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J. Biol. Chem., Vol. 275, Issue 40, 30864-30872, October 6, 2000
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From the Lilly Research Laboratories, the Eli Lilly and Company,
Indianapolis, Indiana 46285
Received for publication, May 24, 2000, and in revised form, July 13, 2000
Acyl carrier protein synthase (AcpS) is an
essential enzyme in the biosynthesis of fatty acids in all bacteria.
AcpS catalyzes the transfer of 4'-phosphopantetheine from coenzyme A
(CoA) to apo-ACP, thus converting apo-ACP to holo-ACP that serves as an acyl carrier for the biosynthesis of fatty acids and lipids. To further
understand the physiological role of AcpS, we identified, cloned, and
expressed the acpS and acpP genes of
Streptococcus pneumoniae and purified both products to
homogeneity. Both acpS and acpP form operons
with the genes whose functions are required for other cellular
metabolism. The acpS gene complements an Escherichia coli mutant defective in the production of AcpS and appears to be
essential for the growth of S. pneumoniae. Gel filtration
and cross-linking analyses establish that purified AcpS exists as a
homotrimer. AcpS activity was significantly stimulated by apo-ACP at
concentrations over 10 µM and slightly inhibited
at concentrations of 5-10 µM. Double reciprocal analysis
of initial velocities of AcpS at various concentrations of CoA or
apo-ACP indicated a random or compulsory ordered bi bi type of reaction
mechanism. Further analysis of the inhibition kinetics of the product
(3',5'-ADP) suggested that it is competitive with respect to CoA but
mixed (competitive and noncompetitive) with respect to apo-ACP.
Finally, apo-ACP bound tightly to AcpS in the absence of CoA, but CoA
failed to do so in the absence of apo-ACP. Together, these results
suggest that AcpS may be allosterically regulated by apo-ACP and
probably proceeds by an ordered reaction mechanism with the first
formation of the AcpS-apo-ACP complex and the subsequent transfer of
4'-phosphopantetheine to the apo-ACP of the complex.
The biosynthesis of fatty acids is known to be required
for the growth of bacteria as fatty acids are essential components of
bacterial membrane lipids and lipopolysacharides (1, 2). The fatty acid
biosynthetic pathway in bacteria is well characterized (1, 2). Bacteria
utilize the type II or dissociated, fatty acid synthase system
for fatty acid synthesis (1, 2). The type II fatty acid synthase system
consists of individual enzymes that are encoded by separate genes (1,
2). On the other hand, the type I fatty acid synthase system, almost
exclusively present in eukaryotes, is characterized by the presence of
a multifunctional protein that possesses all the catalytic activities
required for fatty acid synthesis (3). In both systems, fatty acids are synthesized by using a repeated cycle of condensation, reduction, dehydration, and reduction reactions (1-3). In these reactions, holo-acyl carrier protein
(holo-ACP)1 plays an
essential role as an acyl carrier for fatty acid precursors, growing
acyl intermediates, and nascent fatty acid products (1-5).
ACP is a small acidic protein in bacteria (1, 2) or a small domain of
the type I fatty acid synthase in eukaryotes (3). ACP in
Escherichia coli is encoded by the acpP gene (1,
2). The newly synthesized ACP, or apo-ACP, is not functional in fatty acid synthesis. The conversion of apo-ACP to holo-ACP by ACP synthase (AcpS) is required for its functionality (1, 2, 4, 5). AcpS catalyzes
the transfer of the 4'-phosphopantetheine moiety from coenzyme A (CoA)
onto a serine residue of apo-ACP, thereby converting apo-ACP to
holo-ACP (1, 2, 4-7). The holo-ACP formed then mediates the transfer
of acyl intermediates by the covalent attachment of all acyl
intermediates via their carboxyl group to the thiol group of the
4'-phosphopantetheine group of holo-ACP (1-8). Thus, AcpS also plays
an essential role in fatty acid biosynthesis.
Homologues of AcpS and ACP have been identified in many bacterial
genomes sequenced to date (9-14). E. coli AcpS has been well studied (4-8, 15). The acpS gene from E. coli forms an operon with the upstream gene, pdxJ,
whose function is required for vitamin B6 biosynthesis (16,
17). The acpS gene was originally identified as
dpj (downstream of pdxJ) whose
function, although unknown, was required for the growth of E. coli (16, 17). Later, the landmark biochemical study by Lambalot
and Walsh (5) led to the identification of Dpj as AcpS. E. coli AcpS is a small, highly basic protein of about 14 kDa (5).
The E. coli enzyme has been purified and characterized (5).
The purified AcpS appears to be a homodimer (5). The enzyme exhibits a
broad substrate specificity and can utilize a variety of ACPs that are required for many diverse aspects of cellular metabolism (5-8, 15,
18-22). These results indicate that AcpS may be able to participate in
other metabolism besides fatty acid biosynthesis in the cell. Purified
AcpS also exhibits activity with a number of CoA derivatives (15).
Finally, AcpS is a very low abundance protein in E. coli (4, 5). In contrast, ACP is a very abundant protein that was
estimated to be present at 25,000-60,000 molecules/cell (1, 2, 23,
24). The majority of ACPs present in the cell are found to be holo-ACPs
(1, 2, 25, 26).
Although E. coli AcpS is well studied, the reaction
mechanism of AcpS remains unknown. In addition, only the AcpS from
E. coli, a rod-shaped, Gram-negative bacterium, has been
thoroughly characterized to date. It still remains to be determined
whether AcpS from Gram-positive bacteria would play the same
physiological role. Finally, AcpS appears to possess all of the
features necessary for a good antibacterial target, such as its
essential nature, widespread existence in bacteria, and unique
catalytic position in a pathway (fatty acid biosynthesis). Thus, AcpS
might be a valuable antibacterial target for identifying novel
antimicrobial agents. To better understand the function of AcpS in
Streptococcus pneumoniae, a sphere-shaped, Gram-positive
bacterium and also a major human pathogen of the upper respiratory
tract, and to explore AcpS as an antibacterial target, we first cloned
and expressed the acpS and acpP genes of S. pneumoniae and characterized both gene products. The results of
this study show that S. pneumoniae AcpS shares many
biochemical properties with E. coli AcpS but also exhibits
major differences with respect to their native structures and substrate
regulations. In addition, the results of this study suggest that AcpS
proceeds by an ordered reaction mechanism with the first formation of
the enzyme-apo-ACP intermediate from apo-ACP followed by the transfer
of 4'-phosphopantetheine from CoA to the apo-ACP of the complex.
Finally, both acpS and acpP form complex operons
with the genes whose functions are not required for fatty acid biosynthesis.
Materials--
Unless specified otherwise, all fine chemicals
were purchased from Sigma. All fast protein liquid chromatography
resins and columns used for protein purification and strains and
reagents for construction, expression, and purification of GST-fused
proteins were obtained from Amersham Pharmacia Biotech. Luria Bertani
(LB) broth medium was purchased from Bio 101, Inc. (Vista, CA). All polyacrylamide gels and reagents were purchased from Novex (San Diego,
CA). SYPRO Orange and Bradford protein assay reagents were purchased
from Bio-Rad, and ethylene glycolbis(succinimidylsuccinate) (sulfo-EGS)
was obtained from Pierce. [3H]CoA (specific activity, 1.5 Ci/mmol) was custom-synthesized by NEN Life Science Products.
Cloning and Expression of the acpS and acpP Genes of S. pneumoniae (hex
To clone the acpP gene, the following PCR primers were used
for amplification: the 5' PCR primer
(5'-CGCGGATCCCATATGACAGAAAAAGAAATTTTTGACCGTATTG -3') and the 3'
PCR primer (5'-CGCGGATCCGAATTCCTATTTTCCTTGAATGATTTTAACCACATC-3'). Using
these primers, acpP was PCR-amplified from S. pneumoniae as described above. The PCR products were digested with
BamHI. The BamHI-digested PCR fragment was cloned
into pCZA342. The pCZA342 clone was digested with NdeI and
BamHI. The NdeI-BamHI DNA fragment containing acpP was subcloned into pET-11a (Novagen),
resulting in pRBP-16.
Purification of AcpS and ACP of S. pneumoniae--
LY128
(E. coli BL21 (pLysS)/pRBP-19) was first grown at 35 °C
overnight in LB broth medium supplemented with 100 µg/ml ampicillin. The overnight culture (40 ml) was then inoculated into 1000 ml of LB
medium supplemented with ampicillin and grown at 33 °C with shaking
at 250 rpm until an A590 of 0.5-0.6 was
reached. The culture was induced with 1 mM
isopropyl-1-thio-
LY135 (E. coli XL1 Blue (mRF')/pRBP-20) was grown, induced,
harvested, disrupted, and centrifuged as above. The supernatant fraction was applied to a glutathione-Sepharose 4B column (10 ml) that
had been equilibrated with 100 ml of PBS. The column was washed with
100 ml of PBS, and the GST-AcpS fusion protein was eluted with 10 mM glutathione in PBS. Fractions were analyzed by SDS-PAGE
(12% glycine), and those fractions containing GST-AcpS were pooled,
dialyzed against 50 mM Tris-HCl, pH 7.0 (4 liter), adjusted
with glycerol to a final concentration of 15% (v/v), and stored at
LY140 (E. coli BL21 (pLysS)/pRBP-16) was grown, induced,
harvested, and disrupted as described above. The resulting cell extract was centrifuged as described above. The supernatant fraction was collected and applied to a 15S Source Q column (2.5 × 8 cm) that had been equilibrated with 50 mM Tris-HCl, pH 8.0, 100 mM KCl (buffer C). The column was washed with 100 ml of
buffer C and eluted with a linear gradient of 0.0-1.0 M
KCl in buffer C. Fractions (7 ml each) were collected, and the presence
of apo-ACP in the fractions was detected by SDS-PAGE as described above
(16% tricine gels). The fractions containing apo-ACP were pooled and
applied to a S-100 Sepharose gel filtration column (5 × 60 cm)
equilibrated with 50 mM Tris-HCl, pH 7.0, 100 mM KCl. The column was eluted with the same buffer.
Fractions (10 ml each) containing apo-ACP were collected, analyzed by
electrospray mass spectrometry, and stored at Analysis of AcpS by Gel Filtration Column Chromatography--
To
determine the native structure of AcpS, a purified AcpS preparation
(375 µg) was applied to a S-75 Superdex gel filtration column (HR
1.0 × 30 cm), equilibrated with 50 mM Tris-HCl, pH 7.0, 50 mM KCl, 10 mM MgCl2. The
column was calibrated with the protein molecular weight standards
(Sigma). The effect of detergent or salt on the native structure of
AcpS was analyzed by treating AcpS with 6 mM CHAPS or
50-500 mM KCl before and during column chromatography.
Sedimentation centrifugation analysis of AcpS was carried out using an
XLA ultracentrifuge (Beckman Instruments, Fullerton, CA). A purified
AcpS preparation (adjusted to 0.2 and 0.4 mg/ml) was centrifuged at
16,000 rpm for 24 h at 22 °C. The absorbance at 280 nm as a
function of radius after the system reaches equilibrium was analyzed
using XL-A/XL-1, a nonlinear least squares fit data analysis program.
Partial specific volume of AcpS was calculated to be 0.721 ml/g based
on its amino acid sequence. The molecular weight of AcpS was determined
using a global fit of the two data sets collected with 0.2 and 0.4 mg/ml samples.
Cross-linking experiments were performed as follows. Purified AcpS and
apo-ACP preparations (1 ml each) were dialyzed against 2 liters of 20 mM potassium phosphate buffer, pH 7.0, at 4 °C for
18 h. The dialyzed AcpS (163 µM) and apo-ACP (94 µM) preparations were mixed without or with 19.5 and 9.4 mM sulfo-EGS, respectively, and the mixtures were incubated
at room temperature for 30 min. The reactions were stopped by the
addition of 50 mM Tris-base followed by incubation at room
temperature for 30 min. The resulting AcpS and apo-ACP preparations
treated without or with the cross-linker (10 µl) were mixed with an
equal volume of Tricine sample buffer and analyzed by SDS-PAGE (16%
Tricine gels).
To determine whether AcpS binds directly to apo-ACP or CoA in the
absence of the other substrate, a purified AcpS preparation (27 µM) was first mixed with 10 mM
MgCl2 and then either 100 µM apo-ACP or 50 µM CoA. The mixture was incubated at room temperature for
30 min and subjected to gel filtration column chromatography (S-75
Superdex) under the conditions described above. The fractions containing the AcpS-apo-ACP complex and unbound apo-ACP were analyzed by SDS-PAGE (16% Tricine gels), SYPRO Orange staining, and mass spectrometry.
Enzyme Assay and Kinetics--
Unless otherwise indicated,
reaction mixtures contained 50 mM Tris-HCl, pH 7.0, 10 mM MgCl2, 2.5-50 µM CoA,
0.25-6.0 µM purified apo-ACP of S. pneumoniae, and 3.7 nM purified AcpS of S. pneumoniae and were incubated at 37 °C for 9 min. Reactions
were stopped by the addition of 50 mM EDTA. The formation
of holo-ACP was determined by an HPLC or a trichloroacetic acid
precipitation method (see below). An HPLC-based assay was adapted from
the method as described previously (31). This assay monitors the
conversion of apo-ACP to holo-ACP. Reaction mixtures (100 µl each)
were injected into an analytical HPLC column (Vydac protein C4
reverse-phase; P. J. Cobert Associates, Inc., St. Louis, MO)
equilibrated with 45% acetonitrile in 0.1% trifluoroacetic acid. The
column was eluted with an 8-ml linear gradient of 45-80%
acetonitrile. The column elution profiles were monitored at 220 nm.
Under these conditions, holo-ACP migrated faster than apo-ACP. The
amount of holo-ACP formed is estimated by comparing the peak area of
the holo-ACP formed with those of both apo- and holo-ACP.
AcpS activity was also assayed by using a trichloroacetic acid
precipitation method (5). This method measures the incorporation of the
3H-labeled 4'-phosphopantetheine group from
[3H]CoA into apo-ACP. Reaction conditions used were the
same as those described for the HPLC assay except that
[3H]CoA (specific activity, 1.5 Ci/mmol; NEN Life Science
Products) was used alone or in combination with CoA. Reactions were
stopped by the addition of 0.9 ml of cold 10% trichloroacetic acid
followed by 37 µg of bovine serum albumin. Precipitated protein was
collected by centrifugation (a microcentrifuge at 14,000 rpm) for 5 min and washed twice with ice-cold 10% trichloroacetic acid. The protein collected was resuspended in 150 µl of 1 M Tris base and
0.1% Triton X-100. The resulting suspension (100 µl) was mixed with 2.5 ml of Ready Protein+ scintillation fluid (Beckman) and
counted using a LS 6000IC scintillation counter (Beckman).
To examine the substrate specificity of AcpS, a variety of CoA
derivatives (acetyl-CoA, malonyl-CoA, acetoacetyl-CoA, desulfo-CoA, and
dephospho-CoA) were tested. All reaction mixtures contained 1.5 µM apo-ACP, 20 µM CoA, or CoA derivative,
and 19 nM AcpS, and the formation of holo-ACP was
determined by the HPLC method.
For determination of Km and
Vmax (kcat) of AcpS for
apo-ACP, reaction mixtures (in quadruplicate) contained 20 µM CoA, 0.25-100 µM apo-ACP, and 3.7 nM AcpS. The formation of holo-ACP was measured by the HPLC
method or the trichloroacetic acid precipitation method. To determine
Km for CoA, the reaction conditions used were the
same as those described above except that the concentration of apo-ACP
was 2.0 µM and the concentrations of CoA were 5-600 µM.
To analyze the kinetic mechanisms of AcpS, AcpS activity was measured
at different concentrations of both substrates. Reaction mixtures (in
triplicate) contained 2.5-40 µM CoA, 0.25-2.0
µM apo-ACP, and 3.7 nM AcpS. The formation of
holo-ACP was analyzed by the HPLC assay.
To evaluate the inhibition of AcpS activity by 3',5'-ADP with respect
to CoA, reaction mixtures contained 1-60 µM 3',5'-ADP, 2.5-60 µM CoA, 1.0 µM apo-ACP, and 3.7 nM AcpS. To evaluate the inhibition of AcpS activity by
3',5'-ADP with respect to apo-ACP, reaction mixtures contained 1-60
µM 3',5'-ADP, 0.5-6.0 µM apo-ACP, 20 µM CoA, and 3.7 nM AcpS. The formation of
holo-ACP was analyzed by the HPLC method.
Identification and Organization of the acpS and acpP Genes of S. pneumoniae--
To understand the function of AcpS in the biosynthesis
of fatty acids in S. pneumoniae, we cloned and expressed the
acpS gene as well as the acpP gene that encodes a
substrate of AcpS. Both genes were identified from our S. pneumoniae data base (32) using the E. coli acpS and
acpP gene sequences as queries in the BLAST program (33).
The acpS gene, 369 base pairs long, encodes a protein
consisting of 122 amino acid residues with a predicted molecular
mass of 13.7 kDa (GenBankTM accession number
AF276617). The acpS gene appears to be organized into an
operon with the genes in the order aroG-aroF-acpS-alr-recG, since there are long noncoding regions located upstream of
aroG and downstream of recG. Thus, the
acpS operon appears to consist of the genes that are
required in aromatic amino acid biosynthesis (aroF and
aroG encoding 3-deoxy-D-arabino-heptulosonate
7-phosphate synthases), cell wall biosynthesis (alr encoding
D-alanine racemase), and DNA recombination
(recG). In this regard, the genomic organization of
acpS in S. pneumoniae is quite different from
that of acpS in E. coli, since acpS in
E. coli consists of an operon with its upstream
pdxJ gene that is required for vitamin B6
biosynthesis (16, 17).
The acpP gene, 234 base pairs long, encodes a protein
consisting of 77 amino acid residues with a predicted molecular mass of
8.7 kDa (GenBankTM accession number AF276618). The
acpP gene appears to consist of an operon with the genes in
the order hisC-unknown-plsX-acp. There
are very long noncoding regions located in the upstream of
hisC and downstream of acpP. Like the
acpS operon, the genes in the acpP operon are
also involved in different aspects of cellular metabolism such as
histidine biosynthesis (hisC encoding histidinol phosphate
aminotransferase), lipid biosynthesis (plsX, required for
the phenotype of plsB that encodes glycerol 3-phosphate
acyltransferase, an enzyme required for lipid biosynthesis), and
possibly others (unknown function gene). It is known that the
acpP genes in Bacillus subtilis, E. coli, Pseudomonas aeruginosa, and Vibrio
harveyi are organized into operons with other fatty acid
biosynthetic genes (20, 34-36). Thus, the operon organization of the
acpP gene in S. pneumoniae is also different from
those of the acpP genes in E. coli and other
organisms (20, 34-36). Finally, it is known that plsX and
acpP, along with other fatty acid biosynthetic genes, are
also located in the same operon in B. subtilis, E. coli, P. aeruginosa, and V. harveyi (20,
34-36). This suggests that a genetic reorganization event might have
occurred during evolution, which resulted in the formation of the
complex operons that currently exist in the organisms such as S. pneumoniae.
The subunits of S. pneumoniae AcpS and apo-ACP exhibit
molecular weights virtually identical to those of E. coli
AcpS and apo-ACP, respectively. Both proteins also share 38%
identities with their counterparts in E. coli. The pI value
of S. pneumoniae AcpS was estimated to be 6.5, which is much
lower than 9.98, the pI value of E. coli AcpS (16, 17).
Therefore, S. pneumoniae AcpS is significantly less basic
than E. coli AcpS. Like other ACPs (1, 2, 20, 34-36),
S. pneumoniae apo-ACP is very acidic, with a pI value of
only 3.4.
Finally, we tested whether the S. pneumoniae acpS gene
complements an E. coli mutant strain, HT253, defective in
the production of AcpS (17). HT253 contains a mini-Tn10
insertion in the pdxJ gene, which is upstream of and forms
an operon with acpS (16, 17). The mini-Tn10
carries two divergent tetracycline-inducible promoters (17). In the
absence of tetracycline, HT253 could not grow on LB plates because the
mini-Tn10 insertion in pdxJ blocks the
transcription of the acpS gene. Thus, the growth of HT253 is
tetracycline-dependent. When the acpS gene
(pRBP123, acpS carried on pGEX-2T) was introduced into
HT253, this mutant strain was able to grow on LB medium without the
supplementation of tetracycline and
isopropyl-1-thio- Expression, Purification, and Identification of the AcpS and ACP of
S. pneumoniae--
The acpS and acpP genes
identified were cloned into expression vectors and expressed in
E. coli (see "Experimental Procedures"). Both AcpS and
apo-ACP were highly expressed in E. coli and exhibited the
molecular weights predicted (Fig. 1). The
overexpressed AcpS was purified to apparent homogeneity in two steps
(Fig. 1A) using Source S-cation exchange and gel filtration
column chromatography. The overexpressed S. pneumoniae
apo-ACP was also purified to apparent homogeneity (Fig. 1B)
in two steps using Source Q-anion exchange and gel filtration column
chromatography.
To confirm the purified proteins as AcpS and ACP, we performed
N-terminal sequencing and mass spectrometric analyses. The first 9 amino acid residues of purified AcpS were determined to be MIVGHGIDI, a
sequence that is identical to the predicted amino acid sequence for the
protein encoded by the cloned acpS gene. This encoded
protein is predicted to have a molecular weight of 13,388. Consistent
with this predicted value, mass spectrometric analysis showed that
purified AcpS had a molecular weight of 13,390. Thus, the purified
protein is S. pneumoniae AcpS.
N-terminal sequencing analysis also showed that purified apo-ACP
exhibited the predicted amino acid sequence (data not shown). When
subjected to mass spectrometric analysis, purified apo-ACP was found to
exhibit two peaks, the major peak with a molecular mass of 8834 Da
(about 80% of the total protein) and the minor peak with a molecular
mass of 8861 Da (20%) that is 26 Da larger than that of the major
species. The predicted molecular weight for S. pneumoniae
apo-ACP is 8706, thus in agreement with the results of mass
spectrometric analysis. Mass spectrometric analysis further showed that
both apo-ACPs were converted to holo-ACP upon their reaction with AcpS,
since the molecular weights of both ACPs were increased by 341 Da,
corresponding to the molecular weight of the 4'-phosphopantetheine
group (data not shown). Finally, mass spectrometric analysis showed
that the presence of holo-ACP was not detectable in the apo-ACP
preparations (data not shown).
The mobility of apo-ACP and holo-ACP was examined by native gel
electrophoresis followed by staining with SYPRO Orange (see "Experimental Procedures"). Holo-ACP was found to migrate more slowly than apo-ACP (data not shown). The complete conversion of
apo-ACP to holo-ACP was confirmed as evidenced by the fact that the
molecular weight of ACP was increased from 8,834 Da (apo-ACP) to 9,174 Da (holo-ACP) upon the treatment of apo-ACP with AcpS, CoA, and
Mg2+. Thus, unlike E. coli holo-ACP (5),
S. pneumoniae holo-ACP migrates more slowly than
apo-ACP.
Determination of the Native Structures of S. pneumoniae AcpS and
ACP--
To determine the molecular weight of native AcpS, we
subjected a purified AcpS preparation to gel filtration column
chromatography analysis. AcpS was eluted in the fractions corresponding
to a molecular mass of 38 kDa (Fig.
2A, peak
B). This result suggests that AcpS is a homotrimer with a
predicted molecular mass of 41 kDa (GenBankTM
accession number AF276617). To confirm this further, a purified AcpS
preparation was subjected to sedimentation analysis. This analysis
showed that purified AcpS had a molecular mass of 39 kDa, which is
consistent with that of gel filtration analysis. Finally, when a
purified AcpS preparation was subjected to cross-linking (see
"Experimental Procedures") followed by SDS-PAGE analysis, two
protein bands were observed (Fig. 3). The
two bands had molecular masses of 10.4 and 28.2 kDa, respectively, thus
corresponding to the monomeric and trimeric forms of AcpS (Fig. 3,
lane 2). Taken together, these results show that
the AcpS of S. pneumoniae is a trimeric enzyme. The trimeric
structure of AcpS appears to be stable, since AcpS still retained its
native structure in the presence of 6 mM CHAPS or 50-500
mM KCl during gel filtration column chromatography (data
not shown).
When apo-ACP was also subjected to gel filtration column
chromatography, it was eluted in the fractions corresponding to a molecular mass of 17 kDa, thus indicating that apo-ACP may exist as a
dimer (Fig. 2A). Apo-ACP has been shown to behave abnormally on gel filtration columns due to its molecular asymmetry in shape (39,
40). To examine further whether apo-ACP is a dimer, purified apo-ACP
was subjected to cross-linking followed by SDS-PAGE analysis. Only one
protein band was observed, which had a molecular mass of 5.6 kDa (Fig.
3B). Thus, this result shows that apo-ACP is a monomeric
protein. The result of the gel filtration column analysis was
consistent with the previously reported anomalous behavior of apo-ACP
on gel filtration columns (39, 40).
Kinetic Characterization of S. pneumoniae AcpS--
To elucidate
the reaction mechanism of AcpS, we examined its substrate specificity
and kinetics. The purified AcpS of S. pneumoniae, when
assayed by the HPLC method, exhibited an optimal activity at
45-50 °C and pH 6.5 and was stable at 22-65 °C. AcpS was able to utilize a number of CoA derivatives as substrates and exhibited the
following relative activities: 100 (CoA), 91 (acetyl-CoA), 76 (desulfo-CoA), 65 (acetoacetyl-CoA), 12 (malonyl-CoA), and 0 (dephospho-CoA). Thus, like E. coli AcpS and B. subtilis Sfp protein, S. pneumoniae AcpS utilizes
different CoA derivatives as substrates (15, 41).
S. pneumoniae AcpS appears to exhibit Michaelis-Menten
kinetics when assayed at various CoA concentrations and apo-ACP
concentrations lower than 10 µM (Fig.
4A). AcpS activity increased
in a dose-dependent manner at the apo-ACP concentrations of
0.5-5 µM (Fig. 4A). Then, when the
concentration of apo-ACP approached 10 µM, AcpS activity decreased (Fig. 4A). This result is consistent with the
observation that apo-ACP is inhibitory to AcpS at higher concentrations
(4-6, 15). However, a further increase of apo-ACP concentrations (>10 µM) was accompanied with a significant increase in AcpS
activity (Fig. 4A). As a result, two separate substrate
saturation curves were obtained at low and high concentrations of
apo-ACP (Fig. 4, B and C). The double reciprocal
plot analyses indicated that AcpS had Km (for
apo-ACP) values of 0.5 ± 0.08 and 109 ± 6.8 µM and Vmax values of 2439 ± 243 (kcat = 1.7 ± 0.17 s
When a fixed apo-ACP concentration and various CoA concentrations were
used, a hyperbolic substrate saturation curve was obtained for AcpS
(Fig. 5A). The apparent
Km and Vmax values of AcpS
were determined to be 11.5 ± 0.9 µM (for CoA) and
3976 ± 73 nmol/min/mg (kcat = 2.7 ± 0.05 s
Since the trichloroacetic acid precipitation method has often been used
for the assay of AcpS activity (4-6), we also characterized the
kinetic properties of the enzyme using this assay method. This assay
utilizes [3H]CoA as a substrate for AcpS. The apparent
Km values of the enzyme for apo-ACP and CoA were
determined to be 1.3 ± 0.7 and 7.1 ± 0.4 µM,
respectively. The Vmax
(kcat) values determined were 4179 ± 182 (2.8 ± 0.04 s
Although E. coli AcpS has been extensively studied (4-7),
the kinetic mechanism of the enzyme is unknown. To further elucidate the kinetic mechanism of S. pneumoniae AcpS, we analyzed the
double reciprocal plots of the initial velocities of the enzyme at
fixed concentrations of one substrate versus various
concentrations of the other substrate (42). This analysis yielded
Km and Vmax values that are
similar to those determined before (data not shown). As shown in Fig.
6A, the double reciprocal
plots of the initial velocities of AcpS obtained at the various CoA and fixed apo-ACP concentrations yielded an intersecting pattern. The same
pattern was obtained when various concentrations of apo-ACP and fixed
concentrations of CoA were used (Fig. 6B). Together, these
results suggest that AcpS proceeds by a random or compulsory ordered bi
bi type but not a ping-pong (double displacement) type of reaction
mechanism (42).
To differentiate these two possible reaction mechanisms, we analyzed
the kinetics of product inhibition. AcpS activity was examined in the
presence of 3',5'-ADP. As shown in Fig.
7A, when various CoA
concentrations were used, the double reciprocal plots yielded a simple
competitive pattern with a Ki of 6.0 µM (Fig. 7C). However, when various apo-ACP
concentrations were used, the double reciprocal plots yielded a linear
mixed pattern with a Ki of 2.5 µM
(Fig. 7, A and D). Since the patterns of
inhibition with respect to CoA and apo-ACP are competitive and mixed,
respectively, these results suggest that apo-ACP is probably the first
substrate to bind to the enzyme, which is followed by CoA (Ref. 42; see
"Discussion").
The Binding of apo-ACP and CoA to AcpS--
To determine the order
of substrate binding to AcpS, we analyzed the binding of CoA and
apo-ACP to purified AcpS by gel filtration column chromatography, mass
spectrometry, or filter binding assays. We reasoned that if CoA binds
to AcpS first and forms an enzyme-substrate complex that is required
for the next reaction with apo-ACP, then a stable enzyme-substrate
complex should be detectable. When a mixture of CoA and purified AcpS
that had been incubated at room temperature for 30 min (see
"Experimental Procedures") was subjected to gel filtration column
chromatographic and mass spectrometric analyses, the presence of CoA
was not detectable in the fractions containing purified AcpS (data not
shown). Thus, CoA did not appear to bind to AcpS in the absence of
apo-ACP. To further examine the binding of CoA to AcpS, a mixture of
purified AcpS and [3H]CoA that had been incubated under
the same conditions was subjected to a filter binding assay (see
"Experimental Procedures"). There was no evidence that
[3H]CoA was bound to AcpS, since the radioactivity of
[3H]CoA was not detectable after washing (data not
shown). Thus, CoA does not appear to bind to AcpS in the absence of
apo-ACP.
To examine whether apo-ACP binds to AcpS in the absence of CoA, we
subjected a mixture of AcpS and apo-ACP (apo-ACP/AcpS = 5:1) to
gel filtration column chromatography and analyzed the column fractions
by SDS-PAGE (see "Experimental Procedures"). Two protein peaks were
observed (Fig. 2A). The leading peak (peak A) had a molecular mass of approximately 53 kDa as judged by
gel filtration analysis (Fig. 2A). Since AcpS exists as a
trimer with a molecular mass of approximately 41 kDa, this leading peak
probably represented a complex between apo-ACP and AcpS (Fig.
2A). Consistent with the formation of the AcpS-apo-ACP
complex, the presence of apo-ACP was also detected in the fractions
containing purified AcpS (Fig. 2B). Together, these results
show that apo-ACP can bind to AcpS in the absence of CoA.
In this study, we have identified the acpS and
acpP genes from S. pneumoniae and purified and
characterized their gene products. Sequencing analysis shows that both
acpS and acpP form complex operons with the genes
whose functions are required for other cellular metabolism. We have
established, by using a variety of biochemical approaches, that
purified AcpS and apo-ACP existed as a homotrimer and monomer,
respectively, and that apo-ACP bound tightly to AcpS, but CoA failed to
do so. Kinetic analysis suggests that AcpS proceeds by an ordered
reaction mechanism with the first formation of the enzyme-apo-ACP
intermediate from apo-ACP and the subsequent transfer of the
4'-phosphopantetheine group from CoA to apo-ACP.
The biochemical identification of dpj as acpS (5)
is significant. This work has led to the subsequent identification of a
number of AcpS-like enzymes in different bacterial species that are
required for the biosynthesis of polyketides, enterobactin siderophore,
and others (7) and has established the cross-functionality of ACP and
AcpS in different biosynthetic systems (6, 7, 15, 18-22, 41). The AcpS
from E. coli, a rod-shaped, Gram-negative bacterium, is the
best characterized enzyme among the AcpS-like enzymes identified. It is
a small, basic protein with a molecular weight of 13,922 that can
utilize a variety of CoA derivatives as substrates and is inhibited by
apo-ACP at higher concentrations (>10 µM) (4-6, 15).
This apparent inhibition results from the electrostatic interaction
between the basic AcpS enzyme and the acidic apo-ACP (6). Likewise, the
AcpS from S. pneumoniae, a sphere-shaped, Gram-positive
bacterium, is also a small protein with a virtually identical size, can
use different CoA derivatives, and is slightly inhibited by apo-ACP at
similar concentrations although stimulated at higher concentrations.
Both E. coli and S. pneumoniae AcpS enzymes also
appear to exhibit similar kinetic properties. Purified E. coli AcpS had Km values of 1.5 and 50 µM for apo-ACP and CoA, respectively, and a
kcat value of 1-2 s One of the major differences between E. coli and S. pneumoniae AcpS enzymes appears to be their native structures.
E. coli AcpS was reported to be a homodimer with a molecular
mass of 28 kDa (5). S. pneumoniae AcpS is shown, in this
study, as a homotrimer. Several lines of evidence support the
conclusion that S. pneumoniae is a trimeric enzyme. The gel
filtration analysis showed that purified AcpS had a molecular mass of
38 kDa. Since AcpS has a molecular mass of 13.7 kDa based on its DNA
sequence, a homotrimer of AcpS is predicted to have a molecular mass of
41 kDa, which is consistent with the result of gel filtration analysis.
In addition, the sedimentation analysis showed that purified AcpS had a
molecular mass of 39 kDa. Finally, the results of our cross-linking
experiments clearly demonstrated the presence of two predominant
protein species with molecular masses of 10.4 and 28.2 kDa,
respectively, as resolved by SDS-PAGE. Taken together, these results
establish that S. pneumoniae AcpS does exist as a
homotrimer. In light of this finding, it is possible that AcpS enzymes
from Gram-positive bacteria may differ from those of Gram-negative
bacteria with respect to their native structures. It also remains
possible that E. coli AcpS might exist as a homotrimer. It
should be noted that E. coli AcpS is significantly more
positively charged than S. pneumoniae AcpS. It is possible
that E. coli AcpS might migrate faster than S. pneumoniae AcpS during gel filtration column chromatography. As a
result, E. coli AcpS appears to behave as a dimer as judged by gel filtration analysis (5).
The other difference between E. coli and S. pneumoniae AcpS enzymes appears to lie in their regulation by the
substrate, apo-ACP. Both enzymes are inhibited by apo-ACP at similar
concentrations (6). However, the activity of S. pneumoniae
AcpS, but not that of E. coli AcpS, is significantly
stimulated by apo-ACP at even higher concentrations (Fig.
4A). The stimulation by apo-ACP (Fig. 4A)
suggests that S. pneumoniae AcpS may be allosterically
regulated by apo-ACP at higher concentrations. As a result, at higher
apo-ACP concentrations (>15 µM), the affinity of
S. pneumoniae AcpS for apo-ACP is significantly decreased
(200-fold), and its catalytic activity is also increased (5-fold) (Fig.
4, B and C). These changes in the affinity and
activity of the enzyme at high apo-ACP concentrations might have
relevant physiological significance. Under the conditions where apo-ACP
is overproduced in the cell, an AcpS with increased Km for apo-ACP and Vmax
clearly could be significantly more efficient in the conversion of
apo-ACP to holo-ACP, thereby preventing the accumulation of high levels
of apo-ACP in the cell that is known to be toxic (47).
Among all AcpS-like enzymes, E. coli AcpS enzyme has been
the most extensively studied biochemically. Studies have been primarily focused on the substrate specificity and cross-functionality of the
enzyme in other analogous systems (4-7, 15, 18-22, 41). Despite the
extensive characterization of the E. coli AcpS, the reaction
mechanism of the enzyme has not been determined. In this study, we
attempted to elucidate the reaction mechanism of the enzyme. The
analysis of the initial velocities of AcpS obtained at fixed
concentrations of one substrate and various concentrations of another
reveals that AcpS probably proceeds by a random or ordered compulsory
bi bi reaction mechanism, because an intersecting pattern was obtained
regardless of which substrate (CoA or apo-ACP) was the fixed one or the
varied one (42) (Fig. 6). To further investigate the possible
mechanism, the inhibition kinetics of 3',5'-ADP, one of the reaction
products, was examined. This analysis indicates that AcpS appears to
proceed by an ordered reaction mechanism with the first formation of
the AcpS-apo-ACP intermediate and the subsequent transfer of
4'-phosphopantetheine from CoA onto apo-ACP. The mode of inhibition by
3',5'-ADP with respect to CoA is competitive when apo-ACP is the fixed
substrate and CoA is the varied substrate (Fig. 7). The competitive
inhibition with respect to CoA indicates that CoA only binds to the
enzyme-apo-ACP intermediate. The mode of inhibition by 3',5'-ADP with
respect to apo-ACP is mixed, i.e. a combination of
competitive and noncompetitive inhibition when apo-ACP is the varied
substrate and CoA is the fixed substrate (Fig. 7B). The
mixed type of inhibition by 3',5'-ADP with respect to apo-ACP suggests
that 3',5'-ADP binds to the free enzyme and the enzyme-apo-ACP
intermediate. Thus, inhibition was competitive with respect to apo-ACP
when 3',5'-ADP bound to the free enzyme and noncompetitive with respect
to apo-ACP when 3',5'-ADP bound to the enzyme-apo-ACP intermediate.
This proposed reaction mechanism for AcpS is consistent with the
results of the substrate-binding experiments. Under the conditions
tested, apo-ACP bound tightly to AcpS in the absence of CoA, but CoA
failed to do so in the absence of apo-ACP. Taken together, these
results suggest that the reaction mechanism of AcpS is ordered rather
than random and that the formation of the enzyme-apo-ACP intermediate
occurs first before the transfer of 4'-phosphopantetheine from CoA onto
apo-ACP.
We thank Dr. Malcolm E. Winkler for
suggestions and stimulating discussions. We also thank Mel Johnson for
N-terminal sequencing, John Richardson for mass spectrometric analysis,
Jirong Lu for sedimentation analysis, and Dr. Raymond Gilmour for
critical review of the manuscript.
*
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.
Published, JBC Papers in Press, July 19, 2000, DOI 10.1074/jbc.M004475200
2
P. Treadway, unpublished results.
The abbreviations used are:
ACP, acyl carrier
protein;
AcpS, acyl carrier protein synthase;
CoA, coenzyme A;
PAGE, polyacrylamide gel electrophoresis;
HPLC, high performance liquid
chromatography;
PCR, polymerase chain reaction;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
sulfo-EGS, ethylene glycolbis(succinimidylsuccinate);
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
Biochemical and Molecular Analyses of the Streptococcus
pneumoniae Acyl Carrier Protein Synthase, an Enzyme Essential
for Fatty Acid Biosynthesis*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) R6--
The acpP and
acpS genes were cloned from S. pneumoniae by PCR
as described before (27). All of the reagents, plasmids, and cell lines
used for cloning and expression were the same as those described before
(27). Based on the initial sequence of acpS, it was thought
that the acpS open reading frame started at the second Met codon (see GenBankTM accession number
AF276617). Thus, the primers corresponding to this sequence were
designed and used for cloning the S. pneumoniae acpS gene
(see below). However, we have recently realized that the open reading
frame of the acpS gene probably starts at the first Met
codon rather than the second codon (GenBankTM
accession number AF276617). Thus, the acpS gene cloned and expressed might lack the first two codons encoding the amino acid residues of Met-Arg. To clone the acpS gene, the following
PCR primers were designed and used to amplify the acpS gene
for cloning into E. coli expression systems. The 5' PCR
primer (5'-CGCGGATCCCATATGATAGTTGGACACGGAATTG-3') was designed at the
ATG start codon of acpS and contains BamHI and
NdeI sites for cloning purposes. The 3' PCR primer
(5'-CGCGGATCCCTAGCTTTCATGAATTTCCTCC-3') was designed at
the stop codon of acpS and contains a BamHI site after the stop codon. Using these primers, acpS was
PCR-amplified from S. pneumoniae for 25 cycles under the
conditions as described before (27). Five PCR products were combined,
and a portion of the pooled PCR products was digested with
BamHI. The BamHI-digested PCR fragment was cloned
into pCZA342, a low copy number plasmid (28) that had been digested
with BamHI and dephosphorylated with calf intestinal
alkaline phosphatase. acpS from several pCZA342 clones was
sequenced, and a clone containing the consensus acpS gene
sequence was used for constructing expression systems. This pCZA342
clone was digested with NdeI and BamHI. The
NdeI-BamHI DNA fragment containing
acpS was subcloned into pET-11a (Novagen). The resulting
construct was designated as pRBP-19. The pCZA342 clone was also
digested with BamHI, and the BamHI fragment of acpS was subcloned into pGEX-2T, resulting in pRBP-20.
-D-galactopyranoside for 3 h.
Cells were harvested by centrifugation at 4500 × g at
4 °C for 8 min, washed twice in phosphate-buffered saline (PBS),
resuspended in 50 mM citrate phosphate, pH 6.0, and
disrupted by passing twice through a French pressure cell. The
resulting cell extract was centrifuged at 160,000 × g
for 40 min at 4 °C. The supernatant fraction was collected and
applied to a 15S Source S column (2.5 × 8 cm) that had
been equilibrated with 50 mM citrate phosphate, pH 6.0 (buffer A). The column was washed with buffer A and eluted with a
linear gradient of 0-1.0 M KCl in buffer A. Fractions (7 ml each) were collected. The presence of AcpS in the fractions was
detected by SDS-PAGE analysis (16% Tricine gels) (29). The fractions
containing AcpS were pooled and applied to a S-100 Sepharose preparative gel filtration fast protein liquid chromatography column
(5.0 × 60 cm) equilibrated with 50 mM Tris-HCl, pH
7.0, 100 mM KCl. The fractions containing AcpS were
collected, adjusted with glycerol to a final concentration of 15%
(v/v), and stored in small aliquots at 
70 °C. Protein
concentration was determined using a protein assay kit (Bio-Rad) with
bovine serum albumin as a standard (30).
70 °C as described above.
70 °C as described above.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside. Apparently, the
basal level expression of acpS without
isopropyl-1-thio--D-galactopyranoside induction was
sufficient for the complementation of HT253. This result clearly shows
that the S. pneumoniae acpS gene complements the E. coli mutant deficient in the production of AcpS. Attempts to
inactivate the acpS gene of S. pneumoniae through
genetic insertional mutagenesis failed
(28),2 indicating that
acpS is essential for growth. Since recG and alr, downstream to acpS, are not essential genes
(37, 38), we conclude that the acpS gene is essential for
the growth of S. pneumoniae. Together, these results have
established the identity of the gene as acpS and that the
function of acpS is essential for the growth of bacterial cells.
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Fig. 1.
SDS-PAGE analysis of purified S. pneumoniae AcpS and apo-ACP. Both AcpS and apo-ACP were
expressed in E. coli and purified as described under
"Experimental Procedures." The purified AcpS (A) and
apo-ACP (B) were analyzed by SDS-PAGE (16% Tricine gels)
and stained with Coomassie Blue R-250. A, each
lane contained 5 µg of protein. Lane
M, prestained molecular weight markers; lane
1, E. coli crude extract containing overexpressed
S. pneumoniae AcpS; lane 2, pooled
fractions from 15S Source S column; lane
3, pooled fractions from S-100 Sepharose column.
B, each lane contained 10 µg of protein.
Lane M, prestained molecular weight markers;
lane 1, E. coli crude extract
containing overexpressed S. pneumoniae ACP; lane
2, pooled fractions from Source 15Q column; lane
3, pooled fractions from S-100 Sepharose column.
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Fig. 2.
Analysis of the native structure of S. pneumoniae AcpS and apo-ACP by gel filtration column
chromatography. Both AcpS and apo-ACP were purified as described
under "Experimental Procedures." Purified AcpS (27 µM) and apo-ACP (100 µM) were subjected to
gel filtration column (S-75 Sephadex) chromatography, and their
molecular weights were determined as described under "Experimental
Procedures." A, gel filtration column chromatograph.
Peak A contained both AcpS and apo-ACP and had an
elution volume of 10.5 ml with an estimated molecular mass of 53 kDa.
Peak B only contained AcpS and had an elution
volume of 11.5 ml with an estimated molecular mass of 38 kDa. The
arrow C points to the area where apo-ACP was
eluted. Since apo-ACP does not absorb at 280 nm, there was no apparent
protein peak observed. B, SDS-PAGE analysis of the column
fractions. All relevant fractions collected from A were
subjected to SDS-PAGE analysis. The gels were stained with SYPRO Orange
and analyzed by a FluorImager (see "Experimental
Procedures").
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Fig. 3.
Analysis of the S. pneumoniae
AcpS and apo-ACP native structures by cross-linking. AcpS
and apo-ACP were purified (see "Experimental Procedures"). Purified
AcpS (163 µM) and apo-ACP (94 µM) were
treated without or with 19.5 and 9.4 mM sulfo-EGS,
respectively. The resulting AcpS (A) and apo-ACP
(B) preparations were analyzed by SDS-PAGE (16% Tricine
gels). A, lane 1, the prestained
molecular weight marker; lane 2, AcpS untreated
with sulfo-EGS; lane 3, AcpS treated with
sulfo-EGS. B, lane 1, the prestained
molecular weight marker; lane 2, apo-ACP
untreated with sulfo-EGS; lane 3, apo-ACP treated
with sulfo-EGS.
1) and 13,659 ± 1290 (kcat = 9.3 ± 0.9 s1) nmol/min/mg at the low and high
concentrations of apo-ACP, respectively. Thus, at higher apo-ACP
concentrations, the affinity of AcpS for apo-ACP was significantly
decreased (approximately 200-fold), but its catalytic activity was
significantly increased (5-fold). Together, these results indicate that
the S. pneumoniae AcpS may be allosterically regulated by
its substrate, apo-ACP.
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Fig. 4.
Kinetic analysis of the effect of apo-ACP
concentrations on the AcpS activity of S. pneumoniae. AcpS activity was measured using the HPLC
method under the conditions where the CoA concentration was fixed at 20 µM and the apo-ACP concentration was varied from 0.5 to
100 µM (see "Experimental Procedures"). A,
the substrate (apo-ACP) saturation curve of AcpS. B and
C, the double reciprocal plots of the initial velocities of
the enzyme versus the various apo-ACP concentrations (<5
and >15 µM, respectively).
1), respectively (Fig. 5B).
The kcat values determined for AcpS at low
apo-ACP concentrations thus were in good agreement (1.7 versus 2.7 s1).
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Fig. 5.
Kinetic analysis of the effect of CoA
concentrations on the AcpS activity of S. pneumoniae. AcpS activity was measured by the HPLC
method under the conditions where the CoA concentrations were varied
(2.5-600 µM) and the apo-ACP concentration was fixed (2 µM) (see "Experimental Procedures"). A,
the substrate (CoA) saturation curve of AcpS. B, the double
reciprocal plot of the initial velocities of AcpS versus CoA
concentrations.
1) nmol/min/mg. Thus, the
kinetic parameters determined by the trichloroacetic acid precipitation
method are in general agreement with those obtained by the HPLC method.
However, we did notice that the trichloroacetic acid precipitation
method tended to generate variations significantly higher than those of
the HPLC method, especially when apo-ACP was below 1 µM.
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Fig. 6.
Analysis of the mechanism of the
AcpS-catalyzed reaction. AcpS activity was measured using HPLC
methods (see "Experimental Procedures"). A,
double-reciprocal plots of the initial velocities of AcpS
versus the various CoA concentrations (2.5-30
µM) and the fixed apo-ACP concentrations (0.25 ( 
),
0.35 (
), 0.5 (
), and 0.6 µM (
)). B,
double-reciprocal plots of the initial velocities of AcpS
versus the various apo-ACP concentrations (0.25-0.6
µM) and the fixed CoA concentrations (2.5 (), 5.0 (), 10 (), and 20 µM ()).
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Fig. 7.
Analysis of the inhibition kinetics of
3',5'-ADP with respect to apo-ACP and CoA. AcpS activity was
measured in the absence ( ) or presence of 3',5'-ADP (5 () and 10 µM ()). A and B, the
double-reciprocal plots of the initial velocities of AcpS
versus the various CoA concentrations and the fixed apo-ACP
concentration and the various apo-ACP concentrations and the fixed CoA
concentrations (see "Experimental Procedures"), respectively.
C and D, Dixon plots of A and
B, respectively. The CoA concentrations used in C
were 10 () and 40 µM ().
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
(5, 6). At low apo-ACP concentrations, purified S. pneumoniae AcpS had Km values of 0.5 and 11.3 µM for apo-ACP and CoA, respectively, and a
kcat value of 1.8-2.5 s1.
Clearly, both enzymes have significantly higher (20-30-fold) affinities for apo-ACP than for CoA. It is known that the intracellular CoA and ACP concentrations vary depending on the stage of growth and
carbon sources (43-45). The CoA and total ACP pools were estimated to
be 20-90 and 4-10 pmol/108 cells, respectively (23, 24,
26, 43-45). Thus, the CoA and total ACP concentrations in the cell are
approximately 400-1800 and 90-200 µM, respectively, if
the cell volume is assumed to be 0.5 µm3 (46).
Interestingly, the relative affinities of both AcpS enzymes determined
for their substrates apparently correlate with the estimated in
vivo concentrations of CoA and ACP. These kinetic properties and
the in vivo concentrations suggest that under physiological conditions, the concentrations of both CoA and ACP are probably over
the Km values of AcpS even if apo-ACP is a small fraction of the total ACP pool. Therefore, both substrates might not be
a rate-limiting factor in the conversion of apo-ACP to holo-ACP in the
cell. This may explain why ACP exclusively exists as holo-form (1).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: The Lilly Research
Laboratories, Infectious Diseases Research, Eli Lilly and Company, DC
0438, Indianapolis, Indiana 46285.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Cronan, J. E., Jr.,
and Rock, C. O.
(1996)
in
Escherichia coli and Salmonella: Cellular and Molecular Biology
(Neidhardt, F. C.
, Curtiss, R., III
, Ingramham, J. L.
, Lin, E. C. C.
, Low, K. B.
, Magasanik, B.
, Reznikoff, W. S.
, Riley, M.
, Schaechter, M.
, and Umbarger, H. E., eds)
, pp. 612-636, American Society for Microbiology Press, Washington, D. C.
2.
Rock, C. O.,
and Cronan, J. E., Jr.
(1996)
Biochem. Biophys. Acta
1302,
1-16
3.
Smith, S.
(1994)
FASEB J.
8,
1248-1259
4.
Elovson, J.,
and Vagelos, P. R.
(1968)
J. Biol. Chem.
243,
3603-3611
5.
Lambalot, R. H.,
and Walsh, C. T.
(1995)
J. Biol. Chem.
270,
24658-24661
6.
Flugel, R. S.,
Hwangbo, Y.,
Lambalot, R. H.,
Cronan, J. E., Jr.,
and Walsh, C T.
(2000)
J. Biol. Chem.
275,
959-968
7.
Lambalot, R. H.,
Gehring, A. M.,
Flugel, R. S.,
Zuber, P.,
LaCelle, M.,
Marahiel, M. A.,
Reid, R.,
Khosla, C.,
and Walsh, C. T.
(1996)
Curr. Biol.
3,
923-936
8.
Majerus, P. W.,
Alberts, A. W.,
and Vagelos, P. R.
(1965)
Proc. Natl. Acad. Sci. U. S. A.
53,
410-417
9.
Blattner, F. R.,
Plunkett, G., III,
Bloch, C. A.,
Perna, N. T.,
Burland, V.,
Riley, M.,
Collado-Vides, J.,
Glasner, J. D.,
Rode, C. K.,
Mayhew, G. F.,
Gregor, J.,
Davis, N. W.,
Kirkpatrick, H. A.,
Goeden, M. A.,
Rose, D. J.,
Mau, B.,
and Shao, Y.
(1997)
Science
277,
1453-1462
10.
Cole, S. T.,
Brosch, R.,
Parkhill, J.,
Garnier, T.,
Churcher, C.,
Harris, D.,
Gordon, S. V.,
Eiglmeier, K.,
Gas, S.,
Barry, C. E.,
Tekaia, F.,
Badcock, K.,
Basham, D.,
Brown, D.,
Chillingworth, T.,
Connor, R.,
Davies, R.,
Devlin, K.,
Feltwell, T.,
Gentles, S.,
et al..
(1998)
Nature
393,
537-544
11.
Himmelreich, R.,
Hilbert, H.,
Plagens, H.,
Pirkl, E.,
Li, B.-C.,
and Herrmann, R.
(1996)
Nucleic Acids Res.
24,
4420-4449
12.
Kalman, S.,
Mitchell, W.,
Marathe, R.,
Lammel, C.,
Fan, J.,
Hyman, R. W.,
Olinger, L.,
Grimwood, J.,
Davis, R. W.,
and Stephens, R. S.
(1999)
Nat. Genet.
21,
385-389
13.
Kuns, F.,
Ogasawara, N.,
Moszer, I.,
Albertini, A. M.,
Alloni, G.,
Azevedo, V.,
Bertero, M. G.,
Bessieres, P.,
Bolotin, A.,
Borchert, S.,
Borriss, R.,
Boursier, L.,
Brans, A.,
Braun, M.,
Brignell, S. C.,
et al..
(1997)
Nature
390,
249-256
14.
Tomb, J.-F.,
White, O,
Kerlavage, A. R.,
Clayton, R. A.,
Sutton, G. G.,
Fleischmann, R. D.,
Ketchum, K. A.,
Klenk, H. P.,
Gill, S.,
Dougherty, B. A.,
Nelson, K.,
Quackenbush, J.,
Zhou, L.,
Kirkness, E. F.,
Peterson, S.,
et al..
(1997)
Nature
388,
539-547
15.
Gehring, A. M.,
Lambalot, R. H.,
Vogel, K. W.,
Drueckhammer, D. G.,
and Walsh, C. T.
(1997)
Chem. Biol.
4,
17-24
16.
Lam, H.-M.,
Tancula, E.,
Dempsey, W. B.,
and Winkler, M. E.
(1992)
J. Bacteriol.
174,
1554-1567
17.
Takiff, J. E.,
Baker, T.,
Copeland, T.,
Chen, S-M.,
and Court, D. L.
(1992)
J. Bacteriol.
174,
1544-1553
18.
Carreras, C. W.,
Gehring, A. M.,
Walsh, C. T.,
and Khosla, C.
(1997)
Biochemistry
36,
11757-11761
19.
Crosby, J.,
Sherman, D. H.,
Bibb, M. J.,
Revill, W. P.,
Hopwood, D. A.,
and Simpson, T. J.
(1995)
Biochim. Biophys. Acta
1251,
32-42
20.
Kutchma, A. J.,
Hoang, T. T.,
and Schweizer, H. P.
(1999)
J. Bacteriol.
181,
5498-5504
21.
Tropf, S.,
Revill, W. P.,
Bibb, M. J.,
Howood, D. A.,
and Schweizer, M.
(1998)
Chem. Biol.
5,
135-146
22.
Zhou, P.,
Florova, G.,
and Reynolds, K. A.
(1999)
Chem. Biol.
6,
577-584
23.
Jackowski, S.,
and Rock, C. O.
(1984)
J. Bacteriol.
158,
115-120
24.
Vallari, D. S.,
and Jackowski, S.
(1988)
J. Bacteriol.
170,
3961-3966
25.
Jackowski, S.,
and Rock, C. O.
(1983)
J. Biol. Chem.
258,
15186-15191
26.
Heath, R. J.,
and Rock, C. O.
(1996)
J. Biol. Chem.
271,
1833-1836
27.
Zhao, G.,
Meier, T. I.,
Peery, R. B,
Matsushima, P.,
and Skatrud, P. L.
(1999)
Microbiology
145,
791-800
28.
Baltz, R. H., Matsushima, P., Peery, R. B., Hoskins, J.,
Young, M., Norris, F. H., DeHoff, B. S., Rockey, P., Porter,
G., Burgett, S. G., Rosteck, P. R., Skatrud, P. L., and
Jaskunas, S. R. (1997) Abstracts of the 37th ICAAC,
p. 391(S-46), Interscience Conference on Antimicrobial Agents
and Chemotherapy, Toronto, Canada
29.
Laemmli, U. K.
(1970)
Nature
227,
680-685
30.
Bradford, M. M.
(1976)
Anal. Biochem.
72,
248-254
31.
Lambalot, R. H.,
and Walsh, C. T.
(1997)
Methods Enzymol.
279,
254-262
32.
Baltz, R. H.,
Norris, F. H.,
Matsushima, P.,
DeHoff, B. S.,
Rockey, P.,
Porter, G.,
Burgett, S.,
Peery,
Hoskins, J.,
Braverman, L.,
Jenkins, I.,
Solenberg, P.,
Young, M.,
McHenry, M. A.,
Skatrud, P. L.,
and Rosteck, P. R., Jr.
(1998)
Microb. Drug Resistance
4,
1-9
33.
Altschul, S. F.,
Gish, W.,
Miller, W.,
Myers, E. W.,
and Lipman, D. J.
(1990)
J. Mol. Biol.
215,
403-410
34.
Morbidoni, H. R.,
Mendoza, D. D.,
and Cronan, J. E., Jr.
(1996)
J. Bacteriol.
178,
4794-4800
35.
Rawlings, M.,
and Cronan, J. E., Jr.
(1992)
J. Biol. Chem.
267,
5751-5754
36.
Shen, Z.,
and Byers, D. M.
(1996)
J. Bacteriol.
178,
571-573
37.
Kullik, I.,
Jenni, R.,
and Berger-Bachi, B.
(1998)
Gene (Amst.)
219,
9-17
38.
Lloyd, R. G.,
and Low, K. B.
(1996)
in
Escherichia coli and Salmonella: Cellular and Molecular Biology
(Neidhardt, F. C.
, Curtiss, R., III
, Ingramham, J. L.
, Lin, E. C. C.
, Low, K. B.
, Magasanik, B.
, Reznikoff, W. S.
, Riley, M.
, Schaechter, M.
, and Umbarger, H. E., eds)
, pp. 2236-2255, American Society for Microbiology Press, Washington, D. C.
39.
Cooper, C. L.,
Boyce, S. G.,
and Lueking, D. R.
(1987)
Biochemistry
26,
2740-2746
40.
Rock, C. O.,
and Cronan, J. E., Jr.
(1979)
J. Biol. Chem.
254,
9778-9785
41.
Quadri, L. E. N.,
Weinreb, P. H.,
Lie, M.,
Nakano, M. M.,
Zuber, P.,
and Walsh, C. T.
(1998)
Biochemistry
37,
1585-1595
42.
Copeland, R. A.
(1996)
Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis
, Wiley-VCH, Inc., New York
43.
Jackowski, S.,
and Rock, C. O.
(1981)
J. Bacteriol.
148,
926-932
44.
Jackowski, S.,
and Rock, C. O.
(1986)
J. Bacteriol.
166,
866-871
45.
Vallari, D. S.,
Jackowski, S.,
and Rock, C. O.
(1987)
J. Biol. Chem.
262,
2468-2471
46.
Kubitschek, H. E.,
and Friske, J. A.
(1986)
J. Bacteriol.
168,
1466-1467
47.
Keating, D. H.,
Carey, M. R.,
and Cronan, J. E., Jr.
(1995)
J. Biol. Chem.
270,
22229-22235
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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