Department of Biochemistry, Molecular
Biology, and Cell Biology, Northwestern University, Evanston,
Illinois 60208-3500
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INTRODUCTION |
The biosynthesis of
D-alanyl-lipoteichoic acid (LTA) requires the 56-kDa
D-alanine-D-alanyl carrier protein
ligase (AMP) (Dcl) and the 8.8-kDa D-alanyl carrier
protein (Dcp) (11, 12). Heaton and Neuhaus
(12) showed that D-alanyl-Dcp donates its
D-alanyl residue to the poly(Gro-P) moiety of
membrane-associated LTA. Neither the mechanism nor the topology of this
D-alanylation reaction is known. To address this key
reaction, several groups have identified the genes in a number of
organisms containing the dlt operon (11, 21,
23). In addition to the genes encoding Dcl (dltA) and Dcp (dltC), dltB and dltD encode a
putative transport protein (20) and a protein which
facilitates the binding of Dcl and Dcp for ligation with
D-alanine and has thioesterase activity for mischarged
D-alanyl acyl carrier proteins (ACPs) (5),
respectively. A gene encoding an enzyme which catalyzes the transfer of
the D-alanine residue from D-alanyl-Dcp to
membrane-associated LTA has not been identified.
Dcp provides the essential link between the ligase (Dcl) and the
incorporation of D-alanine into LTA. This carrier protein is a homologue of those ACPs which function in fatty acid biosynthesis and metabolism (4, 26). However, it was unexpected to find that Dcl will ligate D-alanine to ACPs from
Escherichia coli, Vibrio harveyi, Saccharopolyspora
erythraea, and Bacillus subtilis (12).
Nevertheless, only Dcp participates in the D-alanylation of
LTA. These observations suggested that there are at least two determinants for interaction of Dcp with its cognate partners, one of
which is recognized by Dcl and one of which is recognized by the
membrane acceptor LTA. It is this second determinant which is the focus
of structural studies on Dcp (B. F. Volkman, Q. Zhang, D. Debabov, E. Rivera, G. Kresheck, and F. C. Neuhaus, unpublished results), and the
biochemical studies to be reported here.
For Staphylococcus aureus, it was found that growth in the
presence of NaCl resulted in a lower D-alanine ester
content in LTA (8). The mechanism by which the ester
content was reduced during growth in NaCl is unknown and was not
correlated with the reaction catalyzed by DltD (5).
Instead, an NaCl-activated, thioesterase-like activity specific for
D-alanyl-Dcp which is distinct from that catalyzed by DltD
was discovered (5, 20).
Our goal here was to establish the mechanism of D-alanine
transfer from D-alanyl-Dcp to LTA. To accomplish this goal,
the thioesterase-like activity of LTA specific for
D-alanyl-Dcp was examined in incubations containing LTA in
different microenvironments: (i) purified, (ii) membrane associated,
and (iii) membrane associated (salt treated). The results suggested
that complex formation between D-alanyl-Dcp and LTA is one
of the features resulting either in the transfer of
D-alanine from D-alanyl-Dcp to LTA when this
amphiphile is membrane associated or in the hydrolysis of
D-alanyl-Dcp when the LTA is not membrane associated.
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MATERIALS AND METHODS |
Strains and growth of bacteria.
Lactobacillus
casei 102S was generously provided by Bruce Chassy (University of
Illinois). L. casei 102S dltD::cat
was from Debabov et al. (5). These strains were grown in
Lactobacilli MRS broth (Difco Laboratories).
Chemicals and reagents.
Purified samples of LTA prepared by
the procedure of Fischer et al. (9) were generously
provided by Werner Fischer (Universitat Erlangen-Nürnberg): These
samples were isolated from S. aureus DSM20231 (Ala/P 0.43),
B. subtilis JH542 (Ala/P 0.32), Enterococccus faecium MT9 (not purified by hydrophobic chromatography) (Ala/P 0.47; Glc/P 0.38; Glc2/P 0.05), Streptococcus
sanguis DSM 20567 (Ala/P 0.36), Enterococcus hirae (ca.
60% of Gro glycosylated Glc, Glc2, Glc3, and
Glc4), and Lactobacillus garvieae [glycosylated by Gal(
1-2)].
The concentrations of LTA are presented as micromolar concentrations in
phosphorus, and thus in the results the concentrations are designated
as micromolar concentrations of LTA-phosphorus. Since the poly(Gro-P)
chains of LTA are heterogeneous in length, it is not possible to
extrapolate LTA-phosphorus to micromolar concentrations of LTA. In the
case of S. aureus, the average number of Gro-P residues is
25, with a variation between 4 and 30 (7).
E. coli fatty acid synthase ACP and holo-ACP synthase were
generously provided by Ralph H. Lambalot, Roger S. Flugel, and Christopher T. Walsh (Harvard University) (16).
D-[14C]alanine (43 mCi/mmol) was the product
of ICN Biochemicals, Inc. Metricel filter membranes (GN-6) and
Econo-Safe scintillation cocktail were purchased from Gelman Sciences
and RPI Corp., respectively. Nanosep centrifugal concentrators (10K and
30K) were the products of Pall Filtron Corp.
Expression and purification of Dcp and Dcl.
Dcp was
expressed, purified, and converted to holo-Dcp using recombinant
holo-ACP synthase (4). The expression of Dcl and its
purification from inclusion bodies were as previously described (5, 11).
Preparation of D-[14C]alanyl-Dcp and
D-[14C]alanyl-ACP.
For the preparation
of either D-[14C]alanyl-Dcp or
D-[14C]alanyl-ACP, reaction mixtures
contained either 15 µM recombinant holo-Dcp or holo-ACP, 0.23 mM
D-[14C]alanine (43 mCi/mmol), 15 U of
recombinant Dcl, 30 mM bis-Tris (pH 6.5), 10 mM ATP, 10 mM
MgCl2, and 1 mM dithiothreitol (DTT). The mixture was
incubated at 37°C for 90 min, and proteins with a mass of >30 kDa
were separated from D-[14C]alanyl-Dcp or
D-[14C]alanyl-ACP with the Nanosep (30K)
concentrator. D-[14C]alanyl-Dcp or
D-[14C]alanyl-ACP in the filtrate was
desalted and separated from ATP, DTT, MgCl2, and
D-[14C]alanine using four cycles of
filtration with the Nanosep (10K) concentrator. During the course of
this process the buffer was changed to 5 mM sodium acetate (pH 4.5).
Preparation of membrane-associated
D-[14C]alanyl-LTA.
Membranes from
L. casei 102S and L. casei 102S
dltD::cat were prepared according to a
previous procedure (12) using a French pressure cell for
the disruption of bacteria. The incorporation of
D-[14C]alanine from
D-[14C]alanyl-Dcp into
membrane-associated D-[14C]alanyl-LTA was
carried out as previously described (4, 12) in 50 µl
with 100 µg of membranes prepared from Lactobacillus rhamnosus 102S and 5 nmol of recombinant
D-[14C]alanyl-Dcp (43 mCi/mmol). After
incubation for 90 min, the labeled membranes were separated from
D-[14C]alanyl-Dcp using Nanosep (30K)
centrifugal concentrators and used without further washing before
storage at 
80°C.
Assay of D-[14C]alanyl-Dcp and
D-[14C]alanyl-ACP.
D-[14C]alanyl-Dcp was determined by
precipitation with 10% trichloroacetic acid (TCA) and filtration
through 0.45-µm-pore-size Metricel filters, which retained 97% of
the radiolabeled carrier protein. The filters were dissolved in 3.5 ml
of ethyl acetate and assayed for radiolabel. In the hydrolytic
reactions of D-[14C]alanyl-Dcp in the
presence of LTA, the amount of D-alanine was expressed as
the difference between the added D-alanyl-Dcp and the
remaining D-alanyl-Dcp at the termination of the reaction.
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RESULTS |
Hydrolysis of D-alanyl-Dcp in the presence of LTA.
Heaton and Neuhaus (12) showed that
D-alanyl-Dcp donates its D-alanyl substituent
to membrane-associated LTA in the absence of Dcl. To simplify this
system, attempts were made to transfer D-alanine from
D-alanyl-Dcp to isolated, purified LTA in the absence of
membranes. However, instead of transacylation to LTA, incubation of
D-alanyl-Dcp with pure LTA resulted in the time-dependent
hydrolysis of D-alanyl-Dcp (Fig.
1). In contrast, D-alanyl-ACP
was not hydrolyzed in the presence of LTA. D-Alanyl-ACP, a
homologue of D-alanyl-Dcp, apparently does not provide the
necessary determinant for interaction with LTA for the cleavage of the
D-alanyl substituent. On the basis of these observations we
hypothesized that D-alanyl-Dcp forms a complex with the
poly(Gro-P) moiety of LTA and that within this complex a
thioesterase-like activity occurs.
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FIG. 1.
Hydrolysis of
D-[14C]alanyl-Dcp in the presence of LTA. The
reaction mixture contained 4.4 µM LTA-phosphorus from S. aureus, 8 µM D-[14C]alanyl-Dcp (6,700 cpm/nmol), and 30 mM bis-Tris (pH 6.5) in a total of 350 µl. Samples
(50 µl) were removed and added to 950 µl of 10% TCA. The amount of
radiolabeled D-alanyl-Dcp was determined as described in
Materials and Methods, and the amount of D-alanine released
was determined by subtraction.
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A variety of LTAs was examined in the hydrolysis reaction for
D-alanyl-Dcp (Table 1). Using
a fixed-time assay (5 min), we found that the most effective samples of
LTA are those which are nonglycosylated. Prior removal of the
D-alanyl ester residues from the LTA resulted in a 25%
increase of D-alanyl-Dcp hydrolysis activity (Table 1,
S. aureus and S. sanguis). The addition of 5 mM
MgCl2 to these reaction mixtures had no effect on the
velocity of D-alanine formation. For the studies presented
here, LTA with its D-alanine ester content of 0.43 from
S. aureus prepared by the method of Fischer et al.
(9) was used. Because of the polydispersity of LTA,
micromolar concentrations of LTA-phosphorus were used for comparisons.
The putative formation of a complex between LTA and
D-alanyl-Dcp implied that saturation kinetics should be
observed between D-alanyl-Dcp and LTA (Fig.
2). Increasing the concentrations of D-alanyl-Dcp gave saturation kinetics from which an
apparent Km of 7.5 µM for
D-alanyl-Dcp was established. The calculation of this value
assumes that the LTA has a fixed number of binding sites for
D-alanyl-Dcp. Since the velocity of
D-alanyl-Dcp hydrolysis is high relative to the estimated
concentration of LTA, it would appear that LTA turnover occurs. Thus,
while it is not clear how this catalysis is effected, it is apparent
that the reaction might be catalyzed by LTA and that
D-alanyl-Dcp is the substrate.
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FIG. 2.
Effect of increasing D-alanyl-Dcp on the
velocity of hydrolytic cleavage. The reaction mixture contained
increasing concentrations of
D-[14C]alanyl-Dcp in the presence of 4.4 µM
LTA-phosphorus in the reaction mixture described in the legend to Fig.
1. The mixtures were incubated for 5 min prior to termination with 10%
TCA, and the amounts of D-alanyl-Dcp remaining were
determined for calculating the initial velocities of
D-alanine released. In the inset a double-reciprocal plot
is presented from which Vmax (5.0 µM/min) and
Km (7.5 × 10 6 M) were
calculated.
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Increasing concentrations of LTA in the presence of a fixed
concentration of D-alanyl-Dcp gave a sigmoidal response
with a defined saturation curve (Fig. 3).
The Km for LTA is roughly 2 µM LTA-phosphorus.
This response implies a fixed number of sites in LTA for binding
D-alanyl-Dcp. Samples of glycosylated LTA gave lower
velocities of hydrolysis (Table 1) and, thus, it was concluded that the
glycosyl substituents have an effect on the interaction of
D-alanyl-Dcp and LTA. Since D-alanyl-ACP was
not hydrolyzed in this reaction (Fig. 3), it is concluded that ACP and
LTA do not form a complex.
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FIG. 3.
Effect of increasing LTA-phosphorus on the hydrolysis of
D-[14C]alanyl-Dcp. The reaction mixtures (50 µl) contained the indicated concentrations of LTA-phosphorus from
S. aureus, 8 µM
D-[14C]alanyl-Dcp (6,700 cpm/nmol), and
30 mM bis-Tris (pH 6.5). The amounts of D-alanine released
were determined as described in the legend to Fig. 1.
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If Dcp has a specific site for binding LTA, apo-Dcp will inhibit the
hydrolysis reaction while apo-ACP will not. As shown in Fig.
4, the addition of apo-Dcp to the
reaction mixture inhibited the hydrolytic cleavage of
D-alanyl-Dcp, while the addition of apo-ACP had no effect
on the cleavage. For comparison, it is observed that
D-alanyl-Dcp in the absence of LTA is stable in the
reaction mixture. Thus, apo-Dcp binds to LTA in an interaction that is not dependent on the phosphopantetheine prosthetic group.
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FIG. 4.
Inhibition of D-alanyl-Dcp hydrolysis.
The reaction mixture contained 4.4 µM LTA-phosphorus and 8 µM
D-[14C]alanyl-Dcp (6,700 cpm/nmol) in
the presence of either 85 µM apo-Dcp or 85 µM
apo-ACP. Samples were removed at the indicated times, and
the amounts of D-alanyl-Dcp or D-alanyl-ACP
were determined as described in Materials and Methods. The
amounts of D-alanine released were determined as described
in the legend to Fig. 1.
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Variation of pH on the hydrolysis of D-alanyl-Dcp in the
presence of LTA gave a pH optimum of 6.5 (Fig.
5A). However, both the acidic and the
basic limbs of the pH response are nontraditional, i.e., the hydrolytic
rate at the extremes of the pH curve do not approach the control
velocity. It would appear that both an ionizable group(s) and a binding
cleft are functional in Dcp. Additional pH studies may provide clues to
those interactions which are essential for this cleavage reaction. The
effect of temperature (Fig. 5B) on the hydrolytic rate was complex and
would appear to reflect more than one process. The velocity of
D-alanyl-Dcp hydrolysis was dependent on LTA and specific
for Dcp from 10 to 45°C.
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FIG. 5.
(A) Effect of pH on the hydrolysis of
D-alanyl-Dcp in the presence of LTA. (B) Effect of
temperature on the specific and nonspecific hydrolyses of
D-alanyl-Dcp and D-alanyl-ACP in the presence
of LTA. The reaction mixtures for panel A contained either 30 mM sodium
acetate (pH 3.0 to 5.0), bis-Tris (pH 6.0 to 7.0), or Tris-HCl (pH 7.5 to 9.5) in the presence of 4.4 µM LTA-phosphorus and 8 µM
D-[14C]alanyl-Dcp. The reaction mixtures for
panel B contained 30 mM bis-Tris (pH 6.5), either 8 µM
D-[14C]alanyl-Dcp or
D-[14C]alanyl-ACP, and 4.4 µM
LTA-phosphorus where indicated. The reaction mixtures for panel A were
incubated for 5 min at 37°C and at the indicated temperature for
panel B. The amounts of D-alanyl-Dcp remaining were
determined as described in Materials and Methods. In panel A, 100%
activity is given by the velocity at pH 6.5 in the presence of LTA. In
panel B, 100% activity is given by the amount of
D-[14C]alanyl-Dcp added to the reaction
mixture. The amount of specific hydrolysis is the difference between
the hydrolyses observed for D-alanyl-Dcp (LTA) and
D-alanyl-Dcp (no LTA).
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Transfer of D-[14C]alanine from
D-[14C]alanyl-LTA to Dcp in membranes.
A
second approach for studying the putative thioesterase-like activity of
the Dcp-LTA complex was to test whether Dcp will stimulate the
hydrolytic cleavage of the D-alanyl esters of
membrane-associated D-[14C]alanyl-LTA.
However, incubation of Dcp with membrane-associated D-[14C]alanyl-LTA revealed a
new facet of the D-alanine incorporation system. Instead of
hydrolysis, D-[14C]alanyl-Dcp was formed
(Fig. 6). When increasing concentrations of Dcp were incubated with membrane-associated
D-[14C]alanyl-LTA, increasing amounts of
D-[14C]alanyl-Dcp are formed according to
reaction 1. In contrast, these radiolabeled membranes do not transfer
the D-alanyl residues to ACP membrane as follows (reaction
1):
![<UP><SC>d</SC>-</UP>[<SUP><UP>14</UP></SUP><UP>C</UP>]<UP>alanyl-Dcp + membrane-associated LTA</UP>](/content/vol183/issue6/fulltext/2051/img003.gif)
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Debabov et al. (5) proposed that DltD facilitates the
binding of Dcp and Dcl for ligation of Dcp with D-alanine.
Thus, it was essential to establish whether DltD is responsible for the
reverse reaction illustrated in reaction 1. As shown in Fig. 6,
membranes prepared from L. casei 102S
dltD::cat also catalyze the synthesis of
D-[14C]alanyl-Dcp from membrane-associated
D-[14C]alanyl-LTA and Dcp to approximately
the same extent as the parental membranes. Thus, DltD plays no role in
catalyzing the transfer of D-alanine from
membrane-associated D-alanyl-LTA to Dcp.
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FIG. 6.
Effect of Dcp concentration on the formation of
D-alanyl-Dcp from membrane-associated
D-alanyl-LTA. The reaction mixture contained 20 µg of
membrane-associated D-[14C]alanyl-LTA (6,700 cpm/nmol), the indicated concentration of Dcp or ACP, and 30 mM
bis-Tris buffer (pH 6.5) in a total volume of 15 µl. It was incubated
for 30 min at 37°C. The amounts of
D-[14C]alanyl-Dcp formed were quantified by
nondenaturing polyacrylamide gel electrophoresis by the method of
Heaton and Neuhaus (12).
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NaCl-stimulated thioesterase-like activity of membranes for
D-alanyl-Dcp.
Growth of S. aureus in the
presence of high concentrations of NaCl resulted in a lower
D-alanine ester content in LTA (8). While the
precise mechanism of the NaCl effect is unknown, we have used this salt
effect to modulate the thioesterase-like activity for
D-alanyl-Dcp in membranes. The addition of increasing
concentrations of NaCl to membranes stimulated the thioesterase-like
activity for D-alanyl-Dcp (Fig.
7). It is hypothesized that NaCl
increases the accessibility of the endogenous membrane-associated LTA,
making it available for participation in the hydrolytic cleavage
specific for D-alanyl Dcp. To establish whether DltD
participates in this NaCl-stimulated hydrolysis of
D-alanyl-Dcp, membranes from the L. casei 102S
dltD::cat were also examined in the
salt-induced thioesterase-like reaction. As shown in Fig. 7, these
membranes were not different from parental membranes in their ability
to cleave D-alanyl-Dcp in the presence of increasing
concentrations of NaCl. The fact that DltD is not responsible for the
NaCl-stimulated thioesterase-like activity for D-alanyl-Dcp
suggests that a different mechanism is responsible for the cleavage of
D-alanyl-Dcp than that for the DltD-catalyzed hydrolysis of
D-alanyl-ACP.
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FIG. 7.
Hydrolysis of
D-[14C]alanyl-Dcp by membranes in the
presence of increasing concentrations of NaCl. Because
D-alanyl-Dcp can transfer its activated
D-alanine to membrane-associated LTA, the ordinate
represents the aggregate of D-alanyl-Dcp and
D-alanyl-LTA. The reaction mixtures contained 30 mM
bis-Tris (pH 6.5) and 0.25 mg of membranes from either L. casei 102S or the L. casei 102S
dltD::cat mutant (5) per ml in the
presence of increasing concentrations of NaCl in a volume of 50 µl.
The reaction time was 30 min at 37°C. The amount of hydrolysis was
quantified by the procedure described in Materials and Methods.
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NaCl sensitivity of D-alanyl-Dcp formation from
membrane-associated D-alanyl-LTA.
Because the salt
sensitivity of D-alanyl-Dcp hydrolysis (Fig. 7) and the
salt sensitivity of the D-[14C]alanyl-Dcp
formation utilizing D-[14C]alanyl-LTA and Dcp
are similar, a relationship may exist between these activities. For
example, the addition of increasing concentrations of NaCl to a series
of reaction mixtures containing membrane-associated D-[14C]alanyl-LTA and Dcp results in the
formation of lower amounts of D-alanyl-Dcp in the reverse
reaction (Fig. 8). The concentration of
salt which gave 50% inhibition is 0.30 M. This is essentially the same
concentration of NaCl as that required to observe the salt-activated
thioesterase-like activity for D-alanyl-Dcp (Fig. 7; 0.25 M
for 50% stimulation). The correlation of these activities to salt
sensitivity provided support for the suggestion that NaCl enhances the
accessibility of membrane-associated LTA and hence makes the LTA
available for catalyzing the hydrolytic cleavage of
D-alanyl-Dcp. This cleavage is similar to that described
with purified LTA and D-alanyl-Dcp (Fig. 1 and Table 1).
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FIG. 8.
Effect of increasing concentrations of NaCl on the
formation of D-[14C]alanyl-Dcp from
membrane-associated D-[14C]alanyl-LTA and
Dcp. The reaction mixture contained 30 mM bis-Tris (pH 6.5) and 20 µg
of D-[14C]alanyl-LTA (6,700 cpm/nmol) in a
volume of 15 µl. The reaction time was 20 min at 37°C. The reaction
mixtures were desalted using Nanosep microconcentrators. In the
reaction mixture designated "Dialyzed" the
D-[14C]alanyl-LTA was treated with 1.0 M NaCl
for 20 min and then dialyzed before incubation with Dcp by using the
Nanosep concentrator (10K). It was incubated in the reaction
mixture described above for the indicated time. The amount of
D-alanyl-Dcp formed was quantified by nondenaturing
polyacrylamide gel electrophoresis (12).
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DISCUSSION |
The results of these experiments, together with previous
observations (12, 20), describe three reactions. These are
as follows: (i) the hydrolysis of D-alanyl-Dcp in the
presence of isolated LTA, (ii) the formation of
D-alanyl-Dcp from membrane-associated D-alanyl-LTA and Dcp, and (iii) the transacylation of the
activated D-alanyl residue from D-alanyl Dcp to
membrane-associated LTA. We propose that each of these activities is,
in fact, the result of a previously unrecognized feature of Dcp and
LTA, i.e., the binding of this carrier protein to LTA. Binding of
D-alanyl-Dcp with LTA can mimic an enzyme reaction and, for
the purposes of the present study, LTA will be designated an "enzyme
mimic" and D-alanyl-Dcp will be designated the
"substrate." The specificity for the substrate suggests the
presence of a specific binding site on Dcp for its acceptor target LTA.
To consider the mechanisms underlying these reactions, three
illustrations are presented in Fig. 9. In
each case the nucleophilic acceptor generated with a common proton
acceptor is shown: HO:, R-S:, and R-O: (Fig. 9A, B, and C,
respectively). In panel A, the binding of D-alanyl-Dcp to
LTA followed by nucleophilic attack of HO: from water on the
electrophilic carbonyl results in hydrolysis. In panel B, nucleophilic
attack of the R-S: of the phosphopantetheine prosthetic group of Dcp on
the electrophilic carbonyl of the D-alanyl residue of the
membrane-associated LTA results in the transacylation of the
D-alanyl residue to Dcp. In panel C, nucleophilic
attack of the R-O: of the Gro-P residue on the electrophilic
carbonyl of the D-alanyl residue results in the
transacylation of D-alanine to the membrane-associated
LTA. Depending on the nucleophilic acceptor, each of these activities
is correlated with the specific interaction of the carrier protein, Dcp
or D-alanyl-Dcp, with LTA.
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FIG. 9.
Proposed mechanisms for the hydrolysis of
D-alanyl-Dcp in the presence of purified LTA (A), the
formation of D-alanyl-Dcp from membrane-associated
D-alanyl-LTA (B), and the formation of membrane-associated
D-alanyl-LTA from D-alanyl-Dcp (C).
B·· and :B indicate an unknown
proton acceptor for generating the nucleophile. The electrostatic
interaction between Dcp phosphodiester anion may be the result of
Arg-64 (see Discussion). Events up to the tetrahedral intermediate
stage are shown.
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The addition of NaCl to the reaction mixture apparently changes the
accessibility of the membrane-associated LTA. In the native state, the associated LTA is thought to be sequestered from the bulk
solvent. Membranes in the presence of NaCl resulted (i) in the
inhibition of transacylation of the activated
D-alanine from D-alanyl-Dcp to the R-OH of LTA
and (ii) in the stimulation of the hydrolytic cleavage of the
D-alanyl thioester. Alternatively in the reverse reaction,
the addition of NaCl inhibited the formation of
D-alanyl-Dcp from membrane-associated
D-alanyl-LTA and Dcp and stimulated hydrolysis. It is
proposed that membrane-associated LTA in the presence of NaCl provides
access to the bulk solvent, resulting in the hydrolysis of
D-alanyl-Dcp. As shown in Fig. 8, removal of the NaCl by
dialysis allowed the LTA to assume its original organization and
sequestration from the aqueous solvent.
The functional specificity of acyl carrier proteins is a hallmark of
this protein family. For example, in the case of the rhizobial carrier
proteins, four ACPs each have specific nodulation functions
(17). Thus, each of these carrier proteins is targeted to
their respective site of action. In the case of Dcp from L. rhamnosus, a specific binding cleft for LTA would appear to exist on the protein surface, which may allow for this targeting (B. F. Volkman et al., unpublished). While the cleft is not completely defined
at this time, there are candidate residues which may play a role in
this binding. For example, in the case of the Dcp, one of the
candidates for the electrostatic attraction of Dcp with LTA is Arg-64.
It is found in the highly conserved Dcp motif
R64KEW67D. The conserved Trp-67 plays an
important role in positioning helix III and hence Arg-64 in a unique
orientation for its putative interaction with LTA. This Arg
residue is part of a crescent of positive charges not found in E. coli ACP. All attempts to use ACPs in the
D-alanine incorporation system failed, even though all
could be ligated with D-alanine by Dcl in the absence of
DltD. These included ones from V. harveyi, S. erythraea, and B. subtilis (12, 20). Thus,
the experiments described here further address the unique functional
specificity of Dcp.
There are at least two examples where an ACP catalyzes a reaction
(13, 27). In the first the type II polyketide (PKS) ACP
catalyzes self-malonylation with malonyl-coenzyme A, and in the second
PKS ACP catalyzes the malonylation of ACP involved in type II fatty
acid biosynthesis. While these findings were unexpected, it
demonstrated that the PKS ACP has the ability to catalyze a reaction in
the presence of its cognate donor and acceptor. Thus, the
hydrolysis-transacylation reactions observed in the present work with
D-alanyl-Dcp may have some similarity to those in the PKS system.
The reversibility of the transacylation reaction, i.e., the formation
of D-alanyl-Dcp from D-alanyl-LTA and Dcp,
implies that Dcp and LTA can effect the transacylation of the
D-alanyl residues between LTA molecules, as well as between
wall teichoic acid and LTA. Haas et al. (10) showed that
under in vivo conditions the D-alanine esters of LTA are
the precursor of the D-alanine esters of wall teichoic
acid. In addition, the D-alanyl esters of LTA from either
S. aureus (15) or L. casei
(2) are randomly distributed along the poly(Gro-P) moiety.
The results presented here also provide a possible mechanism for the
migration and redistribution of these residues. Thus, it is proposed
that D-alanyl-Dcp is translocated to the extramembranal
site of LTA acylation, and it is further suggested that Dcp can be
ligated with D-alanine from preexisting D-alanyl-LTA for subsequent transacylation to adjacent LTA
as well as WTA.
The enhanced reactivity of the D-alanyl ester residues of
LTA was recognized by Shabarova et al. (24) and correlated
with the presence of the vicinal phosphodiester links. The ability of
Dcp to effect the transacylation of these ester residues in the cell
wall matrix could provide a mechanism for modulating surface charge and
hence ligand binding and autolysin activity. While transacylation of
D-alanyl esters was reported between short-chain LTA and
long-chain LTA (2, 19), the rate of this reaction was
relatively slow. Thus, it is hypothesized that transacylation in the
presence of Dcp is accelerated and hence Dcp plays a major role in
distributing D-alanine esters from one location of the wall
matrix to another.
One of the caveats to this suggestion concerns the topology of these
processes. While a putative channel (DltB) for the translocation of
D-alanyl-Dcp has been proposed (20), proof for
its role in the secretion of D-alanyl-Dcp is lacking. Thus,
it is not known whether the D-alanylation of LTA takes
place during the course of LTA assembly or whether
D-alanylation takes place in the wall matrix in concert
with D-alanyl-Dcp. The fact that D-alanylation of WTA takes place at the expense of D-alanyl-LTA
(10, 15) argues that these events may take place in the
extramembranal cell wall matrix.
The importance of the dlt operon in the physiology of the
gram-positive organism is illustrated by the many phenotypes of mutants
which have been observed. For example, inactivation of dltC
in Streptococcus mutans resulted in a loss of acid tolerance (1). In Streptococcus gordonii DL1 (Challis),
inactivation of dltA resulted in a loss of intrageneric
coaggregation and in the formation of a variety of pleomorphs
(3). In B. subtilis, deletion of either
dltA, -B, -C, or -D
resulted in mutants with enhanced autolytic activity (26).
Additional results with this organism revealed that these deletions in
the dlt operon partially suppressed the secretion deficiency
resulting from a defective PrsA protein (14). In
Lactococcus lactis (6), mutants defective for
DltD have enhanced UV sensitivity. Insertional inactivation of this
gene in Lactobacillus casei 102S resulted in increased cellular length and enhanced antimicrobial activity to
cetyltrimethylammonium bromide and chlorhexidine (5). In
the case of S. aureus, inactivation of the dlt
operon confers sensitivity to defensins, protegrins, and other cationic
peptides (23). This deficiency in D-alanine esters also increased its sensitivities to vancomycin and lysostaphin (22). In S. mutans, a knockout mutation in the
promoter of the dlt operon resulted in the defective
synthesis of intracellular polysaccharides (25). It is
apparent from these different phenotypes that the D-alanyl
esters of LTA play an important role in the physiology of the
gram-positive organism and, thus, an understanding of the
D-alanylation mechanism is essential.
This research was supported by Public Health Service grant RO1
GM51623 to F.C.N.
We are especially grateful to Werner Fischer for the characterized
samples of LTA from six organisms and for discussions of these results.
We also thank Michael P. Heaton for a critical reading of the
manuscript and discussions and Richard B. Silverman for comments on the mechanisms.
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Abstract
Introduction
![<UP>Membrane-associated <SC>d</SC>-</UP>[<SUP><UP>14</UP></SUP><UP>C</UP>]<UP>alanyl-LTA + Dcp⇌</UP>](/content/vol183/issue6/fulltext/2051/img002.gif)
This paper is dedicated to the memory of Werner Fischer of the
Universität Erlangen-Nürnberg.
Present address: Department of Biochemistry and Molecular Biology,
University of Maryland, Baltimore, MD 21201-1503.
