activity; but a large excess of lipid X appears to complete with lipid IVA, partially blocking its effects. Mutants of the 70Z/3 line that are unresponsive to lipopolysaccharide also fail to respond to lipid IVA. The somatic cell system described here, coupled with the use of chemically defined agonists and antagonists, offers a new approach to understanding the early events in lipopolysaccharide/animal cell interactions.
Biosynthesis of lipid A in Escherichia coli: identification of UDP-3-O-[(R)-3-hydroxymyristoyl]-alpha-D-glucosamine as a precursor of UDP-N2,O3-bis[(R)-3-hydroxymyristoyl]-alpha-D-glucosamine.
The lipid A disaccharide of the Escherichia coli envelope is synthesized from the two fatty acylated glucosamine derivatives UDP-N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D- glucosamine (UDP-2,3-diacyl-GlcN) and N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D-glucosamine 1-phosphate (2,3-diacyl-GlcN-1-P) [Ray, B. L., Painter, G., & Raetz, C. R. H. (1984) J. Biol. Chem. 259, 4852-4859]. We have previously shown that UDP-2,3-diacyl-GlcN is generated in extracts of E. coli by fatty acylation of UDP-GlcNAc, giving UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc as the first intermediate, which is rapidly converted to UDP-2,3-diacyl-GlcN [Anderson, M. S., Bulawa, C. E., & Raetz, C. R. H. (1985) J. Biol. Chem. 260, 15536-15541; Anderson, M. S., & Raetz, C. R. H. (1987) J. Biol. Chem. 262, 5159-5169]. We now demonstrate a novel enzyme in the cytoplasmic fraction of E. coli, capable of deacetylating UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc to form UDP-3-O-[(R)-3-hydroxymyristoyl]glucosamine. The covalent structure of the previously undescribed UDP-3-O-[(R)-3-hydroxymyristoyl] glucosamine intermediate was established by 1H NMR spectroscopy and fast atom bombardment mass spectrometry. This material can be made to accumulate in E. coli extracts upon incubation of UDP-3-O-[(R)-3- hydroxymyristoyl]-GlcNAc in the absence of the fatty acyl donor [(R)-3-hydroxymyristoyl]-acyl carrier protein. However, addition of the isolated deacetylation product [UDP-3-O-[(R)-3-hydroxymyristoyl] glucosamine] back to membrane-free extracts of E. coli in the presence of [(R)-3-hydroxymyristoyl]-acyl carrier protein results in rapid conversion of this compound into the more hydrophobic products UDP-2,3-diacyl-GlcN, 2,3-diacyl-GlcN-1-P, and O-[2-amino-2-deoxy-N2,O3- bis[(R)-3-hydroxytetradecanoyl]-beta-D-glucopyranosyl]-(1----6)-2-amino- 2-deoxy-N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D- glucopyranose 1-phosphate (tetra-acyldisaccharide-1-P), demonstrating its competency as a precursor. In vitro incubations using [acetyl-3H]UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc confirmed release of the acetyl moiety in this system as acetate, not as some other acetyl derivative. The deacetylation reaction was inhibited by 1 mM N-ethylmaleimide, while the subsequent N-acylation reaction was not. Our observations provide strong evidence that UDP-3-O-[(R)-3-hydroxymyristoyl]glucosamine is a true intermediate in the biosynthesis of UDP-2,3-diacyl-GlcN and lipid A.
Gram-negative endotoxin: a biologically active lipid.
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Lipid X protects mice against fatal Escherichia coli infection.
Lipid X, the major monosaccharide precursor of lipid A, is nontoxic and has previously been shown to protect mice and sheep from the harmful effects of endotoxin. To test the hypothesis that lipid X could be therapeutic against infections with gram-negative organisms, neutropenic ICR mice were infected by intramuscular inoculation of Escherichia coli and subsequently treated with lipid X alone or in combination with the antibiotic ticarcillin. Lipid X slightly prolonged survival; treated mice had a significantly improved rate of survival 18 h after intramuscular inoculation as compared with controls (P less than 0.025). By 24 h, however, this difference disappeared. When lipid X was combined with ticarcillin, survival differences were both significant and prolonged. Treatment of mice with one to two doses of lipid X for a total dose of 1 mg intravenously and with 1,200 mg of ticarcillin per kg every 6 h improved survival over a 48-h treatment period from 5 to 23% (P less than 0.0025). Treatment with lipid X and ticarcillin over a broad range of antibiotic dosages in 362 mice demonstrated improved survival of two- to fourfold (P less than 0.0001 at 24 h after inoculation, P less than or equal to 0.0005 at 48 h, and P less than or equal to 0.0001 at 5 days). Lipid X enabled the dose of ticarcillin necessary to protect 50% of mice from death to be reduced by two- to fivefold. Pretreatment with lipid X was not necessary to improve survival: 16 of 17 (94%) infected and visibly ill animals that received lipid X and ticarcillin 6 h after thigh inoculation survived versus 30 of 44 (68%) control animals treated with ticarcillin alone (P less than 0.0001). Lipid X had no antimicrobial activity in vitro. Lipid X is a novel agent that enhances survival in an animal model of severe infection with gram-negative organisms.
Identity of the 17-kilodalton protein, a DNA-binding protein from Escherichia coli, and the firA gene product.
The 17-kilodalton protein, a DNA-binding protein encoded by the skp gene of Escherichia coli, was found to be identical to histonelike protein I, the product of the firA gene. This conclusion was reached after chromosomal localization, using the recently constructed high- and low-resolution E. coli restriction maps, and by direct comparison of the N-terminal amino acid sequence of histonelike protein I and the 17-kilodalton protein.
First committed step of lipid A biosynthesis in Escherichia coli: sequence of the lpxA gene.
The min 4 region of the Escherichia coli genome contains genes (lpxA and lpxB) that encode proteins involved in lipid A biosynthesis. We have determined the sequence of 1,350 base pairs of DNA upstream of the lpxB gene. This fragment of DNA contains the complete coding sequence for the 28.0-kilodalton lpxA gene product and an upstream open reading frame capable of encoding a 17-kilodalton protein (ORF17). In addition there appears to be an additional open reading frame (ORF?) immediately upstream of ORF17. The initiation codon for lpxA is a GUG codon, and the start codon for ORF17 is apparently a UUG codon. The start and stop codons overlap between ORF? and ORF17, ORF17 and lpxA, and lpxA and lpxB. This overlap is suggestive of translational coupling and argues that the genes are cotranscribed. Crowell et al. (D.N. Crowell, W.S. Reznikoff, and C.R.H. Raetz, J. Bacteriol. 169:5727-5734, 1987) and Tomasiewicz and McHenry (H.G. Tomasiewicz and C.S. McHenry, J. Bacteriol. 169:5735-5744, 1987) have demonstrated that there are three similarly overlapping coding regions downstream of lpxB including dnaE, suggesting the existence of a complex operon of at least seven genes: 5'-ORF?-ORF17-lpxA-lpxB-ORF23-dnaE-ORF37-3 '.
Accumulation of lysophosphatidylinositol in RAW 264.7 macrophage tumor cells stimulated by lipid A precursors.
N2,O3-Diacylglucosamine 1-phosphate (lipid X), a monosaccharide precursor of Escherichia coli lipid A, was used to stimulate RAW 264.7 macrophage tumor cells, and the effects on macrophage phospholipid metabolism were examined. The addition of E. coli lipid X to the medium of cells that had been uniformly labeled with 32Pi resulted in a 4-8-fold increase in the level of lysophosphatidylinositol. This effect was maximal at 5 microM lipid X. Lysophosphatidylinositol levels reached a maximum 45 min after stimulation, followed by a gradual decline to near normal levels within 2 h. The formation of lysophosphatidylinositol was dependent upon extracellular calcium and was almost completely inhibited when cycloheximide was added at the time of stimulation. The addition of the disaccharide lipid A precursor IVA, commercial lipopolysaccharide (1 microgram/ml), phorbol 12-myristate 13-acetate (10(-7) M), or calcium ionophore A23187 (10(-6) M) to these cells resulted in a similar increase in lysophosphatidylinositol levels, but phosphatidic acid was inactive. The stimulation by IVA and phorbol myristate acetate was blocked by cycloheximide, but the stimulation by lipopolysaccharide was only partially blocked. The stimulation by A23187 was unaffected by cycloheximide. The increase in lysophosphatidylinositol levels might be related to the stimulation of arachidonate release and prostaglandin synthesis that is also observed in cells treated with lipid A precursors. The disaccharide precursor, IVA, was at least 100 times more effective than lipid X at stimulating lysophosphatidylinositol formation and prostaglandin release. The relative ability of lipid X and IVA to stimulate these cells correlated well with their effects on other lipopolysaccharide-responsive systems. Macrophage tumor cells also had the ability to inactivate lipid X by dephosphorylating it.
Phospholipids chiral at phosphorus. Steric course of the reactions catalyzed by phosphatidylserine synthase from Escherichia coli and yeast.
The steric courses of the reactions catalyzed by phosphatidylserine (PS) synthase from Escherichia coli and yeast were elucidated by the following procedure. RP and SP isomers of 1,2-dipalmitoyl-sn-glycero-3-[17O,18O]phosphoethanolamine ([17O,18O]DPPE) were synthesized with slight modification of the previous procedure [Bruzik, K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754] and converted to (RP)- and (SP)-1,2-dipalmitoyl-sn-glycero-3-[16O,17O,18O]phosphoric acid ([16O,17O18O]DPPA), respectively, by incubating with phospholipase D. Condensation of [16O,17O,18O]DPPA with cytidine 5'-monophosphomorpholidate in pyridine gave the desired substrate for PS synthase, [17O,18O]cytidine 5'-diphospho-1,2-dipalmitoyl-sn-glycerol ([17O,18O]CDP-DPG), as a mixture of several isotopic and configurational isomers. Incubation of [17O,18O]CDP-DPG with a mixture of L-serine, PS synthase (which converted [17O,18O]CDP-DPG to phosphatidylserine), and PS decarboxylase (which catalyzes decarboxylation of phosphatidylserine) gave [17O,18O]DPPE. The configuration and isotopic enrichments of the starting [17O,18O]DPPE and the product were analyzed by 31P NMR following trimethylsilylation of the DPPE. The results indicate that the reaction of E. coli PS synthase proceeds with retention of configuration at phosphorus, which suggests a two-step mechanism involving a phosphatidyl-enzyme intermediate, while the yeast PS synthase catalyzes the reaction with inversion of configuration, which suggests a single-displacement mechanism. Such results lend strong support to the ping-pong mechanism proposed for the E. coli enzyme and the sequential Bi-Bi mechanism proposed for the yeast enzyme, both based on previous isotopic exchange experiments.
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.