From the Department of Chemistry, College of
Environmental Science and Forestry, State University of New York,
Syracuse, New York 13210 and the ¶ Department of Biology, Virginia
Polytechnic Institute, Blacksburg, Virginia 24061
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dictyostelium discoideum (Amoebidae) secretes cell-lysing enzymes: esterases, amidases, and glycosylases, many of which degrade soil bacteria to provide a source of nutrients. Two of these enzymes, fatty-acyl amidases FAA I and FAA II, act sequentially on the N-linked long chain acyl groups of lipid A, the lipid anchor of Gram-negative bacterial lipopolysaccharide. FAA I selectively hydrolyzes the 3-hydroxymyristoyl group N-linked to the proximal glucosamine residue of de-O-acylated lipid A. Substrate specificity for FAA II is less selective, but does require prior de-N-acylation of the proximal sugar, i.e. bis-N-acylated lipid A is not a substrate. We have synthesized a 14C-labeled substrate analog for FAA II and used this in a novel assay to monitor its purification. Inhibitory studies indicate that FAA II is not a serine protease, but may have a catalytic mechanism similar to metalloprotein de-N-acetylases such as LpxC. Interestingly, rhizobial Nod factor signal oligosaccharides that induce root nodules on leguminous plants have many of the structural requirements for substrate recognition by FAA II. In vitro evidence indicates that Rhizobium fredii Nod factors are selectively de-N-acylated by purified FAA II, suggesting that the enzyme may reduce the N2-fixing efficiency of Rhizobium-legume symbioses. In contrast, N-methylated Nod factors from transgenic R. fredii carrying the rhizobial nodS gene were resistant to FAA II, suggesting a mechanism by which Nod factors may be protected from enzymatic de-N-acylation. Since FAA II and Nod factors are both secreted, and Nod factors that lack the N-acyl group are unable to induce nodules, dictyostelial FAA II may decrease the efficiency of symbiotic nitrogen fixation in the environment by reducing the available biologically active nodule inducer signal.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dictyostelium discoideum secretes hydrolytic enzymes that degrade bacterial cell walls, enabling it to utilize soil-borne bacteria as a source of nutrition. Many of these enzymes have been implicated in the degradation of bacterial lipopolysaccharide, which is found exclusively in the cell walls of Gram-negative bacteria (1, 2). Two enzymes, fatty-acyl amidases FAA1 I and FAA II, are of particular interest and play a concerted role in the deacylation of lipid A (3), a molecule that anchors lipopolysaccharide to the bacterial outer membrane (Fig. 1). FAA I is highly specific in its substrate requirements and selectively hydrolyzes N-linked fatty acids attached to the proximal sugar of de-O-acylated lipid A (1). This monodeacylated lipid A is then further hydrolyzed by the action of FAA II, releasing fully deacylated lipid A oligosaccharide (1, 3). FAA II is less specific than FAA I, but requires that the lipid A substrate is mono-N-acylated only on the distal sugar and that the fatty acid is longer than three carbons and 2-N-linked to a glucosaminyl residue. There is no requirement for the fatty acid to be 3-hydroxylated (as it is in lipid A) or for the glucosaminyl moiety to be phosphorylated or glycosidically linked (1). Consequently, the minimum structures for substrate recognition by FAA II are 2-N-acylamino-2-deoxy-D-glucopyranoses.
|
Nitrogen-fixing bacteria of the genus Rhizobium secrete Nod factor signal molecules that induce symbiotic root nodules on certain leguminous plants (4). Significantly, these Nod factor lipochito-oligosaccharides have many of the structural requirements for substrate recognition by FAA II (see Fig. 3). The long chain fatty acid moiety (typically C16-C18) is 2-N-linked to the distal glucosamine residue of Nod factors and, unlike bis-N-acylated lipid A, is not blocked by further acylation of the oligosaccharide core. Nod factor formation proceeds by NodC-catalyzed biosynthesis of chito-oligosaccharides, which are subsequently modified by the presence of host-specificity nodulation functions. The N-acyl chain is a common feature of all Nod factor structures described to date and is essential for their biological activity (5, 6). However, certain Nod factor structures are modified at the N-acylated terminus by conjugated double bonds (7, 8) or by N-methylation of the nonreducing residue (9-12). This N-methylation is catalyzed by NodS protein that is present in Rhizobium sp. NGR234, Rhizobium etli, and certain Bradyrhizobium species, but not in Rhizobium fredii USDA257 (13). Potentially, N-methylation or the presence of conjugated polyunsaturation may stabilize the acyl amide bond and therefore protect Nod factors against degradation by FAA II.
We have synthesized N-palmitoyl[1-14C]glucosamine (GlcN-C16) as a synthetic substrate and found it to be readily de-N-acylated by the action of FAA II. This has been utilized in a novel assay for FAA II and used to purify the protein to homogeneity from the culture medium of a Dictyostelium nagA knockout (14). Susceptibility to various inhibitors suggests that FAA II has mechanistic similarities to zinc-containing acetamidases, such as the 3-O-acyl-GlcNAc de-N-acetylase LpxC (15), rather than to serine proteases. The activity of purified FAA II has also been assessed on metabolically radiolabeled Nod factors from R. fredii USDA257, and the results indicate that these also become de-N-acylated. Since Nod factors lacking the N-acyl chain are biologically inactive, the presence of FAA II in soil may reduce the ability of Rhizobium to nodulate host leguminous plants. Interestingly, Nod factors from a transconjugate of USDA257 expressing the nodS N-methyltransferase gene from Rhizobium sp. NGR234 are found to be more resistant to FAA II, suggesting that N-methylation may protect Nod factors against deacylation by Dictyostelium in soil.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials-- D. discoideum strain Ax-3 (ATCC 28368) was obtained from American Type Culture Collection (Rockville, MD). The nagA-deleted strain (HL101) was obtained from William Loomis (University of California, San Diego, CA). Rhizobium strains were obtained from Steve Pueppke (University of Missouri, Columbia, MO). Radiolabeled D-[1-14C]glucosamine (specific activity of 45-60 mCi/mmol) and sodium [2-14C]acetate (540 mCi/mmol) were obtained from NEN Life Science Products. Serine protease inhibitors were obtained from Sigma, as were buffers, chemicals, and chromatography supplies. Kodak X-Omat AR-5 was purchased from S & W X-Ray (Syracuse, NY). Buffer A contained 20 mM Tris-HCl (pH 7.8) plus 0.1 mM EDTA, and Buffer B contained 5 mM sodium acetate (pH 4.0).
Synthesis of the Radiolabeled Substrate
N-Palmitoyl[1-14C]glucosamine--
[1-14C]Glucosamine
(0.5 µl, 50 nCi) was spotted at the origin on a normal-phase Silica
Gel 60 thin-layer chromatography plate, dried under a stream of warm
air, and overspotted with palmitoyl chloride (2 µl). After an 18-h
reaction at room temperature, the plate was developed with chloroform,
methanol, and aqueous ammonium hydroxide (50:40:5 by volume). Untreated
[1-14C]glucosamine was run in a control lane. The plate
was dried until free of solvent and autoradiographed for 2 days.
Radiolabeled N-palmitoylglucosamine was recovered from the
plates by extraction with methanol. Following scintillation counting,
aliquots were evaporated to dryness in Eppendorf tubes and stored at
20 °C.
Isolation and Purification of FAA II--
Culture supernatant
(14 liters) from D. discoideum strain Ax-3 was concentrated
by passage through a 30-kDa cutoff Amicon filter, and the concentrated
supernatant (1 liter) was stirred overnight with DEAE-cellulose (50 g,
4 °C). The ion exchanger was recovered by filtration through a
cotton pad, washed with NaCl (150 mM) in cold Buffer A (125 ml), and eluted with NaCl (1 M) in Buffer A (187 ml).
Following precipitation with ammonium sulfate (70%), and
centrifugation (15,000 × g, 15 min, 4 °C), the
protein pellet was redissolved in Buffer B (3 ml) and dialyzed overnight against Buffer B. It was further fractionated on a
DEAE-cellulose column (20 × 1 cm) as follows: 1) washed with
Buffer B (5 bed volumes) and 2) eluted with a gradient of NaCl
(150-500 mM) in Buffer B. Fractions were assayed for
protein using the BCA assay (16) and for FAA II activity as described
below. Active fractions were stored at 20 °C prior to purification
by high performance liquid chromatography.
High Performance Liquid Chromatography (HPLC)-- The HPLC system consisted of two Waters 512 pumps, a Waters 486 UV detector, and a U6K injector. Reverse-phase chromatography was achieved on a Brownlee Spheri-5 RP-18 column (250 mm, 5-µm particle size) in series with a Waters RP-18 column (150 mm, 5-µm particle size). A gradient from 0.01% trifluoroacetic acid in 10% aqueous acetonitrile to 0.05% trifluoroacetic acid in 100% acetonitrile was run for 30 min at 0.6 ml/min. Gel-filtration HPLC was done on a Phenomenex Biosep-Sec-S-3000 column (300 × 7.8 mm) protected by a Phenomenex Biosep-Sec-S guard column (7.5 × 7.8 mm) using a mobile phase of 50 mM sodium phosphate buffer (pH 6.8; 1 ml/min). In either case, detection was achieved by absorbance at 210 nm. A standard protein ladder was run as a molecular mass marker. Fractions were collected from the columns and assayed for protein and FAA II activity. SDS-polyacrylamide gel electrophoresis was run as per Laemmli (17).
Enzyme Assay and Inhibition Study of the Amidase FAA II-- Enzyme assays consisted of N-palmitoyl[U-14C]glucosamine (a minimum of 10 nCi/assay) and protein (1-10 mg/ml) in 0.2% Triton X-100 in Buffer B at a final volume of 100 µl. The reaction was allowed to proceed to completion (18 h, 26 °C), after which it was stopped by the addition of aqueous NaCl (10% (w/v), 100 µl). Unreacted substrate was extracted by partitioning with ethyl acetate/hexane (90:10 (v/v), 300 µl). Following microcentrifugation, radioactivity was assessed in the upper solvent layer (200 µl) and lower aqueous layer (100 µl) by scintillation counting. A minimum of three replications was analyzed per assay. Controls were identical, but lacked protein exudate. Stock solutions of the inhibitors phenylmethylsulfonyl fluoride (50 mM) and EDTA (1 M) were prepared in acetonitrile and Buffer B, respectively, and, when used, were added prior to the protein addition.
The FAA II assay was modified for testing on Nod factors so that activity could be monitored by TLC/autoradiography. Metabolically radiolabeled Nod factors (typically 30 nCi) were incubated with FAA II (10 mg/ml, 18 h, 30 °C) in Buffer B (30 µl) containing 0.2% Triton X-100. Control reactions lacked the enzyme. The reaction was stopped by the addition of methanol (50 µl), and the precipitated protein was pelleted by brief centrifugation (15,000 rpm, 15 min). Aliquots of the methanolic supernatant (30 µl) were separated on silica TLC plates eluted with butanol/ethanol/water (5:3:2 by volume). Plates were air-dried and exposed to x-ray film for 6 days.Preparation and Testing of Metabolically 14C-Labeled
Nod Factors--
Rhizobia were grown aerobically at 30 °C on
rhizobial minimal medium (RMM) in the presence of succinate (12 mM) and glutamate (6 mM) as described
previously (11). Cultures (10 ml) were radiolabeled by the inclusion of
[1-14C]glucosamine (40-60 mCi/mmol, 1 µCi/ml) at the
start of the growth period. After 4 h of growth
(A550 = 0.3), the nod genes were
induced by the addition of the species-specific flavonoid apigenin
(5 × 104 M). After 18 h, the
bacteria were removed by centrifugation (10,000 × g,
4 °C), and radiolabeled Nod factors were recovered from the culture
supernatant by solid-phase extraction with
C18-functionalized silica (Waters) (18). After washing with
water (5 ml), radiolabeled metabolites were eluted with methanol and
evaporated to dryness.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Substrate Specificity and Development of an Enzyme Assay-- We prepared and purified, by thin-layer chromatography with autoradiographic detection (Fig. 2), a radiolabeled synthetic substrate for FAA II, N-palmitoyl[1-14C]glucosamine (Fig. 3). Conditions were optimized for two-phase partitioning of N-palmitoyl[1-14C]glucosamine from the free sugar [1-14C]glucosamine, making use of ethyl acetate/hexane (90:10, v/v) and aqueous sodium chloride (10%, w/v). This allowed selective recovery of the radiolabeled monosaccharide in the aqueous layer. Thus, after treatment of N-palmitoyl[1-14C]glucosamine with FAA II in aqueous Buffer B and the addition of sodium chloride (to 10%, w/v), selective counting of the aqueous and organic phases is an indication of the reaction progress. The salt also served the dual purpose of stopping the reaction. Solvent extraction of radiolabeled GlcN-C16 was optimized such that 98% of the radioactivity from the enzyme-free control partitioned into the solvent phase (Table I). Following treatment with FAA II (10 mg/ml, 18 h, 30 °C), 35.4% of the counts remained in the aqueous buffer as deacylated [1-14C]glucosamine. The degree of activity in secreted dictyostelial extracts was monitored, allowing for complete purification and a mechanistic study of FAA II.
|
|
|
Purification and Mechanistic Studies of FAA II-- Purification was undertaken on a secreted fraction from the culture supernatant of Dictyostelium strain Ax-3 after passage through a 30-kDa cutoff filter. Activity was monitored by the two-phase partitioning assay. Enzyme assays were typically run at 10 mg/ml total protein, giving a 56.3% decrease in counts in the organic phase relative to controls after a 18-h reaction time. A concomitant increase in counts was recorded in the aqueous extraction buffer. Lower concentrations of FAA II or shorter reaction times resulted in decreased activity. Final purity of the enzyme was ascertained by SDS-polyacrylamide gel electrophoresis and reverse-phase HPLC (Fig. 4). Verret et al. (2) were unable to fully purify FAA II free from acetylhexosaminidase A (NagA), but estimated its relative mass to be in the range of 60-80 kDa. Here the molecular mass of FAA II was determined by gel electrophoresis and gel-filtration HPLC. The protein eluted on gels with a molecular mass of 66,000 (Fig. 4). By gel filtration, the same purified fraction eluted as a single peak, but in this case, the relative molecular mass was estimated at 32,985 Da.
|
FAA II Selectively Deacylates Rhizobial Nod Factors-- Rhizobial Nod factors were radiolabeled metabolically by culturing rhizobial strains on minimal medium in the presence of precursor [1-14C]glucosamine and apigenin, an appropriate flavonoid nod gene inducer (18). Alternatively, optimal incorporation of radiolabel from acetate was attained by incubation with sodium [2-14C]acetate as the sole carbon source prior to chasing with succinate (12 mM) and glutamate (6 mM). The secreted hydrophobic fractions containing the Nod factors were separated from excess radiolabeled precursor and other labeled polar metabolites on a reverse-phase cartridge. The induction of Nod factors, as assessed by TLC/autoradiography, was clearly dependent on the presence of apigenin (Fig. 5). Inducible Nod factor bands of similar RF were revealed by acetate labeling, which, when overspotted, coeluted with the inducible [14C]glucosamine-labeled band.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fatty-acyl amidases play a concerted role in the deacylation of
bacterial lipid A (Fig. 1) by selective removal of the
N-linked fatty acid chains. The presumed natural substrate
for FAA II is generated by degradation of de-O-acylated
lipid IVA by FAA I to form
4-phosphoryl-N--hydroxymyristoyl-D-glucosaminyl-
-1,6-glucosamine 1-phosphate. Removal of the O-acyl chains and the
2-deoxyketooctulosonic acid residues is presumed to occur prior to this
due to the action of other secreted hydrolases (3). Interestingly,
neither amidase requires the C-1 or C-4' phosphate group for substrate
recognition, and although FAA II requires a deacylated proximal sugar,
the positive charge on the free 2-amino group is not obligatory since 2-N-formylation does not significantly inhibit FAA II (1). For many natural endotoxins, the distal fatty acid amide is
-3-hydroxylated, although the 3-hydroxy group is also unnecessary
for substrate recognition by FAA II. Furthermore, the
-1,6-glycosidic linkage is also nonessential, indicating that
N-acyl-D-glucosaminyl monosaccharides are
appropriate as alternate substrates.
Based on this information, we synthesized
N-palmitoyl-D-[1-14C]glucosamine
as a radiolabeled substrate for FAA II. This was prepared by selective
acylation of D-[1-14C]glucosamine HCl with
palmitoyl chloride and was subsequently used in a novel radioenzyme
assay to purify FAA II to homogeneity. The radiolabeled substrate and
the depalmitoylated product were effectively separated by solvent
partitioning (Table I). Using this enzyme assay, a five-step
purification procedure afforded purified FAA II. Partially purified
fractions also contained secreted -N-acetylhexosaminidase
A, the product of the nagA gene. Because
-N-acetylhexosaminidase A might degrade
-1,4-glucosidic linkages, such as are found in Nod factors, it was
important that this activity was removed, particularly since the NagA
protein co-migrated with FAA II both on reverse-phase HPLC columns and
by SDS gel electrophoresis. To circumvent these problems, we made use
of a nagA-deleted strain of Dictyostelium strain
Ax-3, HL101 (14), such that subsequent protein purifications were made
from the culture medium of this knockout strain. Consequently and in
addition to earlier work, we were able to purify FAA II to homogeneity
free from
-N-acetylhexosaminidase A. Interestingly, a
large discrepancy was found for the relative molecular mass of FAA II
as obtained by SDS gel electrophoresis (66 kDa) or size-exclusion HPLC
(33 kDa). This may be attributed to post-translational modification of
FAA II particularly if polyanionic groups such as sulfate or methyl
phosphate predominate, as has been found for other secreted
dictyostelial proteins (21, 22).
The enzyme mechanism of FAA II has not been studied previously, although the activity is suggestive of either a serine (or cysteine) protease or a zinc-containing metalloprotein. Serine proteases have a bimolecular mechanism that proceeds via a covalent acyl-enzyme intermediate. Thus, for FAA II, this would suggest a two-step displacement involving the sugar moiety followed by the acyl group. By contrast, a one-step metalloprotein mechanism, such as for carboxypeptidase A, would initially give rise to a noncovalent zinc-acyl transition state in which the acyl carbonyl oxygen is coordinated to the zinc atom. Significantly, the protease activity of carboxypeptidase A is selective for the N-linked terminal amino acid, analogous to the sugar moiety of GlcN-C16, but is nonselective with regard to the peptide chain. To test these mechanisms, we assayed FAA II in the presence of various protease inhibitors. FAA II was unaffected by either a serine protease inhibitor (phenylmethylsulfonyl fluoride) or a sequestering agent (EDTA). The latter was surprising in view of previous work involving the bacterial lipopolysaccharide deacetylase LpxC (15). LpxC is a lipid A biosynthetic enzyme that de-N-acetylates the substrate UDP-3-O-acyl-GlcNAc in a reaction analogous to FAA II amide hydrolysis. LpxC is a zinc-containing metalloprotein and has been assigned to a unique class of metalloamidases. Although LpxC from Pseudomonas aeruginosa is sensitive to inhibition by EDTA, the analogous protein from Escherichia coli is unaffected presumably because the zinc atom is buried within the protein (15). FAA II is an extracellular protein that most probably requires additional stability to maintain a folded conformation. It too may have an inaccessible buried metallo-center so that enzymatic activity is retained in soil.
We have shown that Nod factors are selectively de-N-acylated
by the action of the fatty-acyl amidase FAA II. Since non-acylated Nod
factors lack nodule-promoting biological activity, deacylation may well
have an effect upon the ability of rhizobia to nodulate host plants.
Moreover, we propose that Nod factors that are modified by the presence
of conjugated double bonds (such as those from Rhizobium
meliloti and Rhizobium leguminosarum) or by
N-methylation of the nonreducing residue (such as, for
example, Rhizobium NGR234) may be altered in their
sensitivity to enzymatic de-N-acylation by FAA II.
Consequently, these modifications may protect Nod factors against
degradation by D. discoideum in soil. Truchet et
al. (23) have shown that nodulation efficiency is
concentration-dependent and that nodule induction does not
occur below a threshold Nod factor concentration of
~107 M, even though a legume root hair
deformation response is observed at far lower concentrations. FAA II
de-N-acylation of Nod factors may therefore reduce the
efficiency of legume nodulation by reducing the concentration of
available biologically active Nod factors. Since Nod factors and FAA II
are both secreted by soil-dwelling Rhizobium and
Dictyostelium species, respectively, Nod factor degradation
may also occur in soils by this mechanism, decreasing the efficiency of
nitrogen-fixing Rhizobium-legume symbioses.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Margreet de Boer and Steve Pueppke for rhizobial strains, William Loomis for nagA-deleted Dictyostelium, and Sharon Long and Matt Anderson for criticism and discussion.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Contributed equally to this work.
Supported by National Institutes of Health Grant AG00678 and
Jeffress Memorial Trust Grant J-222.
** Supported by McIntire-Stennis Grant 210-L124B from the United States Department of Agriculture. To whom correspondence should be addressed. Tel.: 315-470-6858; Fax: 315-470-6856.
1 The abbreviations used are: FAA, fatty-acyl amidase; GlcN-C16, N-palmitoyl[1-14C]glucosamine; HPLC, high performance liquid chromatography; RMM, rhizobial minimal medium.
2 A. E. Tobin and N. P. J. Price, manuscript in preparation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|