(Received for publication, August 9, 1995; and in revised form, August 31, 1995)
From the
Holo-acyl carrier protein synthase (ACPS) transfers the 4`-phosphopantetheine (4`-PP) moiety from coenzyme A (CoA) to Ser-36 of acyl carrier protein (ACP) in Escherichia coli. This post-translational modification renders holo-ACP capable of acyl group activation via thioesterification of the cysteamine thiol of 4`-PP. We have purified E. coli ACPS to near homogeneity by exploiting the ability to refold ACPS and reconstitute its activity after elution from an apo-ACP affinity column under denaturing conditions. N-terminal sequencing of ACPS allowed us to identify dpj, an essential gene of previously unknown function, as the structural gene for ACPS. We report herein the 70,000-fold purification of wild-type ACPS and the overproduction and initial characterization of recombinant ACPS from E. coli.
Acyl carrier protein (ACP) ()is a small acidic
protein (8800 Da) responsible for acyl group activation in fatty acid
biosynthesis. The gene encoding ACP (acpP) has been cloned and
overexpressed(1, 2) , and the solution structure of
ACP has been solved by NMR spectroscopy(3) . Homologs of Escherichia coli ACP exist throughout nature in two forms:
either as an integral domain of a much larger multifunctional enzyme
(type I) or as a discrete protein capable of associating with several
other enzymes constituting a multienzyme synthase complex (type II). In
these two forms ACPs play central roles in a broad range of other
biosynthetic pathways that depend on iterative acyl transfer steps,
including polyketide(4) , non-ribosomal peptide(5) ,
and depsipeptide biosynthesis(6) , as well as in the
transacylation of oligosaccharides (7) and
proteins(8) .
A definitive feature of ACP is the 4`-PP
prosthetic group (Fig. 1)(9) . 4`-PP is attached through
a phosphodiester linkage to a conserved serine residue found in all
ACPs. Acyl groups of the many substrates recognized by type I and type
II ACPs are activated for acyl transfer through a thioester linkage to
the terminal cysteamine thiol of the 4`-PP moiety. The -alanyl and
pantothenate portions of the 4`-PP structure are believed to serve as a
tether between the phosphodiester-ACP linkage and the terminal
thioester, suggesting that 4`-PP may function as a swinging arm,
shuttling growing acyl chains between various active sites, e.g. as in the sequential addition of 11 amino acids by the 1.4-MDa
cyclosporin synthetase(10) .
Figure 1:
ACPS transfers the 4`-PP moiety from
CoA to Ser-36 of apo-ACP to produce holo-ACP and 3`,5`-ADP in a
Mg-dependent reaction. Holo-ACP can then activate
acyl groups for acyl transfer through a thioester linkage to the
terminal cysteamine of the 4`-PP moiety.
Holo-ACP synthase (ACPS)
transfers the 4`-PP moiety from CoA to Ser-36 of apo-ACP to produce
holo-ACP and 3`,5`-ADP in a Mg-dependent reaction.
The ACPS from E. coli was partially purified 780-fold from
crude extracts 27 years ago(11) , and the ACPS from spinach has
been partially purified(12) , but remarkably little has been
shown about the mechanism or specificity of this post-translational
phosphopantetheinylation process. A mutant of E. coli conditionally defective in the synthesis of holo-ACP has been
identified and the mutant phenotype attributed to an altered holo-ACP
synthase activity(13) .
To study the mechanism and specificity of ACP-phosphopantetheinylation, we had as our initial objective the cloning and overproduction of ACPS from E. coli. We now report the 70,000-fold purification and N-terminal sequencing of wild-type ACPS. A search of the translated data bases identified a gene (dpj) of previously unknown function, as the gene which encodes ACPS. Overexpression of dpj allowed the preparation of ACPS in 10-mg quantities.
Figure 2:
A, Coomassie-stained 20% native-PAGE; lane 1, apo-ACP; lane 2, holo-ACP standard (Sigma),
reduced with DTT; lane 3, H-labeled holo-ACP
formed in vitro using recombinant ACPS, reduced with DTT; lane 4, holo-ACP standard, not reduced with DTT; lane
5,
H-labeled holo-ACP formed in vitro using
recombinant ACPS, not reduced with DTT. B, autoradiogram of
gel in A confirms introduction of
[
H]phosphopantetheine into holo-ACP (I),
holo-ACP-CoA mixed disulfide (II), and (holo-ACP)
disulfide (III).
The clarified extract (1.3 liter) was titrated to pH 6.5 with a
saturated MES solution and was then loaded at a flow rate of 10 ml/min
onto a 3 30-cm SP-Sepharose column (Pharmacia Biotech Inc.),
which had been pre-equilibrated with 50 mM MES, 10 mM MgCl
, 5% (w/v) glycerol, pH 6.1 (Buffer A). After the
extract was loaded, the column was washed with 750 ml of Buffer A while
collecting 25-ml fractions. The column was then eluted with a linear
0-1 M NaCl gradient (1 liter) in Buffer A. Active
fractions were pooled to yield 190 ml.
This 190 ml of
SP-Sepharose-purified material was next loaded at a flow rate of 2
ml/min onto a 2.5 4.0-cm Affi-Gel 15 apo-ACP affinity column
(Bio-Rad; prepared following the manufacturer's instructions)
while collecting 25-ml fractions. The column was washed with 100 ml of
Buffer A, and ACPS was then eluted with 50 ml of 6 M guanidinium HCl in 50 mM MES, pH 6.1, while collecting
8-ml fractions. ACPS activity was reconstituted by diluting the
guanidinium HCl to a final concentration
2 M in the assay
mixture. Active fractions were pooled to yield 16 ml, which was then
dialyzed against 2
1 liter of Buffer A to yield
0.2 mg of
protein of apparent 70,000-fold purity (Table 1). Tris-Tricine
SDS-PAGE analysis (16) revealed the presence of only a few
major bands (Fig. 3). Previous purifications had demonstrated
that the 14-kDa band copurified with the ACPS activity (data not
shown). Aliquots (500 µl) of the affinity-purified protein were
concentrated by acetone precipitation. The precipitated protein was
resolved by 16% T, 6% C Tris-Tricine SDS-PAGE and then electroblotted
to a Pro-Blot membrane (Applied Biosystems Inc.) following the
manufacturer's instructions. Proteins were visualized by staining
briefly with 0.1% Amido Black in 1% acetic acid. The 14-kDa protein was
excised and submitted for N-terminal sequencing.
Figure 3: Tris-Tricine SDS-PAGE analysis of fractions from purification of wild-type and recombinant ACPS. Lane 1, crude lysate of E. coli K-12; lane 2, DE-52 supernatant; lane 3, SP-Sepharose pool; lane 4, apo-ACP affinity column pool (0.5-ml sample concentrated 20-fold by acetone precipitation); lane 5, SP-Sepharose-purified recombinant ACPS.
Three 1-liter cultures of E. coli BL21(DE3)pDPJ in 2 YT media supplemented with 50 µg/ml
ampicillin were grown at 37 °C, 250 rpm to an O.D. of 0.8-1.0
before transferring the cultures to 30 °C, 250 rpm and inducing
with 100 µM isopropyl
-D-thiogalactopyranoside. Cultures were grown at 30
°C for an additional 3 h and were then harvested by centrifugation.
Cells were resuspended (5 ml/g, wet cell mass) in 50 mM Tris
HCl, 10 mM MgCl
, 5% glycerol, pH
8.0 (Buffer B), and lysed by two passages through a French pressure
cell at 10,000-15,000 p.s.i. Cellular debris was removed by
centrifugation at 16,000
g for 30 min. The cell free
extract was then treated twice with an equal volume of DE-52 slurry (pH
8.0). The DE-52 supernatant was adjusted to pH 6.5 with a saturated MES
solution and loaded onto a 3
30-cm SP-Sepharose column, which
had been pre-equilibrated with Buffer A. The column was washed with 250
ml Buffer A. ACPS was then eluted with a linear 500-ml, 0-1 M NaCl gradient.
In order to initiate purification of ACPS, we sought a rapid
and reliable assay for monitoring ACPS activity through the
purification process. Of the several methods described for the in
vitro determination of ACPS
activity(11, 12, 13) , we chose the
direct-discontinuous radioassay employing
[pantetheinyl-H]CoA and apo-ACP. The
amount of ACPS activity is measured by monitoring the rate at which
radiolabeled pantetheine gets incorporated into holo-ACP. Radiolabeled
holo-ACP is quantified by co-precipitation with bovine serum albumin
using 10% trichloroacetic acid, followed by liquid scintillation
counting of the protein pellet. The formation of
H-labeled
holo-ACP was confirmed by autoradiography of 20% native polyacrylamide
gels.
Earlier reports had indicated that ACPS is a basic protein,
which would not bind to anion exchange resins(11) . A 3-fold
purification was thereby quickly achieved by batchwise DE-52 treatment.
The DE-52 supernatant was then adsorbed onto the cation exchanger resin
SP-Sepharose. Following an extensive wash, the column was eluted using
a linear NaCl gradient (0-1 M). ACPS activity eluted at
approximately 0.35 M NaCl. The submicromolar K of ACPS for apo-ACP previously measured with 780-fold purified
enzyme (11) suggested a very tight binding interaction suitable
for affinity chromatography. We linked apo-ACP to an Affi-Gel 15 matrix
and found that ACPS activity was indeed tightly retained by the apo-ACP
affinity column. ACPS activity did not elute with either high salt or
low pH, although it did elute with apo-ACP. Unfortunately, apo-ACP
elution was not suitable for subsequent purification steps, since
separation of the apo-ACP from ACPS was difficult and trace
contaminants in the apo-ACP preparation prevented identification of the
low abundance ACPS protein. The elution requirement was satisfied when
we determined that we could refold ACPS and reconstitute its activity
following elution with chaotropes under denaturing conditions by
subsequent dilution of the denaturant. While both urea and guanidinium
HCl proved suitable for this purpose, guanidinium HCl (6 M)
was chosen as the preferred eluant to minimize the risk of N-terminal
carbamylation of the target protein. Guanidinium HCl elution of ACPS
activity from the apo-ACP affinity column yielded an apparent
70,000-fold purified preparation. Tris-Tricine SDS-PAGE analysis (16) revealed the presence of only a few major bands (Fig. 3). Previous purifications had demonstrated that the
14-kDa band copurified with the ACPS activity (data not shown). Using
the standard Tris
glycine SDS-PAGE analysis, the 14-kDa protein
migrated with the buffer front, greatly hampering initial detection of
a candidate band for N-terminal sequence analysis. Tris-Tricine
SDS-PAGE analysis offers greater resolution of low molecular weight
proteins and was therefore used for all subsequent analyses of ACPS
containing fractions. Superdex-75 gel filtration chromatography of the
ACPS preparation indicated a native molecular weight of approximately
30,000, suggesting that the native enzyme is a homodimer (data not
shown). The 14-kDa protein was electroblotted and submitted for
N-terminal sequencing. Twenty-five cycles of N-terminal sequencing
yielded a primary sequence of AILGLGTDIVEIARIEAVIARSGDR. A BLAST search (18) of the non-redundant protein data base revealed that the
14-kDa protein is encoded by dpj ( downstream of pyridoxal j),
the second gene in the pdxJ operon(19) .
The dpj gene was amplified from the E. coli genome by
polymerase chain reaction and subcloned into the NdeI/HindIII restriction site of the pET22b vector
(Novagen). Induction of E. coli BL21(DE3)pDPJ and purification
of the ACPS activity yielded 50 mg of protein with >95% purity and
320 milliunits/mg specific activity (Table 2). This corresponds
to at least one-half the specific activity of the partially pure
wild-type preparation. This difference is most probably due to errors
associated with quantification of the dilute wild-type protein
preparation using the Bradford protein assay. DNA sequencing of the
pDPJ construct confirmed the recombinant sequence was correct
(Dana-Farber Molecular Biology Core Facility, Boston, MA). The 14-kDa
overproduced recombinant protein was blotted and submitted for
N-terminal sequencing, which confirmed the first 10 residues as the dpj gene product. Mass spectrometric analysis indicated a
molar mass of 13,950 within 0.2% of the calculated mass of 13,922.
Incorporation of the [H]phosphopantetheine moiety
into apo-ACP by recombinant enzyme was again confirmed by 20% native
gel electrophoresis, followed by autoradiography (Fig. 2).
Holo-ACP migrates slightly faster than apo-ACP on 20% native-PAGE (14) . Furthermore, mass spectral analysis of unlabeled
enzymatic holo-ACP product indicated a molecular weight of 8841
(calculated 8847) in contrast to an observed molecular weight of 8518
(calculated 8508) for the apo-ACP substrate. Steady-state kinetics on
recombinant ACPS using the [
H]CoA radioassay
yielded a K
value of 50 µM for CoA.
As previously reported with partially purified ACPS(11) , we
observed substrate inhibition at apo-ACP concentrations greater than 2
µM. However, we were able to assign an upper limit of
1 µM to the K
value for apo-ACP.
An apparent k
value of
10 min
was measured at saturating CoA and 50 µM apo-ACP.
Identical K
values were obtained for apo-ACP and
CoA with wild-type ACPS under the same assay conditions. Differences
between our kinetic constants and those reported previously, 0.4
µM and 150 µM for K
(apo-ACP) and K
(CoA),
respectively(11) , are most likely attributable to variations
in the apo-ACP and CoA substrate preparations and the assay conditions
employed.
Takiff et al. identified dpj as an essential gene in E. coli by using mini-Tn10 transposons to isolate conditionally lethal mutants(19) . In the absence of tetracycline, a transposon insertion between an essential gene and its natural promoter leads to a lethal phenotype. Tetracycline in the growth medium induces divergent transcription of the tetA and tetR genes within the transposon. This transcription extends beyond the transposon in both directions into the bacterial genes. In this manner, essential genes can be screened by using a conditional growth phenotype dependent upon the presence of tetracycline. These researchers found that transposon insertion into a pyridoxal biosynthetic gene (pdxJ) induced such a phenotype. Upon further analysis, an essential gene downstream of pdxJ, designated dpj, was identified. Takiff et al. were able to define the pdxJ operon as a complex operon comprised of two genes, pdxJ and dpj. They were unable to find any evidence suggesting dpj was related to pyridoxal biosynthesis; however, they did note that dpj contains rare codons characteristic of E. coli genes that are expressed at low levels. This correlates with the fact that even at 70,000-fold apparent purity ACPS is not homogeneous (Fig. 3).
Lam et
al. (20) further characterized a series of mini-Mud insertions in pdxJ and dpj and identified three
classes of suppressors, including mutations in the protease lon. A model proposed in their study suggested that lon may be proteolyzing a second protein, which complements dpj mutants. Interestingly, at least three proteins are known to be
labeled when E. coli SJ16, a pantetheine auxotrophic strain,
is grown in the presence of
[H]
-alanine(21) . These proteins are
ACP, the serine-activating enzyme in enterobactin biosynthesis (EntF)
which we have previously shown to contain 4`-PP and have L-serine activating function(6) , and a 35-kDa protein
of unknown identity. One could propose that there are several isoforms
of ACPS within E. coli each specific for its own substrate.
Perhaps a mutation in lon prevents proteolysis of an ACPS
isoform with relaxed specificity, which complements mutations in dpj. It is not yet known whether EntF or the 35-kDa protein
are substrates for ACPS.
We have purified wild-type holo-ACP synthase (ACPS) to homogeneity from E. coli and have used N-terminal peptide sequence to identify dpj as the gene which encodes ACPS. ACPS appears to be a homodimer with a native molecular weight of 28,000. Overexpression of dpj has allowed the isolation of > 10 mg of active recombinant ACPS. Surprisingly, a search of the GenBank data bases including the recently reported Haemophilus influenzae genome (22) revealed no known genes that share significant homology with dpj. We anticipate that dpj will serve as a valuable tool for the cloning of other ACPSs and will assist in the heterologous overproduction of appropriately modified 4`-PP requiring enzymes, such as PhbC(21) , Dcp(23, 24) , TcmM(4) , and NodF(7) , thereby greatly facilitating mechanistic studies of acyl activating enzymes in macrolide, polyketide, depsipeptide, and non-ribosomal peptide biosynthesis, as well as ACP-dependent transacylase activities. In order to more accurately reflect its newly determined function, we propose a redesignation of the dpj gene as acpS.