 |
INTRODUCTION |
Lipopolysaccharide, a macromolecular glycolipid found in the outer
membranes of Gram-negative bacteria (1-6), is anchored to the outer
leaflet of the outer membrane by its lipid A moiety (Fig. 1). The
biosynthesis of the lipid A portion of Escherichia coli
lipopolysaccharide is required for cell viability (3, 7, 8).
Additionally, lipid A (endotoxin) causes extreme stimulation of the
innate immune system of animals, resulting in the overproduction
of diverse cytokines, which can cause the syndrome of Gram-negative
sepsis (3, 4, 9, 10). Pharmacological studies have shown that both
phosphate groups, the glucosamine disaccharide, and the correct number
of fatty acyl chains (Fig. 1) are crucial for the cytokine-inducing
activities of lipid A (3, 4, 9).
The structure of lipid A varies slightly among different Gram-negative
bacterial pathogens (1, 11), such as E. coli
versus Pseudomonas aeruginosa (Fig. 1), but most
of the distinguishing structural features are conserved. However, the
lipid A from the nitrogen-fixing bacterium Rhizobium
leguminosarum differs strikingly from that of E. coli
(Fig. 1) (12-15). Both phosphate groups are missing, a galacturonic
acid residue is attached at the 4'-position, and the glucosamine
1-phosphate unit of E. coli lipid A is largely replaced with
an aminogluconate moiety (Fig. 1) (12, 13). In the initial structural
studies by Carlson and co-workers (12, 13), it was further suggested
that R. leguminosarum lipid A does not possess any
acyloxyacyl residues and that it contains a peculiar long fatty acid,
27-hydroxyoctacosanoic acid (Fig. 1) (16). R. leguminosarum
lipid A therefore lacks many of the features thought to be necessary
for stimulation of innate immunity in animals (1, 3, 4, 9).
Conceivably, the unique structure of R. leguminosarum lipid
A might be important for the establishment of successful symbiosis in
plants (17, 18).
Despite the structural diversity of their lipid A moieties, both
E. coli and R. leguminosarum employ the same
seven enzymes to synthesize the key, phosphate-containing lipid A
precursor, Kdo2-lipid
IVA1 (19). A
number of distinct R. leguminosarum enzymes are then required for the alternative processing of Kdo2-lipid
IVA to generate R. leguminosarum lipid A. We
have previously identified a 4'-phosphatase (20), a 1-phosphatase (21),
a long chain acyl transferase (22), a mannosyl transferase (23, 24), a
galactosyl transferase (21, 24), and a special Kdo transferase (24)
that are involved in the unique metabolism of Kdo2-lipid
IVA in extracts of R. leguminosarum. The
biosynthetic origins of the galacturonic acid and the aminogluconate moieties are unknown.
We have recently discovered that lipid A of R. leguminosarum
can be separated into five related molecular species (14,
15),2 two of which are shown
in Fig. 1A (dashed bond at position
3). Structural studies have revealed that some of this heterogeneity can be attributed to lipid A variants lacking the equivalent of the
ester-linked
-hydroxymyristoyl moiety that is usually attached to
the 3-position of lipid A disaccharides (Fig. 1) (14, 15). Unexpectedly, our reevaluation of the structure of R. leguminosarum lipid A also indicates the presence of a single
acyloxyacyl moiety in all five molecular species (14,
15),2 as illustrated in Fig. 1A for two of
the subtypes.
We now describe a divalent cation-dependent deacylase from
R. leguminosarum membranes that selectively removes a single
3-O-linked
-hydroxyacyl chain from certain precursors of
lipid A that are common to both E. coli and R. leguminosarum. The enzyme removes only the ester-linked
-hydroxymyristoyl residue that is attached to the 3-position of the
proximal glucosamine unit (3) of precursors like lipid IVA
(Fig. 1B) and Kdo2-lipid IVA. It is
also capable of cleaving the monosaccharide precursor lipid X at a slow
rate. A similar deacylase is found in membranes of P. aeruginosa, in which the presence of 3-O-deacylated
lipid A species is well established (Fig. 1A) (25, 26).
E. coli K-12 and Rhizobium meliloti do not
contain the deacylase. The enzyme may therefore account for the
presence of the 3-O-deacylated lipid A subtypes found in
R. leguminosarum (Fig. 1A), and it may be a
useful reagent for the preparation of novel endotoxin analogs with
which to study innate immunity.
 |
EXPERIMENTAL PROCEDURES |
Chemicals and Materials--
[
-32P]ATP and
32Pi were obtained from NEN Life Science
Products. Silica gel 60 (0.25-mm) thin layer plates were purchased from EM Separation Technologies. DEAE-cellulose (DE52) was obtained from
Whatman. BAKERBOND octadecyl (C18) reverse phase resin was from J. T. Baker, and Silica Gel DavisilTM (grade 638, 100-200
mesh, 60 Å) was from Aldrich. Triton X-100 and bicinchoninic acid were
from Pierce. Yeast extract and tryptone were purchased from Difco. All
other chemicals were of reagent grade, and were obtained from Sigma or
Mallinckrodt. Deuterated solvents were purchased from Aldrich.
Bacterial Strains and Growth Conditions--
R.
leguminosarum biovar phaseoli CE3 (recently
reclassified as Rhizobium etli) was a gift of K. D. Noel (Marquette University, Milwaukee, WI) (27). R. leguminosarum biovar viciae 8401 (20, 21) was obtained
from J. A. Downie (John Innes Institute, Norwich, United Kingdom),
and mutant 24AR of R. leguminosarum biovar
trifolii was obtained from R. Russa via R. Carlson (Marie
Curie Sklodowska University, Lubin, Poland) (28). R. meliloti 1021 was from S. Long (Stanford University). All other
strains of Rhizobium were purchased from the American Type
Culture collection (ATCC). E. coli strain W3110 was obtained
from the E. coli Genetic Stock Center of Yale University.
P. aeruginosa strain PAO1 was a gift of G. Pier (Harvard),
and P. aeruginosa strain 27853 was obtained from the
American Type Culture Collection.
All Rhizobium strains were grown at 30 °C on TY medium,
which contains 5 g/liter tryptone, 3 g/liter yeast extract, 10 mM CaCl2, and 20 µg/ml nalidixic acid (19).
In addition, 200 µg/ml streptomycin was also added to the medium for
the growth of CE3. E. coli W3110 and P. aeruginosa (PAO1 and 27853) were grown at 30 °C in LB broth,
consisting of 10 g of NaCl, 10 g of tryptone, and 5 g of
yeast extract per liter (29).
Preparation of Radiolabeled
Substrates--
[4'-32P]Lipid IVA was
generated from [
-32P]ATP and the appropriate
tetraacyldisaccharide 1-phosphate acceptor by using the overexpressed 4'-kinase present in membranes of E. coli BLR(DE3)pLysS/pJK2
(30). Kdo2-4'-32P-lipid IVA was
then prepared from [4'-32P]lipid IVA by the
action of the purified E. coli Kdo transferase (31, 32). The
[4'-32P]lipid IVA and the
Kdo2-[4'-32P]lipid IVA were
purified by thin layer chromatography (31, 32) and were stored as
aqueous dispersions at
20 °C in 10 mM Tris chloride,
pH 7.8, containing 1 mM EDTA and 1 mM EGTA.
Prior to use, all lipid substrates were dispersed by sonic irradiation for 1 min in a bath sonicator.
The substrate 32P-lipid X was prepared from
32Pi labeled cells of E. coli strain
MN7, as described previously (33, 34).
Tetraacyldisaccharide-1-32P was made from
32P-lipid X and UDP-2,3-diacylglucosamine using a highly
purified preparation of E. coli disaccharide synthase (34).
Nonradioactive tetraacyldisaccharide 1-phosphate carrier was prepared
in the same way (34). The [4'-32P]lipid IVA
(100 µM, 20,000 cpm/nmol) was hydrolyzed for 90 min at
100 °C in 0.2 M HCl to make
tetraacyldisaccharide-4-32P.
Gal-Man-Kdo2-[4'-32P]lipid IVA
was prepared from Kdo2-[4'-32P]lipid
IVA (20,000 cpm/nmol, 20 µM) with membranes
of R. meliloti 1021/pIJ 1848, which overexpresses both the
mannosyl transferase and the galactosyl transferase (24). The reaction
mixture was incubated with 0.75 mg/ml membrane protein at 30 °C for
90 min. The membranes were then inactivated by heating at 65 °C for
10 min. The deacylase reaction (see below) was subsequently carried out
in the same reaction tube without further purification of the substrate
by adding the additional necessary components.
Lauroyl-Kdo2-[4'-32P]lipid IVA
was synthesized from Kdo2-[4'-32P]lipid
IVA with Kdo-dependent lauroyl transferase
(HtrB), partially purified from MLK1067/pKS12 (35, 36). The reaction
conditions for lauroyl transferase were as follows: 0.35 µg/ml
lauroyl transferase, Kdo2-[4'-32P]lipid
IVA (20,000 cpm/nmol, 20 µM), lauroyl-ACP (30 µM), Triton X-100 (0.2%), MgCl2 (5 mM), and NaCl (50 mM) at pH 7.5 in 50 mM Hepes. The reaction mixture was incubated at 30 °C
for 60 min. The lauroyl transferase was inactivated by heating the
reaction mixture at 65 °C for 10 min. The heat-inactivated reaction
mixture was then used as the substrate for the deacylase reaction
without further purification.
Preparation of Cell-free Extracts and Membranes--
Two liters
of mid-logarithmic phase cells (A550 = 0.6-0.8)
were harvested by centrifugation (7,000 × g for 15 min
at 4 °C) and were resuspended in 50 mM Hepes, pH 7.5, to
give a final protein concentration of 5-10 mg/ml. Cells were broken by
passage through a French pressure cell at 18,000 p.s.i. Remaining
intact cells and large debris were removed by centrifugation at
7000 × g for 15 min. Membranes were prepared by
ultracentrifugation at 149,000 × g for 60 min.
Membrane pellets were resuspended in 50 mM Hepes, pH 7.5, at a protein concentration of ~10 mg/ml. All preparations were
carried out at 4 °C, and samples were stored frozen in aliquots at
80 °C. Protein concentrations were determined with bicinchoninic acid (37), using bovine serum albumin as the standard.
Deacylase Assay Conditions and Thin Layer
Chromatography--
Optimized standard assay conditions for the
deacylase were as follows. The reaction mixture (10-20 µl) contained
50 mM MES, pH 6.25, 1.0% Triton X-100, 2 mM
dithiothreitol, 2 mM EDTA, 20 mM calcium
chloride, and 10 µM [4'-32P]lipid
IVA (20,000 cpm/nmol). The reactions were incubated at 30 °C for 30 min or as indicated. The reactions were terminated by
spotting 2-5-µl samples onto silica gel 60 thin layer chromatography plates. The spots were allowed to dry, and the plates were developed in
the solvent chloroform/pyridine/88% formic acid/water (50:50:16:5, v/v/v/v). When substrates other than lipid IVA or
tetraacyldisaccharide 4'-phosphate were used, a different solvent
system was employed, consisting of chloroform/pyridine/88% formic
acid/water (30:70:16:10, v/v/v/v). These alternative substrates were
Kdo2-[4'-32P]lipid IVA,
Gal-Man-Kdo2-[4'-32P]lipid IVA or
lauroyl-Kdo2-[4'-32P]lipid
IVA. The solvent chloroform/methanol/water/acetic acid (25:15:4:2, v/v/v/v) was used for deacylase reactions in which 32P-lipid X or tetraacyldisaccharide-1-32P
was the substrate.
Radiochemical analyses of thin layer chromatography plates were carried
out with a Molecular Dynamics PhosphorImager 425S, equipped with
ImageQuant software. The percentage of conversion of unreacted
32P-labeled substrates to enzymatic products was calculated
for each reaction tube and could be converted to specific activity (nmol/min/mg) based on the chemical concentration of the substrate in
the assay.
Base Hydrolysis of the Deacylase Product Generated from
[4'-32P]lipid IVA--
Two incubations
(designated 1 and 2) were set up using the standard optimized assay
conditions for the deacylase with 10 µM [4'-32P]lipid IVA as the substrate.
Incubation 1 contained no enzyme, while incubation 2 contained 1 mg/ml
R. leguminosarum (8401) membranes. Both incubations 1 and 2 were incubated for 16 h at 30 °C. Under these conditions, the
conversion of [4'-32P]lipid IVA to the slower
migrating deacylation product was almost complete in incubation
2, whereas no change was seen in incubation 1. For mild base
hydrolysis of the products, a 2-µl portion of each incubation was
mixed with 3 µl of triethylamine (TEA) and 5 µl of H2O,
and the material was then incubated for various times at 37 °C.
After 0, 2, 5, and 120 min, 2-µl samples of the TEA hydrolysis
mixture were added to 2 µl of H2O and spotted onto a
silica gel 60 thin layer plate. For strong base hydrolysis of the
products, 2-µl portions of 1 or 2 were mixed with 18 µl of
chloroform/methanol (2:1, v/v) and 0.4 µl of 10 M NaOH.
This NaOH hydrolysis was carried out at room temperature for 30 min with occasional mixing of the phases. Next, each mixture was further diluted 3-fold with water, and 4-µl portions were spotted onto a
silica gel 60 thin layer plate. The plates were developed with choloroform/pyridine/88% formic acid/water (50:50:16:5, v/v/v/v), dried, and exposed overnight to a PhosphorImager screen.
Large Scale Isolation of the Deacylase Product--
R.
leguminosarum 8401 membranes, prepared by ultracentrifugation as
described above, were enriched for the deacylase activity before being
used for the large scale preparation of the reaction product. Membrane
preparations (25 ml, 10 mg/ml protein) were mixed at 4 °C with 2.5%
Triton X-100 for 90 min, followed by ultracentrifugation at
149,000 × g for 60 min. The pellet, containing the
deacylase activity, was resuspended in 10 ml of 50 mM Hepes
buffer (pH 7.5) to a protein concentration of 7.0 mg/ml. The detergent
extraction process was repeated another time with 1% Triton X-100. In
the pellet (resuspended in 10 ml of 50 mM Hepes buffer, pH
7.5; 5.5 mg/ml protein), more than 90% of the deacylase activity was
recovered. The activity was enriched 2-fold (final specific activity of
0.1 nmol/mg/min), while most of the interfering 4'-phosphatase activity (>80%) was solubilized by the detergent. The procedure was also effective in removing about half of the membrane lipids.
Three 10-ml deacylase reaction mixtures were prepared using 50 µM lipid IVA substrate and 2 mg/ml of Triton
X-100-extracted membrane protein under conditions otherwise similar to
the standard assay. The reaction mixtures were initially incubated at
30 °C for 24 h. Then additional membranes were added to yield a
final protein concentration of 3 mg/ml, and the reactions were
continued for another 24 h. The progress of the reaction was
monitored by thin layer chromatography, as described above for the
assays, but the deacylase product was detected by charring the plates after spraying with 10% sulfuric acid in ethanol. More than 90% of
the substrate was deacylated under these conditions. Prior to product
isolation, the reaction mixtures were stored at
20 °C. After
thawing, the reactions were distributed equally into two 150-ml Corex
bottles. The reactions were diluted with water to yield a final volume
of 20 ml/bottle. The proteins were precipitated by adding 1.25 ml of
CHCl3, 2.5 ml of methanol, and 0.04 ml of concentrated HCl
per ml of the diluted reaction mixtures. The samples were thoroughly
mixed, and then centrifuged at 3,000 × g for 20 min at
room temperature. The supernatant was decanted, and it was converted to
a two-phase system by adding 0.263 ml of CHCl3 and 0.263 ml
of water per ml of supernatant. After mixing, the phases were separated
by centrifugation, as above. The CHCl3-rich lower phase was
removed, and the upper phase was washed twice with fresh,
preequilibrated lower phase (i.e. a lower phase generated by
mixing chloroform, methanol, and 0.1 M HCl in a ratio of
2:2:1.8, v/v/v). The lower phases were pooled, 0.5 ml of pyridine was
added to neutralize residual HCl, and the solvent was removed by rotary evaporation.
The residue was redissolved in ~15 ml of chloroform/pyridine/88%
formic acid/water (70:60:16:3, v/v/v/v) and was loaded onto a 9.5-ml
silicic acid column, equilibrated in the same solvent. The column was
washed with another 20 ml of the same solvent, followed by 60 ml of
chloroform/methanol (95:5, v/v). The lipid IVA-derived
deacylase product was then eluted with ~20 ml of an acidic single
phase Bligh and Dyer mixture, consisting of chloroform, methanol, and
0.1 M HCl (1:2:0.8, v/v/v) (38). The fractions containing
the desired product were identified by thin layer chromatography, followed by charring as described above. The pertinent fractions were
pooled and converted to a two-phase system by the addition of 0.263 ml
of CHCl3 and 0.263 ml of water per ml. The solution was
mixed, and the phases were separated by centrifugation. The lower phase
was collected, and the upper phase was washed twice with
preequilibrated acidic lower phase (see above). The lower phases were
pooled, 5-10 µl of high pressure liquid chromatography grade
pyridine was added, and the solvents were removed by rotary evaporation.
A small amount of contaminating lipid IVA was removed from
the deacylase product by the reverse phase chromatography procedure described by Hampton et al. with minor modifications (39).
The method utilizes a two-solvent system for the resolution of lipid IVA derivatives with octadecylsilane silica gel (C18
silica). Solvent A was 50% (v/v) acetonitrile in water, and solvent B
was 85% isopropyl alcohol in water. Both solvents contained 5 mM tetrabutylammonium phosphate. The dried compound was
redissolved in 3 ml of a 1:1 (v/v) solvent mixture of A and B. The
compound was loaded onto a 0.5-ml C18 silica column equilibrated in the
same solvent ratio. The column was washed with 3 ml of the 1:1 (v/v)
solvent ratio. The column was then washed with 1.5 ml of solvent
consisting of a 1:2 (v/v) ratio of A to B. Finally, the column was
washed with 1.5 ml of solvent consisting of 1:4 (v/v) ratio of A to B. During the chromatography, 0.5-ml fractions were collected. Fractions containing the deacylase product were identified by charring. The
relevant fractions were pooled and diluted 1:1 with
chloroform/methanol/water (2:3:1, v/v/v). The diluted pool was loaded
directly onto a 1-ml DE52 column equilibrated in
chloroform/methanol/water (2:3:1, v/v) to remove the
tetrabutyl-ammonium phosphate. The column was washed with 6 ml of
chloroform/methanol/water (2:3:1, v/v/v), and the compound was then
eluted with ~10 ml of chloroform, methanol, and 480 mM
ammonium acetate (2:3:1, v/v/v). The compound-containing fractions were
again identified by charring and were pooled. The pooled fractions were
converted to a two-phase Bligh and Dyer system by the addition of 0.167 ml of CHCl3 and 0.283 ml of water per ml of pool. The
solution was mixed, and the phases were separated by centrifugation.
The lower phase was collected, and the upper phase was washed twice
with preequilibrated acidic lower phase (see above). The lower phases
were pooled, and the solvents were removed by rotary evaporation. The
isolated deacylase product was stored at
20 °C prior to further analysis.
1H NMR Analysis of the Deacylase Reaction
Product--
The purified enzymatic reaction product (2 mg) was
dissolved in 0.6 ml of
CDCl3/CD3OD/D2O (2:3:1, v/v/v). Its
1H NMR spectrum was recorded on a Varian 600 Unity
spectrometer using a 3779.29-Hz spectral window with the 5-mm probe at
25 °C. Chemical shifts were referenced to the methyl protons of
internal tetramethylsilane (0.00 ppm). A line broadening of 0.2 Hz
before Fourier transformation was used to process the data. The water signal at 4.6 ppm was suppressed by presaturation (satpower = 0).
Two-dimensional 1H correlation (COSY) spectra were recorded
in the absolute value mode over the same spectral region used in the
one-dimensional 1H NMR spectrum. Four hundred time
increments were collected and zero-filled to 2048 points with sine-bell
weighting along both f1 and f2 dimensions. Three hundred twenty
scans were collected per increment, and the relaxation delay was 1 s. Presaturation of the water line was also included in the pulse sequence.
 |
RESULTS |
A Novel Deacylase in Membranes of R. leguminosarum and P. aeruginosa--
As shown in Fig. 2, membranes of R. leguminosarum 8401 and R. leguminosarum/etli CE3 can
convert [4'-32P]lipid IVA to a more
hydrophilic metabolite in the presence of 1% Triton X-100 and 20 mM CaCl2. As will be demonstrated below, this
product, designated metabolite A in Fig. 2, corresponds to a
deacylated derivative of lipid IVA specifically lacking the 3-O-linked
-hydroxymyristoyl residue (Fig.
1B). Membranes of the
nodulation-deficient mutant 24AR of R. leguminosarum biovar trifolii, which were previously shown to lack the 4'- and
1-phosphatases of the R. leguminosarum lipid A pathway (20),
do contain this deacylase. A similar deacylase is also detected in cell
extracts and membranes (Fig. 2) of two
wild type strains of P. aeruginosa (PA1O and 27853).
However, deacylation of [4'-32P]lipid IVA is
not observed in extracts or membranes (Fig. 2) of either E. coli or R. meliloti, as judged by comparison with the
no enzyme control.

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Fig. 1.
Structures of lipid A from three diverse
Gram-negative bacteria and their relationship to the conserved
precursor lipid IVA. A, predominant species
of lipid A found in E. coli K-12 (3), R. leguminosarum (12), and P. aeruginosa (25, 26). The
presence of an acyloxyacyl moiety involving the C28 acyl chain and the
3-O-deacylated forms of R. leguminosarum lipid A
was discovered recently in our laboratory based on new isolation
techniques (14, 15). Molecular species of R. leguminosarum
and P. aeruginosa lipid A may differ by the presence or
absence of a hydroxyacyl chain at position 3, as indicated by the
dashed bond. B, proposed reaction
catalyzed by the 3-O-deacylase of R. leguminosarum with lipid IVA as the substrate. Key
hydrogen atoms used to assign the structure of the product by
1H NMR spectroscopy are labeled in this
representation.
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Fig. 2.
Deacylation of [4'-32P]lipid
IVA in membranes of R. leguminosarum and
P. aeruginosa but not of E. coli or
R. meliloti. Membranes of the indicated strains were assayed
for deacylase activity using the standard conditions. The protein
concentration was 1.0 mg/ml, and the incubations were carried out for
60 min at 30 °C. The products generated from
[4'-32P]lipid IVA were separated by thin
layer chromatography and detected with a PhosphorImager.
Lanes 1, no membrane control; lane
2, E. coli W3110; lane 3,
R. leguminosarum biovar phaseoli CE3;
lane 4, R. leguminosarum biovar
viciae 8401; lane 5, R. leguminosarum biovar trifolii 24AR; lane
6, R. meliloti 1021; lane
7, P. aeruginosa PAO1; lane
8, P. aeruginosa 27853.
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R. leguminosarum biovar viciae 8401 was used as
the source of the enzyme in all subsequent experiments. Strain 8401 (20) lacks the pSym plasmid that carries many of the nodulation genes. All of the deacylase activity in R. leguminosarum 8401 extracts is membrane-associated (Fig. 3).
Similarly, in P. aeruginosa PAO1 and 27853, all of the
deacylase also is membrane-bound (not shown).

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Fig. 3.
Deacylase activity is associated with the
membrane fraction of R. leguminosarum. Deacylase
was assayed under standard conditions with 10 µM
[4'-32P]lipid IVA and crude extract, cytosol,
or membranes as the enzyme source. Reactions were analyzed after the
indicated times by thin layer chromatography and PhosphorImager
analysis, as described under "Experimental Procedures".
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Assay and Catalytic Properties of the R. leguminosarum
Deacylase--
Deacylation of [4'-32P]lipid
IVA by R. leguminosarum membranes proceeds in a
linear fashion for up to 4 h at 30 °C with 1.0 mg/ml protein
(Fig. 4A). After prolonged
incubation, deacylation is nearly complete. Deacylation activity also
increases with increasing membrane protein concentrations, but the
effect is not linear above 0.5 mg/ml (Fig. 4B), perhaps
reflecting the presence of inhibitors in R. leguminosarum
membranes. Optimal deacylase activity is observed between pH 5.5 and
6.5 (not shown).

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Fig. 4.
Time course and protein concentration
dependence of the R. leguminosarum deacylase.
A, membranes of R. leguminosarum 8401 were used
at 1 mg/ml. The deacylase reaction was performed under standard
conditions in 30 µl. At each time point, a 2-µl portion was
withdrawn and analyzed by thin layer chromatography and PhosphorImager
analysis. B, the deacylase reaction was performed under
standard conditions in 10 µl. Membranes of R. leguminosarum 8401 were used as the enzyme source at the indicated
protein concentrations. Reaction mixtures were incubated for 20 or 40 min at 30 °C.
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The deacylation reaction is absolutely dependent upon the presence of a
nonionic detergent. Maximal activity is seen in the presence of 1%
Triton X-100 at membrane protein concentrations below 2 mg/ml. Nonidet
P-40 (1% also) supports deacylase activity, but Tween 20, deoxycholate, CHAPS, and dodecylmannoside are inhibitory (not shown).
The presence of EDTA or EGTA at 2 mM completely inhibits
the deacylase activity, suggesting a divalent metal requirement. Accordingly, the deacylase was assayed in the presence of varying concentrations of calcium, magnesium, or manganese ions (Fig. 5). In this experiment, the concentration
of EDTA was held constant at 2 mM. Among these three
divalent metal ions, calcium is the most effective. Enzymatic activity
increases with increasing concentrations of calcium ions up to 20 mM. Partial stimulation of the activity is also seen with
magnesium or manganese chloride (Fig. 5). However, 5 mM
ferrous and zinc ions completely inhibit the activity (not shown), and
monovalent cations (like sodium and potassium) have no effect.

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Fig. 5.
Dependence of the deacylase reaction on
divalent metal ions. The deacylase was assayed under standard
conditions, except that the indicated concentration of
CaCl2, MgCl2, or MnCl2 was added
together with 2 mM EDTA to the reaction mixtures. R. leguminosarum 8401 membranes were used at 1.0 mg/ml. Reaction
mixtures were incubated for 40 min at 30 °C, after which product
formation was analyzed by thin layer chromatography and PhosphorImager
analysis.
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Under standardized assay conditions with R. leguminosarum
8401 membranes, partial dephosphorylation of the 4'-phosphate of lipid
IVA by the 4'-phosphatase is observed in parallel with
deacylation as judged by the appearance of 32Pi
(Fig. 2, lane 4), but the 4'-phosphatase is
partially inhibited by the presence of 20 mM
CaCl2 and 1% Triton X-100. Since Kdo2-lipid IVA is strongly preferred over lipid IVA by the
4'-phosphatase (20), lipid IVA was generally employed for
the deacylase assay. The 1-phosphatase of the R. leguminosarum lipid A pathway (21) was inhibited completely by 20 mM CaCl2; therefore, its activity was not
apparent under the assay conditions used for the deacylase. Furthermore, the lipid product that is generated by the 4'-phosphatase (20) is not seen under these assay conditions, since it is not radioactive after the removal of the 4'-phosphate residue.
With 25 µM [4'-32P]lipid IVA
under optimized conditions, the specific activity of the deacylase in
crude extracts and membrane preparations of R. leguminosarum
8401 is 0.019 and 0.045 nmol/min/mg protein, respectively. The apparent
Km for lipid IVA in this mixed micelle
system (40) is estimated as 17.9 µM (Fig. 6).

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Fig. 6.
Effect of lipid IVA concentration
on deacylase specific activity. The deacylase was assayed under
standard conditions with membranes of R. leguminosarum 8401 at 1.0 mg/ml. The lipid IVA concentration was varied, as
indicated. Reaction mixtures were incubated for 40 min, after which
product formation was analyzed by thin layer chromatography and
PhosphorImager analysis. The inset shows a double reciprocal
plot of the same data.
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Substrate Specificity of the Deacylase--
As shown in Fig.
7, the deacylase utilizes lipid
IVA and Kdo2-lipid IVA at about the
same rate. Unlike the 4'-phosphatase, the deacylase is not dependent
upon the Kdo domain. However, the rate of deacylation of the
monosaccharide precursor lipid X is about 20 times slower than that of
lipid IVA (Fig. 7), indicating that the distal
diacylglucosamine unit of lipid IVA somehow enhances catalytic efficiency. The biosynthetic precursor
tetraacyldisaccharide-1-32P (3) and the analog
tetraacyldisaccharide-[4'-32P] (prepared by acid
hydrolysis of [4'-32P]lipid IVA) are both
deacylated efficiently (data not shown), indicating that both
phosphates are not required for efficient turnover. Extra core sugars
(mannose and galactose) (21, 24) attached to Kdo2-lipid
IVA do not interfere with deacylation. However, the
presence of an acyloxyacyl group on the distal glucosamine residue of
Kdo2-lipid IVA, as in
lauroyl-Kdo2-lipid IVA generated by HtrB (35)
or in lipid A generated from compound 505 by the 4'-kinase (30),
prevents deacylation (data not shown).

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Fig. 7.
Substrate specificity of the deacylase.
The deacylase was assayed under standard conditions with 10 µM [4'-32P]lipid IVA,
Kdo2-[4'-32P]lipid IVA, or
32P-lipid X (each at about 20,000 cpm/nmol). Membranes (1.0 mg/ml) of R. leguminosarum 8401 were used as the enzyme
source. Product formation was analyzed by thin layer chromatography and
PhosphorImager analysis.
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Chromatographic Characterization of the Deacylase Product Generated
from Lipid IVA--
To characterize the structure of the
material generated by the enzymatic deacylation of lipid
IVA, the reaction product was subjected to mild alkaline
hydrolysis. The substrate [4'-32P]lipid IVA
(not treated with enzyme) (Fig. 8) was
processed in parallel (see "Experimental Procedures"). Treatment
with TEA removes both ester-linked
-hydroxyacyl chains from lipid
IVA. When carried out at 30 °C, TEA hydrolysis of
[4'-32P]lipid IVA proceeds via two distinct
and separable intermediates (designated intermediates
1 and 2 in lanes 3 and
5 of Fig. 8). These eventually collapse to form the same
limiting alkaline hydrolysis product, which lacks both ester-linked
-hydroxyacyl chains (Fig. 8, lane 7).
Intermediates 1 and 2 arise by the loss of either the 3- or the
3'-
-hydroxyacyl chains of lipid IVA. Stronger base (NaOH) hydrolysis of lipid IVA (Fig. 8, lane
9) rapidly removes both O-linked
-hydroxyacyl
chains without accumulation of intermediates 1 and 2 under the
conditions employed.

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Fig. 8.
Mild alkaline hydrolysis and thin layer
analysis of [4'-32P]lipid IVA and of the
deacylase reaction product. As described under "Experimental
Procedures," two incubations (designated 1 and 2) were set up using
the standard optimized assay conditions with 10 µM
[4'-32P]lipid IVA as the substrate.
Incubation 1 contained no enzyme, while incubation 2 contained 1 mg/ml
R. leguminosarum 8401 membranes. After 16 h at
30 °C, portions of these incubations were treated with TEA or 0.1 M NaOH for the times indicated in the figure, after which a
portion of each treated sample was analyzed by thin layer
chromatography and PhosphorImager analysis.
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The more rapidly migrating intermediate 1 derived by TEA treatment of
[4'-32P]lipid IVA (Fig. 8, lanes
3 and 5) is the same as the product made by the
R. leguminosarum deacylase (Fig. 8, lane
2, metabolite A). Consequently, treatment of the deacylase
reaction product with TEA (Fig. 8, lanes 4,
6, and 8) or with NaOH (Fig. 8, lane 10) results in the direct conversion of the deacylase
reaction product to a compound that migrates with the limiting NaOH
hydrolysis product of [4'-32P]lipid IVA. This
finding demonstrates that the deacylase does not remove an
N-linked
-hydroxyacyl chain from lipid IVA,
and it also shows that the deacylase removes only one specific
O-linked
-hydroxyacyl chain from
[4'-32P]lipid IVA. Last, it can be inferred
that the glucosamine disaccharide backbone structure of the deacylase
reaction product is likely to be the same as that of lipid
IVA.
1H NMR Spectroscopy of the Deacylase Reaction Product
Generated from Lipid IVA--
A definitive assignment of
the position (3 or 3') that is attacked by the deacylase in lipid
IVA cannot be made by thin layer chromatography analysis
alone. Accordingly, a 2-mg sample of the deacylase reaction product was
isolated and subjected to 1H NMR spectroscopy under
conditions reported previously (41).
Characterization of the purified reaction product by COSY spectroscopy
established unequivocally that lipid IVA is deacylated by
the enzyme exclusively at the 3-position. While the 1H NMR
spectrum of the substrate, lipid IVA (not shown), displays two downfield overlapping triplets at 5.18 ppm, which are attributed to
H-3 and H-3' of the acylated glucosamine disaccharide (42, 43), the
deacylase product retains only one of these two triplets at 5.17 ppm
(Fig. 9). Based on the COSY spectrum of
the deacylase product, this remaining downfield triplet is assigned to
an axial H-3' (Fig. 9), indicating that the 3'-position is still
acylated in the product (33, 45). However, a new triplet at 3.69 ppm (Fig. 9), which is not detected in the substrate lipid IVA
(42, 43), was observed in the product (Fig. 9). By tracing the
cross-peaks of the COSY spectrum (Fig. 9), the new resonance at 3.69 ppm could be assigned to the glucosamine H-3 of the product, which is
shifted upfield by about 1.5 ppm compared with lipid IVA,
because of the loss of the 3-O-acyl moiety. Deacylation at
the 3-position was also indicated by the slight (~0.2 ppm) upfield
shifts of the H-2 and H-4 signals of the product in comparison with the
H-2 and H-4 resonances of lipid IVA (42, 43). Thus, the
deacylase selectively removes the ester-linked
-hydroxymyristate
chain at the 3-position of the proximal glucosamine unit of lipid
IVA.

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Fig. 9.
Partial COSY spectrum of the deacylase
reaction product generated from lipid IVA. The
one-dimensional spectrum in the region of the relevant carbohydrate
proton resonances is shown along the edges. Proton
cross-peak assignments are indicated, and the atom labeling of the
glucosamine disaccharide protons follows the numbering scheme shown in
Fig. 1B. The distal glucosamine 1H chemical
shifts of the deacylase product are nearly the same as those in the
lipid IVA substrate (43). The most pronounced difference
between lipid IVA and the deacylase reaction product is
seen with H-3 of the proximal glucosamine residue, which is shifted
upfield by 1.5 ppm relative to H-3 of lipid IVA (43)
because of the loss of the acyl chain.
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DISCUSSION |
In previous studies, we described six enzymes unique to R. leguminosarum extracts that convert Kdo2-lipid
IVA (20-24), an intermediate made both by E. coli and R. leguminosarum, to novel compounds that are
precursors of the unusual lipid A of R. leguminosarum (Fig.
1). We have now identified a novel deacylase specific to R. leguminosarum membranes that selectively removes the
O-linked
-hydroxyacyl chain from the 3-position of
Kdo2-lipid IVA as well as from the precursors
lipid IVA and lipid X (Figs. 1, 7, and 9). This
membrane-associated deacylase is present in extracts of all R. leguminosarum strains tested so far, but not of E. coli or R. meliloti (Fig. 2).
Although the deacylase is not dependent upon the Kdo domain of
Kdo2-lipid IVA for activity, it displays a
strong kinetic preference for substrates containing a distal
diacylglucosamine unit (Fig. 7). However, the enzyme selectively
cleaves only the
-hydroxymyristoyl moiety attached to position 3 of
the proximal glucosamine moiety (Figs. 1 and 9). 1H NMR
spectroscopy (Fig. 9) provided unequivocal evidence for the specificity
of the enzyme, as demonstrated by the 1.5-ppm upfield shift of the H-3
of the proximal glucosamine residue in the product compared with the
substrate. The NMR spectrum also revealed that the proton chemical
shifts of the distal glucosamine unit of the product were virtually
unchanged relative to those of lipid IVA (43).
The deacylase does not require the presence of a phosphate group at
either the 1- or the 4'-position for activity, and the attachment of
the inner core sugars (mannose and galactose) to Kdo2-lipid
IVA does not interfere with the deacylation reaction (data
not shown). Interestingly, the enzyme does not deacylate lauroyl-Kdo2-lipid IVA (in which an acyloxyacyl
group is present on the N-linked hydroxymyristate group of
the distal glucosamine unit) (35). These findings suggest that the
deacylase functions after lipid A disaccharide formation but before the
Kdo-dependent acylation of the distal unit to generate an
acyloxyacyl residue (3). The observation that the deacylase strongly
prefers glucosamine disaccharides as substrates (Fig. 7) is reasonable
given that the disaccharide synthase of E. coli (34) is
highly specific for diacylated monosaccharide precursors, like lipid X. If the deacylase were to catalyze rapid cleavage of these
monosaccharide precursors, it would interfere with the functioning of
the disaccharide synthase.
Like many of the lipases that deacylate glycerophospholipids (46), the
R. leguminosarum deacylase requires Triton X-100 and
divalent metal ions for activity (Fig. 5). Whether or not the R. leguminosarum deacylase also utilizes glycerophospholipids as
substrates can only be assessed once the enzyme is purified to
homogeneity. However, the remarkable specificity of the deacylase for
the 3-position of lipid IVA argues against a more general role for this enzyme as a phospholipase. Furthermore, when commercially available lipases from Rhizopus and Pseudomonas
or bovine pancreatic phospholipase A2 was tested under the conditions
optimized for the R. leguminosarum deacylase (or under their
own optimal conditions for the hydrolysis of glycerophospholipids)
(46), no deacylation of [4'-32P]lipid IVA was
observed (data not shown).
The present work therefore represents the first description of a lipase
from a procaryotic organism that deacylates lipid A precursors. While
several deacylases that attack certain lipid A-like molecules have been
reported in eucaryotic systems, these appear to be distinct from the
R. leguminosarum deacylase. For instance, the acyloxyacyl
hydrolase of human leukocytes (47) removes the secondary acyl chains
from the lipid A residues of intact lipopolysaccharide, thereby
reducing the immunostimulatory activity and the toxicity of the lipid A
moiety. Since the R. leguminosarum deacylase does not attack
Kdo2-(lauroyl)-[4'-32P]lipid IVA,
it is obviously not an acyloxyacyl hydrolase (data not shown). Rosner
et al. (48) and Verret et al. (49, 50) identified
two distinct amidases in extracts of the slime mold, Dictyostelium discoideum, an organism that is likely to
scavenge E. coli in nature. These enzymes remove the two
amide-linked
-hydroxymyristoyl residues of lipid A, but only after
complete O-deacylation by prior base treatment. Amidase I of
D. discoideum is inhibited by chitobiose and
N-acetyl-
-glucosamine 1-phosphate (49, 50). The R. leguminosarum deacylase does not cleave either of the amide-linked acyl chains (Fig. 8), and it is not affected by the above compounds up
to 1.0 mM (data not shown). The specificity of our
deacylase is thus completely different from those of the previously
reported lipid A hydrolases (49, 50) of D. discoideum.
Finally, Drozanski et al. (51) reported deacylation of
lipopolysaccharide in Acanthamoeba castellanii crude
extracts with release of both O-linked and
N-linked acyl chains. These early studies were carried out
without the benefit of structurally defined substrates. The putative
enzymes described by Drozanski et al. (51) were not further
characterized. Their specificity and function remain unclear.
The physiological function of the R. leguminosarum deacylase
is not yet known. Elucidation of its biological roles will require genetic studies. It is very likely that the R. leguminosarum
deacylase generates the lipid A species lacking the
3-O-linked
-hydroxyacyl chain that we have recently
identified in R. leguminosarum (14, 15) (Fig.
1A). The related deacylase found in P. aeruginosa membranes (Fig. 2) may likewise explain the structure of the
predominant pentaacylated form of lipid A present in P. aeruginosa lipopolysaccharide (25, 26), in which the
3-O-linked
-hydroxydecanoate group is missing in the
proximal glucosamine unit. Mass spectrometry of lipid A variants
isolated from diverse Gram-negative bacteria suggests that enzymatic
cleavage of 3-O-linked
-hydroxyacyl chains may actually
be a widespread phenomenon (52, 53).
Many studies have confirmed the importance of the structure and
composition of the acyl chains attached to lipid A for biological activity in the stimulation of mammalian immune cells (1, 3, 4, 9). The
deacylase of R. leguminosarum may provide a new tool for the
selective modification and preparation of interesting lipid A analogs.
Given the structure-activity relationships of known lipid A
derivatives, one would expect that many of the unusual chemical
features of R. leguminosarum lipid A (Fig. 1A),
including the partial 3-O-deacylation, would reduce immune
stimulation in animal systems (4, 9).
The distinctive structure of R. leguminosarum lipid A and
its possible lack of immunostimulatory activity might also play a role
the establishment of symbiosis in plants (17, 18). Although not yet
characterized in terms of their ability to respond to lipid A, some
plants have recently been shown to possess systems of innate immunity
(54-56) and to synthesize antibacterial peptides in a manner that is
reminiscent of insects infected with bacteria or fungi (44). The
unusual lipid A of R. leguminosarum might therefore help
bacteroids evade the innate immune response of plants during symbiosis
in root cells, while still allowing the plant to defend itself against
Gram-negative pathogens containing the more typical, phosphorylated
lipid A disaccharide. Isolation of R. leguminosarum mutants
that specifically lack the 3-O-deacylase and the other
unique enzymes recently identified in our laboratory (20, 21, 24) will
be required to validate this hypothesis.