biosynthesis of lipid a precursors in escherichia coli · the journal of biological chemistry 0...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Vol. 262, No. 11, Issue of April 15. pp. 5159-5169,1987 Printed in U. S.A.

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*

(Received for publication, November 25, 1986)

Matt S. Anderson$ and Christian R. H. Raetzt From the Department of Biochemistry, College of Agricultural and Life Sciences, University of Wiseomin-Madison, Madison, Wisconsin 53706

Preliminary studies from our laboratory have sug- gested the existence of a novel set of fatty acyltrans- ferases in extracts of Escherichia coli that attach two R-3-hydroxymyristoyl moieties to UDP-GlcNAc (An- derson, M. S., Bulawa, C. E., and Raetz, C. R. H. (1985) J. Biol. Chem. 260,15536-15541). The resu1ting“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- 3-0-(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 ‘H-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 com- mitted 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.

The lipid A component of lipopolysaccharide is the predominant amphipathic molecule found on the outer surface of the outer membrane of Gram-negative bacteria (1, 2). Lipid A is also the pharmacological agent that is responsible for many of the toxic and immunostimulatory phenomena associated with Gram-negative infections (2-4). Following the identification of the true covalent structure of lipid A (5-9), first

*This research was supported by National Institute of Health Grant AM 19551 (to C. R. H. R.). The mass spectral determination was performed at the Middle Atlantic Mass Spectrometry Laboratory, a National Science Foundation Shared Instrumentation Facility. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Wharton Research Fellow. To whom correspondence should be addressed.

proposed by Takayama et al. in 1983 ( 5 ) , there has been rapid progress with the elucidation of lipid A biosynthesis (10-13) and pharmacology (13-17).

As shown at the bottom of Fig. 1, mature lipid A of Esche- richia coli (and related Gram-negatives) is a D l 4 linked disaccharide of glucosamine that is phosphorylated at positions 1 and 4’ and acylated with R-3-hydroxymyristoyl moieties at positions 2, 3, 2‘, and 3‘ (2, 5-9). Core sugars, starting with KDO,’ are attached to position 6‘ (2,6, 18). Some of the R-3-hydroxymyristoyl moieties are further acylated with nor- mal fatty acids (2, 9, 19), giving rise to so-called acyloxyacyl residues (Fig. 1).

Elucidation of the biosynthesis of lipid A was greatly facil- itated by the discovery of the monosaccharide precursor, 2,3- diacylglucosamine 1-phosphate (lipid X) ( 5 ) , which accumulates together with UDP-2,3-diacylglucosamine (10) at 30 “C in E. coli mutants defective in a gene designated IpxB (Fig. 1) (20-22). Compelling evidence for the pathway by which the p l 4 linkage of lipid A is formed was provided by the identification of an enzyme (11) capable of condensing UDP- 2,3-diacylglucosamine with lipid X to generate a ( 3 1 4 linked tetraacyldisaccharide 1-phosphate intermediate (Fig. 1). The latter product was then shown to be a substrate for a distinct kinase (13, 23) that incorporates the 4”phosphate (Fig. 1). Mutants defective in KDO biosynthesis accumulate the same tetraacyldisaccharide 1,4’-bisphosphate as generated in vitro by the 4’ kinase (24, 25).

The pathways by which the fatty acyl chains are attached to lipid A precursors have not been explored in detail (13). Anderson et al. (12) recently reported that [/3-32P]UDP- GlcNAc is converted to a family of lipophilic derivatives when incubated with an E. coli cytosol in the presence of R-3- hydroxymyristoyl-acyl carrier protein. Some of these substances were found to migrate with standards of UDP-2,3- diacylglucosamine, lipid X, and tetraacyldisaccharide l-phosphate upon thin-layer chromatography (12). In addition, preliminary evidence for an 0-monoacyl-UDP-GlcNAc precursor

The abbreviations and trivial names used are: KDO, 3-deoxy-~- manno-octulosonate; UDP-3-O-(R-3-hydroxymyristoy1)-GlcNAc, UDP-N-acetyl-3-O-[(R)-3-hydroxytetradecanoyl]-~-; dz-glucosamine; 2,3-diacylglucosamine-1-P or lipid X,NZ,03-bis[(R)- 3-hydroxytetradecanoyl]-ol-~-glucosamine 1-phosphate; UDP-2,3- diacyl-GlcN, UDP-N2,03-bis[(R)-3-hydroxytetradecanoyl]-ol-~- glucosamine; tetraacyldisaccharide 1-phosphate, 0-[2-amino-2-deoxy-N2,03-bis-(R-3-hydroxytetradecanoyl)-~-~-glucopyranosyl]- (1~6)-2-amino-2-deoxy-Nz,O3-bis(R-3-hydroxytetradecanoyl)-~- D-glucopyranose 1-phosphate; PEI, polyethyleneimine; FAB, fast atom bombardment; HEPES, 4-(2-hydroxyethyl)-l-piperazineeth- anesulfonic acid; bis-tris, 2-[bis(2-hy&oxyethyl)amino]-2-(hydroxy- methyl)-propane-1,3-diol; ACP, acyl carrier protein; HPLC, high pressure liquid chromatography; NMP, nucleotide monophosphate; NDP-GlcNAc, nucleotide diphospho-N-acetylglucosamine.

5159

5160 ACP-dependent UDP-GlcNAc Acyltransferase

UDP-GIcNAc

HO mn. I 0-

R R R 1 0-6-00

R I

@o-iLonw @ A * 0 ao-"-.w 4 &

NH I YH0-6-oQ R

R R AQ ( T ~ f r o o ~ y l - d ~ r a c c h o r ~ d ~ - I , 4 " b l s - P )

I

L-- -CMP-KDO i . - .~auroyl and rnyrnstoyl molafi*s

I "'Polor rnO,.f1.S

(KDO),

o~:~o~:lNH "o{ H~INno-~;o"-~oQ 0 0

E colt K12 Lnpld A wlfh - K C 0 dtsacchortde

, I I L.---Ofher COW sugars +-- -O-Anftg*n

M o f u r e I tpopoIysucchortd*

FIG. 1. Postulated pathway for the biosynthesis of lipid A in E. coli. Synthesis is initiated by the acylation of the known metabolite, UDP-GlcNAc, at position 3 of the glucosamine ring, as described in the present study. Evidence for the other reactions shown has been presented previously (10-13). In the above figure, R represents an R-3-hydroxymyristoyl moiety, and U represents uridine. The structure shown at the bottom is the predominant one found in E. coli and S. typhimurium, but in S. minnesota an additional palmitoyl residue is attached to the &OH function of the N-linked hydroxymyristoyl moiety of the reducing end sugar.

of UDP-2,3-diacylglucosamine was presented (12), but none of the acylated metabolites of UDP-GlcNAc, generated in vitro, were actually isolated and characterized by spectro- scopic methods.

We now describe conditions to assay the first enzyme involved in the fatty acylation of UDP-GlcNAc, and we have determined the structure of its product using 'H NMR and fast atom bombardment mass spectrometry. As previously hypothesized (12, 13), UDP-GlcNAc acyltransferase incorporates an R-3-hydroxymyristoyl residue at position 3 of the GlcNAc moiety. This product is likely to be a biosynthetic intermediate, since it is an excellent substrate for further fatty acylation when reincubated in the presence of R-3-

hydroxymyristoyl-acyl carrier protein and E. coli cytosol. In the accompanying manuscript (26) we describe a differ-

ent, membrane-bound acyltransferase that incorporates palmitoyl moieties derived from glycerophospholipids into lipid X. The palmitoyltransferase is the first example of an in vitro system for the biosynthesis of an acyloxyacyl moiety.

EXPERIMENTAL PROCEDURES

M~terials-[r-~~P]ATP was obtained from New England Nuclear. Acyl carrier protein (ACP) was isolated from E. coli K12, strain W3106 (Table I), by the method of Rock and Cronan (27). Silica Gel 60 thin layer plates (0.25 mm) were the product of E. Merck, Darmstadt, Germany.

Bacterial Strains-All strains were derivatives of E. coli K12 (Table I). Some assays were performed with strain JB1104 (28), which contains a TnlO insertion in the structural gene for cytidine 5'- diphosphate diglyceride hydrolase (CDP-diglyceride hydrolase). This hydrolase has previously been shown to efficiently cleave the pyro- phosphate bridge of UDP-2,3-diacyl-GlcN to yield 2,3-diacyl-GlcN- 1-P and UMP (10). We have found that this hydrolase can also cleave UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc to form 3-0-R-3-hydroxymyristoyl-GlcNAc-1-P + UMP, neither of which are metabolized further. Accordingly, extracts of JB1104 were used to observe maxi- mal production of the lipid A metabolites, UDP-2,S-diacyl-GlcN, 2,3- diacyl-GlcN-1-P, and tetraacyldisaccharide-1-P, starting either with added UDP-GlcNAc or with UDP-3-O-(R-3-hydroxymyristoyl)- GlcNAc. Strain MClOGl/pSRl contains a plasmid directing overproduction of both UDP-GlcNAc acyltransferase (but) and lipid A disaccharide synthase (IpxB), when induced with 0.5% L-arabinose (22). Its construction and the conditions for induction have been described (22). Strain W3106 was obtained from the E. coli Genetic Stock Center, Yale University, New Haven, CT.

Preparation of E. coli Cell Extracts-Cell extracts were made as described previously (12). Unless otherwise indicated, all assays were performed on membrane-free cell supernatants (12). Protein concentrations were determined by the method of Peterson (30).

UDP-GlcNAc Acyltransferase Assays-All routine UDP-GlcNAc acyltransferase assays were performed in the presence of 1% octyl-8- D-glucoside. The presence of this detergent above its critical micellar concentration efficiently blocks the formation of UDP-2,B-diacyl- GlcN from UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc, but it does not interfere with the formation of the latter metabolite.

Unless otherwise stated, assay mixtures contained [p-3ZP]UDP- GlcNAc (116 p ~ , 4 X 10' cpm/nmol), R-3-hydroxymyristoyl-acyl carrier protein (300 p ~ ) , octyl-/3-D-glucoside (1.0%), HEPES, pH 8.0 (40 mM), and 0.05-1.2 units of enzymatic activity in a final volume of 20 or 50 pl. One unit of activity is that amount of enzyme required to produce 1 nmol of UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc/min at 30 "C. Samples of the assay mixture were removed at various times and spotted directly onto silica thin-layer plates. We have found that identical rates of acylation are observed whether the reactions are first quenched by addition of chloroform and methanol as described previously (22) or are spotted directly onto silica plates. After drying

TABLE I Strains of Escherichia coli K12

Strain Relevant eenotwe Source

W3106 Wild-type strain of E. CGSC" coli K12, F-, X-

MC1061 araD139 A(ara-ku)7697 Ref. 29 hsdR hsdM+

MClOGl/pINGl Strain of MC1061 har- Ref. 29 boring the PING1 expression vector

boring pSR1, a pING1-derived hybrid plasmid carrying the bxA+ and lpxB+ genes of E. coli on a 2.1-kil- obase insert under araC control

MClOGl/pSRl Strain of MC1061 har- Ref. 22

JB1104 cdh-4::TnlO lmA' ~DxB+ Ref. 28

CGSC, E. coli Genetic Stock Center, Yale University.

ACP-dependent UDP-GlcNAc Acyltransferase 5161

of the spots under a cool air stream, the thin layer plate was developed in ch1oroform:methanol:water:acetic acid (25:15:4:2, v/v), dried, and autoradiographed to visualize the lipid products. Next, each product was scraped from the plate and counted in 10 ml of scintillation fluid (12). To obtain an accurate initial rate, it was necessary to analyze samples a t early times (1 or 2 rnin).

Preparation of R,S-3-Hydroxyhurate, R,S-3-Hydroxymyristate, and R,S-3-Hydroxypalmitate-Samples of R,S-3-hydroxylaurate and R,S-3-hydroxypalmitate were prepared by reduction of the corresponding 3-ketomethylesters. Briefly, 5 g of both methyl-3-ketolau- rate and methyl-3-ketopalmitate were synthesized by condensation of Meldrum's acid with decanoylchloride and myristoylchloride, respectively, as described by Oikaya et al. (31). Next, 50-mg portions of each ketoester were reduced with a 2-fold reductive excess of NaBH, in 2 ml of tetrahydr0furan:water (91, v/v) at 25 "C for 30 min. The reaction mixtures were each extracted with three 2-ml portions of diethyl ether. After drying over Na2S04 and evaporation of the ether, the R,S-3-hydroxy derivatives were redissolved in 1 ml of Skelly C:diethyl ether (3:1, v/v) and purified by passing them through 1-ml silica gel Sep Pak cartridges (Waters Associates) equilibrated with the same solvent. After removal of the solvent, the purified R,S-3- hydroxy derivatives were saponified by heating at 55 "C for 30 min in 1 ml of ethanol in the presence of 1.2 eq of KOH. The potassium salts of the acids form precipitates that were collected by filtration, were dried, and were redissolved in 1 ml of water, acidified to pH 2 with HCI. The free acid forms of R,S-3-hydroxylaurate and R,S-3- hydroxypalmitate were then extracted with three 1-ml portions of diethyl ether. The extracts were dried, giving the pure R,S-3-hydroxy acids in an overall yield of 50% relative to the acid chlorides.

R-3-Hydroxymyristic acid was the gift of Drs. L. Anderson and J. Naleway, University of Wisconsin-Madison, and S-3-hydroxymyristic acid was the gift of Drs. F. Unger and I. Macher, Sandoz, Vienna, Austria.

Preparation of [&3ZPJUDP-GlcNAc, R-3-Hydroxymyristoyl-Acyl Carrier Protein, and Other Acyl-Acyl Carrier Proteins-The substrate, [p-32P]UDP-GlcNAc, was synthesized and purified as described previously (12, 32). The analogs, [&32P]UDP-N-propionyl-GlcN and [@-32P]UDP-N-butyryl-GlcN, were synthesized and purified exactly as described for [@-32P]UDP-GlcNAc (12), except that propionic and butyric anhydrides, respectively, were substituted for acetic anhydride. The preparation of R-3-hydroxymyristoyl-acyl carrier protein (12) was modified as follows. Acyl-acyl carrier protein synthetase, solubilized from 200 g of frozen E. coli cells exactly as described previously (12, 33), was immobilized onto 40 ml of blue-Sepharose CL-GB (33) in a batchwise manner by gently swirling the blue- Sepharose together with the solubilized enzyme in a 500-ml Erlen- meyer flask at 4 "C for 12 h. All subsequent washes of the blue- Sepharose were also performed in a batchwise manner on a Buchner funnel. The reaction to make the R-3-hydroxymyristoyl-acyl carrier protein was performed with the same concentrations of all reagents and enzymes previously reported (12, 33), except that the reaction was initiated by the addition of the enzymatically active blue-Seph- arose (40 ml) to the reaction mixture (150 ml) in a 500-ml Erlenmeyer flask. The mixture was gently swirled at 4 'C until the reaction was finished (14 h for 50 mg of ACP). The subsequent workup was also performed as described previously (12, 33) except that all washes were performed batchwise on a Buchner funnel, instead of in a column (33). The eluate was concentrated to about 80 ml using an Amicon YM5 ultrafiltration membrane. Next, the material was dialyzed twice against 4 liters of 0.5 mM bis-tris, pH 6.0, and lyophilized. The solids were redissolved in the same buffer to yield a concentration of approximately 12 mg/ml and analyzed for their thiol content by 5,s'- dithiobis(nitrobenzoic acid) titration before and after exposure to 0.5 M NaOH at 37°C for 30 min. There was never more than 5% underivatized ACPSH in any preparation of R-3-hydroxymyristoyl- ACP. With the above modifications, it is possible to prepare -100 mg of acylated-ACP in 7 days, including the acyl-ACP synthetase isolation. All other acyl-acyl carrier proteins were prepared on a scale that was 10-fold smaller than the one described above.

Partial Purification of UDP-GlcNAe Acyltransferase-UDP- GlcNAc acyltransferase was isolated from the strain MClOGl/pSRl (Table I). These cells were cultured as described previously (22) in LB-fructose medium at 37 "C and induced with 0.5% L-arabinose for 8 h. The harvested cells could be frozen at -80 "C for at least 3 months.

Ten grams of frozen, induced MClOGl/pSRl cells were resus- pended in 100 ml of 10 mM phosphate buffer, pH 7.0, at 0 "C. The cells were disrupted by passage through a French pressure cell at

18,000 p.s.i. Unbroken cells were removed by centrifugation at 5,000 X g for 20 min. This crude cell-free extract was then centrifuged at 150,000 X g for 90 min at 4 "C to remove membranes. UDP-GlcNAc acyltransferase activity was found exclusively in the soluble fraction, which could either be purified further or frozen in a dry ice/acetone bath and stored at -80 "C. At this point, the enzyme preparation was stable a t -80 'C for several months.

Portions of this membrane-free supernatant were further purified as needed. Samples containing 75 mg of protein were adjusted to 1% octyl-,%D-glucoside by addition of a 10% octyl-8-D-glucoside stock solution in 10 mM sodium phosphate buffer, pH 6.0. This fraction was loaded at 5 ml/min onto a 15-ml(2.4 x 3.3-cm) column of DEAE- cellulose (Whatman DE52), equilibrated with 1% octyl-d-D-glucoside in 10 mM sodium phosphate buffer, pH 6.0. The column was washed with 2 column volumes of this buffer and then with another 3 volumes of the same buffer, containing 10 mM NaCl. Next, UDP-GlcNAc acyltransferase was eluted at 5 ml/min with 3 column volumes of the above buffer, containing 100 mM NaCI. Enzyme recovery was 78%. UDP-GlcNAc acyltransferase purified in this manner could be stored at 4 "C for several days with minimal loss of activity or it could be frozen in liquid nitrogen and stored at -80 "C for at least 3 months. However, such samples could not be frozen and thawed repeatedly.

Large Scale Preparation and Purification of UDP-3-O-(R-3-hydrox- ymyristoy1)-GlcNAc-Conditions used in the large scale preparation of UDP-3-O-(R-3-hydroxymyristoyl)-Gl~NAc were altered from those of the standard assay in order to maximize the use of the R-3- hydroxymyristoyl-ACP, with minimal use of enzyme-protein. The final concentrations in a volume of 40 ml were as follows: [p-32P] UDP-GlcNAc, 1.5 mM (170 cpm/mmol); R-3-hydroxymyristoyl-ACP, 150 pM; HEPES, pH 8.0, 40 mM; octyl-0-D-glucoside, 1.5%; and partially purified UDP-GlcNAc acyltransferase, 19 pg/ml. The reaction was initiated by the addition of 12 pg/ml of the acyltransferase preparation, and the system was incubated at 30 "C for 1 h. Next, a further 7 pg/ml of acyltransferase was added, and the incubation continued for a second hour.

The monoacyl-UDP-GlcNAc was isolated immediately, according to the following scheme. The reaction mixture was cooled on ice and diluted with an equal volume of cold 2-propanol. The resulting precipitate was removed by centrifugation. The supernatant was passed over a 20-ml Sephadex-A25 column at 4 "C which had been equilibrated with 10 mM sodium phosphate, pH 6.0. The column was washed to remove detergent and extraneous ions with 5 volumes of 10 mM sodium phosphate, pH 6.0, containing 10 mM NaCl. The monoacyl-UDP-GlcNAc and unreacted UDP-GlcNAc were coeluted with 5 volumes of 10 mM sodium phosphate, pH 6.0, containing 100 mM NaCl. This material was frozen in a dry ice/ethanol bath and concentrated by lyophilization in a round bottom flask for 4 h. The resulting 7 ml of residual solution was fractionated in 1-ml aliquots by reverse phase HPLC. This was accomplished using an Alltech 10- pm C18 reverse phase HPLC column (0.65 X 25 cm) and a mobile phase consisting of 0.05% ammonium acetate, pH 5.4, at a flow rate of 2.5 ml/min. Various components were separated using a 40-min. linear acetonitrile gradient (0-40%), followed by a wash at 40% acetonitrile for 10 min. Further elution was continued linearly to 100% acetonitrile over another 10 min. The effluent was monitored at 260 nm using a Waters Lambda-Max model 400 variable wave- length UV detector interfaced with a Waters model 700 Data Module and a Waters model 710 system controller. In the above method, the putative UDP-3-0-(R-3-hydroxymyristoyl)-GlcNAc eluted as a single peak at about 36% acetonitrile. As this substance emerged, the fractions were titrated with 0.1 volume of 10 mM sodium phosphate, pH 6.0, and stored on ice. The pooled material was lyophilized and stored desiccated at -80 "C. This procedure yielded 0.8 mg of putative UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc. In the above reaction about 50% of the added R-3-hydroxymyristoyl-ACP participated in the desired conversion, and the 0.8 mg of final product represents a 40% recovery of the total material produced during the incubation.

Fast Atom Bombardment Mass Spectrometry and 'H NMR Spec- troscopy-Fast atom bombardment spectrometry was performed in the positive mode, as described previously for lipid X (5). Spectra were acquired using an MS-50 mass spectrometer (AEI/Kratos, Manchester, England) interfaced to a DS-55 data system. Mass assignments were made by computer with an accuracy of +1 atomic mass units.

Proton NMR spectra of UDP-GlcNAc and UDP-3-O-(R-3-hydrox- ymyristoy1)-GlcNAc were taken in D20(pD6), using a Bruker 270- MHz spectrometer. The spectrum of UDP-3-O-(R-3-hydroxymyris- toy1)-GlcNAc was acquired with a PRESAT experiment in order to


diminish the H20 signal. This PRESAT experiment used a 1-s presaturating pulse at the HDO resonance, followed by a 90" acquisition pulse. The chemical shifts, expressed in parts/million downfield from tetramethylsilane, were referenced to acetone a t 2.08 ppm, added as an internal standard (not shown).

Preparation of /fi-:"P]UDP-3-0-(R-3-hydroxymyristoyl)-GlcNAc- Samples of [/3-~"P]UDP-3-0-(R-3-hydroxymyristoyl)-GlcNAc were prepared enzymatically from [P-"P]UDP-GlcNAc under reaction conditions modified from those described above, in order to maximize the acylation of the radiolabeled substrate. In this procedure, 1 mM N-ethylmaleimide was added to the reaction mixture in order to block the further metabolism of [/3-'2P]UDP-3-0-(R-3-hydr~xymyristoyl)- GlcNAc. Reaction mixtures contained 12-30 p~ [P-"'PIUDP-GlcNAc (specific radioactivity -2 X 10" cpm/nmol), 1 mM N-ethylmaleimide, 40 mM HEPES, pH 8.0, a 10-fold excess of R-3-hydroxymyristoyl- ACP relative to UDP-GlcNAc, and 50 units of UDP-GlcNAc acyltransferase, partially purified as described above. Reactions were incuhated a t 30 "C in 0.5 ml. The acyltransferase was added in two portions, as described above for the large scale synthesis of UDP-3- O-(R-3-hydroxymyristoyl)-GlcNAc. After 2 h, the mixture was brought to a volume of 900 pl by addition of the above reverse phase HPLC buffer and chromatographed, as described above for the large scale preparation of this compound. These conditions convert 95% of [/3-:"P]UDP-GlcNAc to the monoacylated compound.

Analysis of Metabolites Formed from [fi-"2P]UDP-3-0-(R-3-hydroxymyristoyl~-GlcNAc-[/3-~*P]UDP-3-0-(R-3-hydroxymyristoyl)-G1c- NAc, 3.1 p~ (2 X IO6 cpm/nmol), was incubated a t 30 "C with 300 p~ R-3-hydroxymyristoyl-ACP, 40 mM HEPES, pH 8.0, and 3.8 mg/ ml cytosolic protein from a JB1104 extract in a final volume of 120 pl. A t various times (2,5, 15, 30, and 60 min), 5- and 1-pl portions of the reaction mixture were removed and spotted directly either onto a Silica Gel 60 plate or onto a PEI-cellulose plate, respectively. Both plates were dried in a cool stream of air. The silica plate was then developed in chloroform/methanol/water/acetic acid (25:15:4:2, v/v). The PEI-cellulose plate was washed for 15 min in methanol, dried, and developed in 0.2 M guanidinium hydrochloride. After chromatography, both plates were dried and autoradiographed to locate radiolabeled metabolites. These spots were scraped and counted in 10 ml of scintillation fluid (12) for quantitation.

At the 60-min time point, the remaining 90 pl of reaction mixture was diluted with 800 pl of the initial reverse phase HPLC buffer, described below for the isolation of [RH-acetyl]NDP-GlcNAcs, also containing 20 nmol each of cold carrier UMP, UDP-GlcNAc, and UDP. This sample was passed through a 0.22-pm Millipore filter and injected directly onto the reverse phase HPLC column. The HPLC system used to elute these metabolites was also identical to that described below. One-minute fractions were collected and 20 pl of each was analyzed by liquid scintillation counting. To confirm the purity of the isolated materials, 5 pl of each radioactive peak was spotted on a PEI-cellulose plate, along with cold carrier UDP, UDP- GlcNAc, or Pi, and developed and autoradiographed as above.

Synthesis of pH-AcetyllNDP-GlcNAcs-All ['H-acetyl]NDP- GlcNAcs were made from ['H-acetyl]GlcNAc-1-P. The latter was synthesized by reacting 5 mCi of ["HJacetic anhydride (50 mCi/mmol) with 100 pmol of glucosamine 1-phosphate in 2 ml of MeOH, 100 mM aqueous NaH,PO, (l:l, v/v), the pH of which was adjusted to 8.4 with saturated NaHC03. After 60 min a t room temperature, 5 eq of excess acetic anhydride were added dropwise to complete the reaction. After 15 min, the reaction was diluted with 2 volumes of distilled water and loaded onto a Dowex AG 1-X2 column (2.3 X 4.5 cm). The column was washed with 50 ml of 10 mM triethylammonium bicarbonate, pH 8.0, and eluted with an 800-ml linear gradient (10-750 mM) of triethylammonium bicarbonate, pH 8.0. The fractions (20 ml each) containing [:'H-acetyl]GlcNAc-l-P were pooled, lyophilized, and used directly in the next step.

NDP-GlcNAcs were synthesized from [RH-acetyl]GlcNAc-l-P (as prepared above) and various nucleotide morpholidates (NMP morpholidates) using the coupling procedure described by Bulawa and Raetz (10) for the synthesis of UDP-2,3-diacyl-GlcN. The NMP morpholidates used included the U, C, T , A, or G derivatives. Briefly, 60 pmol of NMP morpholidate was dissolved with 2 pmol of ["H- ac~tyl]GlcNAc-l-P in 1 ml of anhydrous pyridine and incubated a t 3'7 "C for 20 h. After this time, pyridine was removed with a vacuum pump, and the crude reaction mixtures were redissolved in 800 pI of 30 mM sodium phosphate, pH 6.0, containing 5 mM tetrabutylammonium chloride. The samples were passed through 0.22-pm Gelman filters and purified by HPLC. The ["H-acetyl]NDP-GlcNAcs were chromatographed on a 10-pm C18 reverse phase Alltech column (1 X

25 cm), using a mobile phase of 30 mM sodium phosphate, pH 6.0, containing 5 mM tetrabutylammonium chloride a t a flow rate of 5 ml/min. Separation was achieved with a 60-min linear gradient of increasing acetonitrile (0-40%). Fractions containing the desired radioactive products were loaded onto 3-ml DEAE-cellulose columns that were washed with 5 volumes of 10 mM triethylammonium bicarbonate, pH 7.5, to remove the ion pairing agent. The ['H-acetyl]NDP- GlcNAcs were then eluted with a 10-column volume linear gradient of 0-400 mM triethylammonium bicarbonate, pH 7.5. Peak fractions were combined and lyophilized. Specific radioactivity of the ['H- acetyl]NDP-GlcNAcs was 4200 cpm/nmol.

RESULTS

Formation of Lipid Derivatives of [~-"P]UDP-GlcNAc in the Presence of R-3-Hydroxymyristoyl-ACP-The cytosol of E. coli contains enzymes that convert [P-"PIUDP-GlcNAc to lipophilic derivatives in the presence of R-3-hydroxymyristoyl-ACP. Some of these products migrate with the lipid A precursors UDP-2,3-diacyl-GlcN, 2,3-diacyl-GlcN-1-P, and tetraacyldisaccharide 1-phosphate during thin-layer chromatography (12). A typical time course for these transformations is shown in lanes 2-5 of Fig. 2. Inspection of these lanes reveals the presence a t early times of a less hydrophobic "new metabolite," that may be an 0-monoacylated form of UDP- GlcNAc (12). As shown in lane 6 of Fig. 2, the enzyme forming this metabolite displays considerable specificity for the fatty acyl donor, since palmitoyl-ACP does not substitute for R-3- hydroxymyristoyl-ACP.

The Effect of Octy&3-D-Glucoside on the Acylation of [p-"PIUDP-GlcNAc-While optimizing the conditions for the formation of the lipid derivatives of [P-""PIUDP-GlcNAc, we discovered that addition of octyl-P-D-glucoside, above its critical micellar concentration (0.73%) to the reaction mix-

Solvent front -

Dlsacchande I - P - DloCyl-GlcN-I -P - U D P - D I O C ~ I - G I C N - New Melobollte ".

O r q m (UDP-GlcNAc),

1 2 3 4 5 5 7 8 " J

FIG. 2. Formation of lipid metabolites from [@-"'P]UDP- GlcNAc. This figure is an autoradiograph of a thin-layer plate developed in ch1oroform:methanol:water:acetic acid (25:15:4:2, v/v). The origin, the solvent front, and the mobilities of various standards are indicated. With the exceptions noted, all reactions contained 25 pM [&:'rP]UDP-GlcNAc (2 X lofi cpm/nmol), 300 pM fatty acyl-acyl carrier protein, 40 mM HEPES, pH 8.0, and 3.8 mg/ml membrane- free extract from strain JB1104 (cdh::TnlO, IpxR'), prepared as described under "Experimental Procedures." Samples of each reaction mixture (5 pl) were spotted directly onto the plate at the times indicated. Lane I, [/3-:'2P]UDP-GlcNAc substrate spotted directly onto plate. Lanes 2-5, portions of a complete reaction mixture spotted a t 2', 5', 15', or 30' time points, respectively, using 8-hydroxymyristoyl-acyl carrier protein as the donor. Lane 6, identical to lane 5 but with palmitoyl-acyl carrier protein as the acyl donor. Ianes 7-10, 2', 5', 15'. 30' time points using R-3-hydroxymyristoyl-acyl carrier protein as the acyl donor together with 1.0% octyl-P-D-glucoside.


ture, caused the accumulation of the new metabolite by inhib- iting its further conversion to UDP-2,3-diacyl-GlcN as shown in lanes 7-10 of Fig. 2. However, octyl-p-D-glucoside did not inhibit the UDP-GlcNAc acyltransferaseper se, since the sum of the products formed in 2 min either in the absence or the presence of octyl-P-D-glucoside was virtually the same (Table 11). The rate of UDP-GlcNAc acylation was unaffected by octyl-p-D-glucoside concentrations as high as 3% (w/v). Other nonionic detergents such as 0.1% Triton X-100 were unsuit- able replacements for octyl-8-D-glucoside since they inhibited the formation of the putative 0-monoacyl-UDP-GlcNAc (Ta- ble 11), especially a t higher concentrations (data not shown). Triton X-100 also did not block the conversion of the new metabolite to UDP-2,3-diacyl-GlcN.

A Quantitative Assay for UDP-GlcNAc Acyltransferase- Given the unanticipated effect of octyl-P-D-glucoside and the fact that UDP-GlcNAc is easily separated from acylated UDP-GlcNAc derivatives by thin-layer chromatography, we devised an assay for measuring the rate of formation of the putative monoacylated UDP-GlcNAc (see "Experimental Procedures"). The specific activity of a typical E. coli supernatant is 1.3 nmol/min/mg. The requirements for the reaction in such crude extracts are summarized in Table 11. No product is detected in the absence of enzyme or in the presence of enzyme boiled for 10 min. Treating the enzyme at 60 "C for 20 min, however, does not inhibit enzymatic activity. At 300 ~ L M the level of R-3-hydroxymyristoyl-ACP is well above its K,,, and, despite some hydrolysis, is not significantly depleted during the first 10 min of the incubation.

Fig. 3A shows the dependence of the reaction rate on the concentration of UDP-GlcNAc. This reveals that the K,,, for UDP-GlcNAc is 450 PM. The apparent K,,, for R-3-hydroxymyristoyl-ACP has not been determined but is well below 100 PM. Panel B shows a typical time course for this reaction. The striking nonlinearity is observed at all concentrations of enzyme (data not shown) and may be caused by enzyme insta- bility during the incubation. Consequently, incubations were carried out for 2 min or less. Panel C demonstrates that product formation is proportional to enzyme concentration under these conditions, provided that the protein concentration remains less than 5 mg/ml.

TABLE I1 Requirements for the fatty acylation of UDP-GlcNAc in membrane-

free extracts of E. coli The complete system (see "Experimental Procedures") gave an

average rate of 1.3 nmol X min" X mg-l. Values are the averages of duplicates. Values of <0.1 indicate that no product was detectable under these assay conditions. Extracts of strain JB1104 were employed at 3.8 mg/ml.

Condition Relative rate

% Complete system 100 Omit enzyme <0.1 Enzyme heated a t 60 "C for 20 min 100 Enzyme boiled 10 min <o. 1 Omit R-3-hydroxymyristoyl-ACP <0.1

Substitute R-3-hydroxymyristoyl-CoA (0.1 Substitute palmitoyl-ACP 10.1 Substitute ATP + R-3-hydroxymyristate <o. 1

+ MgCI~" Omit octyl-P-D-glucoside* 90

Substitute 0.1% Triton X-100 for octyl-B- 85 D-glucoside*

Reaction concentrations were: ATP, 100 mM; R-3-hydroxymyris-

'All radiolabeled lipid products (as in Fig. 2) formed were added tic acid, 100 p ~ ; and MgCI2, 100 mM.

together.

2 0 ! i ! L " A Enzymm Addod (ag/mll

FIG. 3. Quantitative assay for UDP-GlcNAc acyltransferase in wild-type E. coli. With the exception ofpanel A, data points in this figure were generated using an assay mixture consisting of [P-32P]UDP-GlcNAc (4 X lo5 cpm/nmol at 116 p ~ ) , 40 mM HEPES, pH 8.0, 300 p~ P-hydroxymyristoyl-acyl carrier protein (saturating), and 1.0% octyl-P-D-glucoside to prevent further metabolism of the monoacylated product. Reaction volumes were 50 pl and reactions were performed at 30 "C. Membrane-free extracts of wild-type (JB1104) E. coli were used in all experiments (see "Experimental Procedures"). UDP-GlcNAc and enzyme concentrations were varied as shown. Panel A, initial velocity as a function of substrate concentration at 3.8 mg/ml protein. Panel B, time course at 3.8 mg/ml protein and 116 p M UDP-GlcNAc. Panel C, initial velocity under standard conditions as a function of enzyme concentration. The results are nonlinear above 5 mg/ml protein.

Partial Purification of UDP-GlcNAc Acyltransferase-The specific activities of UDP-GlcNAc acyltransferase in three relevant strains are shown in Table 111. Extracts of JB1104 have a 2.3-fold lower specific activity than the vector control (MClOGl/pINGl) used to construct the L-arabinose-inducible strain MClOGl/pSRl (22).

UDP-GlcNAc acyltransferase was partially purified from fully induced MClOGl/pSRl as described under "Experimen- tal Procedures." The fold purification and yield obtained at each step are shown in Table IV. The final preparation is


TABLE 111 Specifit activity of UDP-GlcNAc acyltransferase in various strains of

E. coli Specific activities were measured in crude cell-free extracts using

the standard assay described under “Experimental Procedures.” Strain MClOGl/pSRl overproduces UDP-GlcNAc acyltransferase upon addition of 0.5% arabinose to the growth medium (22). Specific activities in strains MClOGl/pINGl and MClOGl/pSRl are those measured after 8 h of arabinose induction, while cells of JB1104 were grown on LB broth in the absence of arabinose.

Strain Specific Relative specific activity activity

nrnoljminlrng JB1104 MClOGl/pINGl

1.3 1

MClOGl/pSRl 3.0 2.3“

560 430 The 2.3-fold difference between strains MClOGl/pINGl and

JB1104 is real. Therefore, the specific activity of strain MC1061/ pSRl is 430 times that found in extracts of strain JB1104.

973-fold enriched in acyltransferase specific activity, relative to crude extracts of the vector control, or 2245-fold when compared to extracts of strain JB1104. Gel electrophoresis of the partially purified enzyme revealed a prominent band at approximately 27 kDa (data not shown), but many other contaminating bands were also visible. This value is consistent with the molecular weight predicted by Crowell et al. (22) on the basis of minicell radiolabeling studies. The nonlinearity of the enzymatic time course, shown in Fig. 3 for crude cellular extracts, was unchanged when examined with this highly purified enzyme (data not shown).

FAB Mass Spectrometry of the Putative UDP-3-O-(R-3- hydroxymyristoy1)-GlcNAc-In order to isolate enough of the new metabolite shown in Fig. 2 to determine its structure, we performed a large scale enzymatic synthesis of this compound using the partially purified UDP-GlcNAc acyltransferase (see “Experimental Procedures”). A portion of this material was analyzed by FAB mass spectrometry in the positive mode in order to verify the presence of only one R-3-hydroxymyristoyl residue and to confirm its covalent attachment to the glucosamine ring. A typical spectrum is shown in Fig. 4. The peaks observed in the high molecular weight range (m/z 834 to 933) can be fully explained by the formation of complexes of the R-3-hydroxymyristoyl-UDP-GlcNAc product (C3’HS3N3. O19P,, having a chemical molecular weight of 833.72) with Na and K ions, presumably carried over from the HPLC system. For instance, we attribute the small peak at m/z 834 to (M + H)+ and the larger one at m/z 857 to (M + Na)’. Furthermore, we assign (M + K)’ at m/z 872; (M - H + 2Na)’ a t m/z 878; (M - H + Na + K)+ at m/z 895; (M - 2H + 3Na)’ at m/z 900; (M - H + 2K)’ at m/z 910; (M - 2H + 2Na + K)’ at m/z 916; and (M - 2H + Na + 2K)+ at m/z 933. Prominent peaks between m/z 427 and m/z 465 are assigned as follows: (UDP + Na)’ at m/z 427; (M - UDP)+ at m/z 430; (UDP +

K)’ at m/z 443; (UDP - H + 2Na)’ at m/z 450; (M - UDP - H + Na)+ at m/z 452; and (UDP - H + Na + K)+ at m/z 465. The fragment at m/z 430 is especially important, since it is an oxonium ion that arises on the GlcNAc ring when UDP is released. Its mass indicates that an R-3-hydroxymyristoyl substituent is present on the GlcNAc moiety. The masses of the ions in the high molecular weight range demonstrate that only one R-3-hydroxymyristoyl residue is present in the enzymatic product.

‘H NMR Spectroscopy of the R-3-Hydroxymyristoyl-UDP- GlcNAc-In order to determine the location of the esterified R-3-hydroxymyristoyl residue on the glucosamine ring, the enzymatically synthesized sample was analyzed by ‘H NMR at 270 MHz. The full spectrum is shown in Fig. 5A, and the spectrum of the starting material, UDP-GlcNAc, shown in Fig. 5B, is in accord with that reported by Lee and Sarma (34). A comparison of these spectra shows the presence of additional protons in the enzymatic product (designated HM in Fig. 5A), that are indicative of the incorporation of a single R-3-hydroxymyristoyl group, as judged by the integration of the signals (data not shown). The acetyl group (designated A in both panels) is present in the product and is observed just upfield of 2 ppm.

The site of attachment of the R-3-hydroxymyristoyl moiety is found by decoupling of the glucosamine ring protons between 3.4 and 5.5 ppm, designated G in Figs. 5A and 6. As shown in Fig. 6A, irradiation of the G-1 proton (i.e. H-1 of the glucosamine ring), indicated by the heavy arrow above the peak, causes a pair of triplets at 4.1 ppm to collapse to a pair of doublets, which may, therefore, be assigned to proton G-2. Next, irradiation of the G-2 proton verifies the assignment of G-1 and establishes the location of the G-3 proton at 5.1 ppm (which collapses to a doublet). All coupling constants and chemical shifts derived from this analysis are summarized in Table V and are contrasted with those of UDP-GlcNAc (Fig. 6B). As previously demonstrated for 2,3-diacyl-GlcN-l-P (5), the acylation of a glucosamine hydroxyl function shifts the ring proton at the acylated position downfield by 1-1.5 ppm. Protons linked to carbons that are immediately adjacent to the position of acylation are shifted downfield by 0.2-0.5 ppm (5, 19). The spectra shown in Figs. 5A and 6A demonstrate that the proton at position three of glucosamine (G-3) has undergone a large downfield shift (1.60 ppm) relative to the substrate UDP-GlcNAc, indicating this position is the site of acylation. Further evidence is provided by the slight downfield shift of protons G-2 and G-4 (0.13 and 0.21 ppm, respectively). An additional proton present in the product at 3.85 ppm is attributed to H-3 of the R-3-hydroxymyristoyl moiety.

Substrate Specificity of the Partially Purified UDP-GlcNAc Acyltransferase-A preliminary survey of the specificity of the enzyme (Table VI) demonstrates that UDP-GlcNAc and R-3-hydroxymyristoyl-ACP are strongly preferred as sub-

TABLE IV Partial purification of UDP-GlcNAc acyltransferase

The specific activities shown are for 8-h arabinose-induced cultures. The purification scheme is described under “Experimental Procedures.”

Fraction Total volume Total protein Specific activity

Purification factor Yield

rnl w nrnollrninjrng %

MClOGl/pSRl extract 100 1280 560 1.0 100 Membrane free supernatant 100 1110 614 1.1 95 DEAE-cellulose 735“ 192 2919 5.2 78

(2245)b

Done in smaller portions as described in the text. Fold increase in specific activity over strain JB1104, the strain used in the experiments of Figs. 2, 3, and 7.

ACP-dependent UDP-GlcNAc Acyltransferase 5 165

roo1 427 /

430 1.443

400 GOO 800 1000 M I Z

FIG. 4. Positive ion fast atom bombardment mass spectrum of the enzymatic product. The sample, containing some sodium and potassium counter ions, was prepared as described under "Ex- perimental Procedures." Mass assignments are discussed in the text.

strates, supporting the biosynthetic scheme of Fig. 1. We have not determined whether these effects reflect changes in K,,, or V,,,. Nevertheless, it is remarkable that no activity is detect- ble when myristoyl-ACP, palmitoyl-ACP or R-3-hydroxymyristoyl coenzyme A are employed (Table VI, A). The small amounts of activity observed with S-3-hydroxymyristoyl-ACP may be due to the presence of residual R enantiomer, but the small activities observed with R,S-3-hydroxylauroyl-ACP and R,S-3-hydroxypalmitoyl-ACP are certainly not due to con- tamination with R-3-hydroxymyristoyl-ACP.

As shown in Table VI, B, UDP-GlcN does not function as a substrate for the purified enzyme, indicating specificity for

A

FIG. 5. 'H-NMR analysis of the enzymatic product at 270 MHz. Panel A, the 'H NMR spectrum of 0.8 mg of product, taken as described under "Experimental Procedures." Panel B, the 'H NMR spectrum obtained from a standard sample (5 mg) of UDP- GlcNAc. Both samples were exchanged twice with D,O before acquisition of the spectra. Abbreviations used refer to protons attached to carbons of the five domains of this molecule (boldface letters shown in the structure). These are: HM, R-3-hydroxymyristate protons; A, acetyl protons; G, glucosamine ring protons; R, ribose ring protons; and U, uracil ring protons.

an acylated nitrogen atom. Although the enzyme functions poorly with the N-propionyl or the N-butyryl analogs, it does not utilize [~-32P]-UDP-N-(R-3-hydroxymyristoy1)-GlcN, a compound that is prepared by mild alkaline hydrolysis of [/3-3ZP]UDP-2,3-diacyl-GlcN.

The specificity of the UDP-GlcNAc acyltransferase for the nucleotide base (Table VI, C) was also examined. Only the uridine and the thymidine derivatives were utilized by the enzyme to an appreciable extent. The rate observed with the guanosine analog is very small, but significant.

Further Acylation and Metabolism of UDP-3-O-(R-3-hy- droxymyristoy1)-GlcNAc in Extracts of E. coli-In order to establish the function of UDP-3-O-(R-3-hydroxymyristoyl)- GlcNAc as a precursor of lipid A, enzymatically synthesized [/3-32P]UDP-3-0-(R-3-hydroxymyristoyl)-GlcNAc was isolated and reincubated with extracts of JB1104 in the presence of R-3-hydroxymyristoyl-ACP or palmitoyl-ACP. Analyses of the reaction products at various time points by thin-layer chromatography and HPLC are shown in Figs. 7 and 8. The former is useful for the identification of more lipophilic metabolites, which include 32P-labeled substances migrating with standards of UDP-2,3-diacyl-GlcN, 2,3-diacyl GlcN-1-P, and tetraacyldisaccharide 1-phosphate (Fig. 7 ) , while the latter is used to identify hydrophilic 32P-labeled products (Fig. 8) that accumulate on the origin in the experiment of Fig. 7 and consist of [P-32P]UDP, 32Pi, and [/3-32P]UDP-GlcNAc (Fig. 8). The concentrations of both the lipophilic and the hydrophilic metabolites formed with time were quantitated by scintillation counting (Fig. 9). The [p-32P]UDP-3-0-(R-3-hydroxy- myristoy1)-GlcNAc was consumed very quickly, while 32P- labeled metabolites migrating with standards of UDP-2,3- diacyl-GlcN, 2,3-diacyl-GlcN-1-P, and tetraacyldisaccharide 1-phosphate appeared in the expected order and a t a rate comparable to that observed when [P-32P]UDP-GlcNAc and R-3-hydroxymyristoyl-ACP were employed as the substrates

HM 5-13

.r I

A

HDO I


A

I G-4

I

l " ' l " ' " ' ' ~ " l " ' " ' ' ~ ' ' ' ~ ' ~ ~ , ~ ' ' , ' ~ ~ ~ 5.4 5.0 4.6 4.2 3.8 3.4

8

I ' " 1 " ' l " ' l ' " l ' " I ' " " " ' " " I ' ' ' [ ' ' ' ~ ~ 5.4 5.0 4.6 4.2 3.8 3.4

8 , PPm FIG. 6. 'H decoupling of the glucosamine ring protons in the

enzymatic product. Panel A, expansion of the region from 3.4 to 5.5 ppm of the 270-MHz 'H NMR spectrum of Fig. 5A. The sample was selectively decoupled at each of the resonances indicated by the heavy arrows, starting with G-1, the anomeric proton. For each decoupling, the changes observed in the spectrum are also indicated. Panel B, expansion of the corresponding region of the UDP-GlcNAc spectrum for comparison. Coupling constants of the relevant glucosamine ring protons of both compounds are summarized in Table V. G is defined in Fig. 5.

TABLE V A summary of the chemical shifts 13) and coupling constants (Hz) of

relevant glucosamine ring protons of the enzymatic product and of the substrate, UDP-GlcNAc

H-1 H-2 H-3 H-4

Enzymatic product 5.42 3.97 5.06 3.61 51.2 = 3.4 5 2 . 3 = 10.8 5 3 . 4 = 10.3 J1.p = 7 J2,p = 3

UDP-GlcNAc 5.37 3.84 3.66 3.40 J1 ,2 3.4 5 2 , s = 10.5 53.4 = 9.4 5, p = 7.6 5 2 p = 2.8

in crude extracts of E. coli (Fig. 2). Although most of the [@-32P]UDP-3-O-(R-3-hydroxymyris-

toy1)-GlcNAc is consumed (Figs. 7 and 9), about 2% appeared to remain unaltered. This residual material appears not to be UDP-3-O-(R-3-hydroxymyristoy1)-GlcNAc, since it is separable from the bulk of the [@-32P]UDP-3-0-(R-3-hydroxymy- ristoy1)-GlcNAc by means of the C18 reverse phase HPLC system, described above. Since mild alkaline hydrolysis (12)

TABLE VI substrate specificity of the partially purified UDP-GlcNAc

acyltransferase Standard assay conditions (see "Experimental Procedures") were

used with the following minor modifications (A) Each acyl-ACP substrate was present at 60 p ~ , except for the R,S derivatives that were employed a t 120 p ~ . (B) R-3-hydroxymyristoyl-ACP (100 PM) was employed together with each UDP derivative at 100 PM. (C) For the [3H-acetyl]NDP-GlcNAcs, the reactions were performed on a 100- p1 scale. After 2 min, the reaction was quenched by addition of 400 pl of ch1oroform:methanol (1:2, v/v). The mixtures were reduced in volume under a stream of nitrogen and dried in a Savant Speed-Vac. The samples were redissolved in 20 pl of ch1oroform:methanol (1:2, v/v) and spotted onto a Silica Gel 60 thin-layer plate. The tubes were rinsed and the plates were spotted two more times in this manner. The plates were developed as described under "Experimental Proce- dures." The regions of interest (i.e. those expected to contain the 0- monoacyl-NDP-GlcNAc product) were then scraped and counted in 10 ml of Patterson and Green (12) scintillation mixture.

Substrate pairs action rates Relative re-

% A. [P-32P]UDP-GlcNAc and

R-3-Hydroxymyristoyl-ACP 100 S-3-Hydroxymyristoyl-ACP 7 R,S-3-Hydroxymyristoyl-ACP 35 R,S-3-Hydroxylauroyl-ACP 1.5 R,S-3-Hydroxypalmitoyl-ACP 1.5 Myristoyl-ACP <o. 1 Palmitoyl-ACP <o. 1 R-3-Hydroxymyristoyl coenzyme A eo. 1

[P-32P]UDP-GlcNAc 100 [/3-32P]UDP-N-propionyl-GlcN 22

[~-32P]UDP-N-(R-3-hydroxymyristoyl)-GlcN 0

B. R-3-Hydroxymyristoyl-ACP and

[fl-32P]UDP-N-b~ty~1-Gl~N 8

[P-32P]UDP-Gl~N 0 C. R-3-Hydroxymyristoyl-ACP and

[3H-Acetyl]UDP-GlcNAc 100 [3H-Acetyl]TDP-GlcNAc 20 [3H-Acetyl]GDP-GlcNAc 0.04 [3H-Acetyl]ADP-GlcNAc <0.001 [3H-Acetyl]CDP-GlcNAc <0.001

of the residual material regenerates [P-32P]UDP-GlcNAc (data not shown), we believe that it may be a positional isomer, such as a 4- or 6-0-monoacyl derivative. Such a compound might form by nonenzymatic migration of the R- 3-hydroxymyristoyl moiety from the 3 position, but this hy- pothesis remains to be established.

Origin of [fi-32P] UDP, 32Pi, and [P-"P] UDP-GlcNAc-The hydrophilic products generated from [/3-32P]UDP-3-0-(R-3- hydroxymyristoy1)-GlcNAc (Figs. 7 and 8) are formed enzymatically, since they are not detected when boiled extracts are employed (data not shown). The identification of these substances by reverse phase HPLC (Fig. 8) as [P-32P]UDP, 32Pi, and [P-32P]UDP-GlcNAc was based on their mobility compared to internal standards. As an additional criterion, each HPLC fraction was subjected to PEI-cellulose thin-layer chromatography together with appropriate standards (not shown).

The [p-32P]UDP arises during the formation of the tetraacyldisaccharide 1-phosphate from [P-32P]UDP-2,3-diacy1- GlcN and lipid X (Fig. 1). 32Pi is presumably formed in the crude extracts employed by the hydrolysis of [P-32P]UDP. The HPLC fractionation of the hydrophilic products formed after 60 min allows calculation of the concentration of UDP (1.38 FM) that would have been formed had there been no UDP hydrolysis. This value is stoichiometric with the concentration of tetraacyldisaccharide 1-phosphate generated after

r I 50

2 0

a ; 100

[L LL

a a w

n

‘0 5 * 50

a V


+3-OH.MYR-ACP + PALM - ACP

Disaccharide I - P

Diacyl-GlcN-I-P - UDP-Diacyl-GlcN -

Origin - 1 2 3 4 5 6 7 8

FIG. 7. Conversion of [~-S2P]UDP-3-0-(R-3-hydroxymyristoy1)-GlcNAc to UDP-2,3-diacylglucosamine and other lipid A precursors. This figure is an autoradiograph of a thin-layer plate developed in ch1oroform:methanol:water:acetic acid (25:15:42, v/v). The origin and the location of various standards are indicated. The reaction mixture contained 3.2 p M [~-3ZP]UDP-3-0-(R-3-hydroxymyristoyl)-G1cNAc (2 X lofi cpm/ nmol), 300 p~ fatty acyl-acyl carrier protein, 40 mM HEPES, pH 8.0, and 3.8 mg/ml protein from strain JB1104, prepared as described under “Experimental Procedures.” Portions of the reaction mixtures (5 pl) were spotted directly onto the plate, just prior to development. Lanes 1-4, 2’, 5‘, 15’, and 30’ time points, respectively, using R- 3-hydroxymyristoyl-acyl carrier protein (+3.OH-MYR-ACP) as the acyl donor. Lanes 5-8, 2’, 5’, 15‘, and 30‘ time points, respectively, using palmitoyl-acyl carrier protein (+PALM-ACP) as the acyl donor.

P. ’ \ UDP-GlcNAc

I 5 20 25 30 IO I5

1

FRACTION NO.

FIG. 8. C18 reverse phase HPLC analysis of the polar metabolites formed from [fl-32P]UDP-3-0-(R-3-hydroxymyristoyl)-GlcNAc after 60 min. The figure is a plot of the radioactivity found in each 1.5-ml fraction eluting off of a C18 reverse phase HPLC column, as described under “Experimental Procedures.” The positions of internal UDP and UDP-GlcNAc standards, monitored a t 260 nm, are as shown. The mobility of Pi was determined using ”Pi in a separate run. All peaks were also examined for purity on PEI- cellulose, as described in the text.

60 min, calculated to be 1.3 p~ from the data shown in Fig. 7.

The formation of [@-32P]UDP-GlcNAc from [@-32P]UDP- 3-0-(R-3-hydroxymyristoyl)-GlcNAc was unexpected. Sub- traction of the amount of tetraacyldisaccharide 1-phosphate formed a t various times from the amount of total hydrophilic metabolites formed at the corresponding times allows construction of a UDP-GlcNAc concentration curve, shown in Fig. 9. I t is apparent from this data that [P-32P]UDP-GlcNAc is produced from [fi-32P]UDP-3-O-(R-3-hydroxymyristoyl)- GlcNAc and not from any of the other metabolites, since it forms only while the [P-32P]UDP-3-0-(R-3-hydroxymyris- toy1)-GlcNAc is still present in the reaction mixture. Impor- tantly, [@-32P]UDP-GlcNAc is not formed by the highly purified UDP-GlcNAc acyltransferase, acting on [P-32P]UDP-3- 0-(R-3-hydroxymyristoyl)-GlcNAc, either in the presence or absence of 0-hydroxymyristoyl-ACP (data not shown). Con- sequently, the [P-32P]UDP-GlcNAc must arise by the action of some unrelated lipase present in E. coli extracts.

As shown in Fig. 7 (right-handpanel) the fatty acylation of [@-32P]UDP-3-0-(R-3-hydroxymyristoyl)-GlcNAc does pro- ceed (albeit relatively slowly) with palmitoyl-ACP as the acyl donor. This is in contrast to the UDP-GlcNAc acyltransferase which does not utilize palmitoyl-ACP to any measurable extent (Fig. 2, lane 6, and Table VI). Conditions inside the cell must prevent palmitoyl-ACP utilization, since N-linked palmitoyl residues are not found in lipid A (2).

DISCUSSION

Our current efforts to demonstrate that UDP-3-0-(R-3- hydroxymyristoy1)-GlcNAc is the first intermediate in lipid A biosynthesis were aided by two discoveries. The first was the finding that octyl-P-D-glucoside in the reaction mixture

ACP-dependent UDP-GlcNAc Acyltransferase

1 i

INCUBATION TIME WIN) FIG. 9. Time course of the formation of the lipid and polar

products generated from [&S2P]UDP-3-O-(R-3-hydroxymy- ristoy1)-GlcNAc by E. coli extract in the presence of R-3- hydroxymyristoyl-ACP. The concentration of each metabolite formed in Fig. 7A was quantitated by scraping and counting each product in 10 ml of Patterson and Green (12) scintillation mixture. Symbols used are: ."--., UDP-3-O-(R-3-hydroxymyristoyl)- GlcNAc; o " 0 , UDP-2,3-diacyl-GlcN; X-X, 2,B-diacyl-GlcN-l- P; D-", tetraacyldisaccharide-1-P A-A, total origin material (UDP, Pi, and UDP-GlcNAc); U, UDP-GlcNAc.

blocked the further metabolism of the putative O-monoacyl- UDP-GlcNAc product, causing it to build up to much higher levels than previously possible (12) and permitting the development of a reliable assay. Secondly, the discovery of a genetic locus (@A), the molecular cloning of which causes massive overproduction of UDP-GlcNAc acyltransferase (22), facili- tated a 1000-fold purification of the enzyme. Since very little of the purified enzyme preparation is required for the formation of milligram quantities of product, it was possible to reisolate the product from an enzymatic reaction mixture and to establish its structure as UDP-3-O-(R-3-hydroxymyris- toy1)-GlcNAc by 'H NMR and FAB mass spectrometry (Figs.

UDP-3-O-(R-3-hydroxymyristoy1)-GlcNAc may well be a true precursor, since it is rapidly converted to UDP-2,3-diacyl- GlcN and other lipid A precursors (as judged by thin-layer chromatography) is the presence of E. coli extract and R-3- hydroxymyristoyl-ACP (Fig. 9). Assays for each of the enzymes involved in the transformation of UDP-3-04R-3-h~- droxymyristoy1)-GlcNAc to UDP-2,3-diacyl-GlcN must still be developed, and the structures of the intervening metabolites remain to be elucidated. Presumably, a deacetylase first removes the acetyl group, whereupon a second acyltransferase attaches the N-linked R-3-hydroxymyristoyl residue (as proposed in Fig. 1). So far, we have not found conditions under which the expected UDP-3-O-(R-3-hydroxymyristoy1)-glucosamine intermediate accumulates, but it may be necessary to

4-6).

fractionate the extracts in order to demonstrate the deacety- lation step. Furthermore, mutants defective in each of the enzymes leading to UDP-2,3-diacyl-GlcN are needed to pro- vide biological evidence that no other schemes exist in the cell for lipid A biosynthesis.

The glucosamine disaccharide backbone of lipid A (Fig. 1) is acylated with four R-3-hydroxymyristoyl moieties, situated at positions 2, 3, 2', and 3' (5-9). According to the pathway (Fig. l ) , the R-3-hydroxymyristoyl residues at positions 3 and 3' are incorporated by the UDP-GlcNAc acyltransferase that we have characterized. The extreme specificity of the enzyme for the R-3-hydroxymyristoyl moiety (as opposed to palmitoyl, myristoyl, R-3-hydroxylauroyl, or R-3-hydroxypalmitoyl residues) is consistent with the fact that greater than 97% of the esterified fatty acyl chains attached to positions 3 and 3' of lipid A and its precursors are R-3-hydroxymyristate (5,9,24). Recently, Takayama et ul. (35) have demonstrated that the esterified fatty acids a t positions 3 and 3' of lipid A from Neisseria gonorrheae are R-3-hydroxylaurate, but the substrate specificity of the UDP-GlcNAc acyltransferase from N. gonorrheue has not yet been examined.

The acyl chain specificity of UDP-GlcNAc acyltransferase is much narrower than that of E. coli glycerol-3-phosphate acyltransferase, which can utilize fatty acids ranging from 12 to 20 carbons in length in vitro, including unsaturated and hydroxylated analogs (36, 37). Remarkably, no detectable R- 3-hydroxymyristate is actually incorporated into glycerophospholipids in vivo (13, 38), suggesting that other mechanisms (such as compartmentalization) might prevent glycerol-3- phosphate acyltransferase from gaining access to R-3-hydroxymyristoyl-ACP. Another possibility, that could be studied by NMR methods (39), is that R-3-hydroxyacyl-ACPs have dif- ferent conformations than nonhydroxylated acyl-ACPs. Per- haps, UDP-GlcNAc acyltransferase is very sensitive to ACP conformation.

UDP-GlcNAc acyltransferase of E. coli also shows consid- erble specificity with regard to the N-linked substituent of UDP-GlcNAc, since it does not act on UDP-glucosamine (12) or UDP-N-(R-3-hydroxymyristoy1)-glucosamine. The latter differs from UDP-GlcNAc by the length of the fatty acyl chain and the presence of a P-OH function, both of which might prevent access to the active site. Furthermore, as shown in Table VI, UDP-GlcNAc acyltransferase functions best with uridine and thymidine derivatives, but not with the cytidine or adenosine analogs. Since the uridine derivative is the most abundant in vivo (10, 40), it is probably of the greatest biological significance, but a role for TDP-GlcNAc cannot be excluded completely, since bacteria have the enzymes to syn- thesize this substance (40).

Unexpectedly, UDP-3-O-(R-3-hydroxymyristoy1)-GlcNAc was also 0-deacylated to regenerate UDP-GlcNAc in the E. coli extracts employed. An enzyme that is separable from UDP-GlcNAc acyltransferase is involved. Whether or not this lipase functions in vivo remains to be explored. If significant, this enzyme could be part of a regulatory scheme involved in the partitioning of UDP-GlcNAc between the synthesis of lipid A (13) and peptidoglycan (41), the two major glucosamine-derived substances in the E. coli envelope.

A provocative aspect of the UDP-GlcNAc acyltransferase is its specificity for acyl carrier protein. De nouo synthesis of fatty acids in E. coli occurs while the nascent acyl chain is attached to ACP (42, 43). The elegant studies of Bloch and co-workers (42, 44, 45) showed that R-3-hydroxydecanoyl- ACP is situated at a major branchpoint leading either to saturated or to unsaturated fatty acids in E. coli. The latter are formed by a P-y-dehydratase (the fubA gene product) (46,


47) that acts on R-3-hydroxydecanoyl-ACP. Our studies demonstrate the existence of a second important branchpoint in fatty acid biosynthesis at the level of R-3-hydroxymyristoyl- ACP, leading either to the incorporation of R-3-hydroxymyristate into lipid A precursors or to the generation of palmitoyl-ACP for glycerolipid synthesis. I t is conceivable that the partitioning of R-3-hydroxydecanoyl-ACP between the @ , y dehydratase (fubA gene product) and the a,@-dehydratase of the fatty acid elongation cycle (42) (genetic locus unknown) may share some common features with the partitioning of R- 3-hydroxymyristoyl-ACP between the UDP-GlcNAc acyltransferase ( I p r A gene product) (22) and the a,@-dehydratase. In both cases a pair of enzymes recognize and compete for a specific R-3-hydroxyacyl-ACP, the levels of which are probably very low but have not actually been determined in vivo (48). Given these considerations, the sequences and three- dimensional structures of the P,y-dehydratase, the UDP- GlcNAc acyltransferase, and the a,@-dehydratase should be compared in depth, since the R-3-hydroxyacyl-ACP recogni- tion domains of these enzymes might play an important role in the regulation of membrane biosynthesis.

Acknowledgments-We thank Dr. John Naleway and Andy Rob- ertson for assistance in taking the 'H NMR spectra. We would also like to thank Dr. Gary Ashley and Dr. Laurens Anderson for many helpful discussions, and Dr. Frank Unger of Sandoz for providing us with S-3-hydroxytetradecanoic acid.

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