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J Biol Chem, Vol. 275, Issue 2, 959-968, January 14, 2000
From the Holo-(acyl carrier protein) synthase (AcpS)
post-translationally modifies apoacyl carrier protein (apoACP) via
transfer of 4'-phosphopantetheine from coenzyme A (CoA) to the
conserved serine 36 Escherichia coli utilizes a repeated cycle of
condensation, reduction, dehydration, and isomerization reactions to
produce saturated and unsaturated fatty acids (1, 2). This biosynthetic mechanism employs multiple enzymatic activities conglomerately termed
the fatty acid synthase. The central coenzyme in all fatty acid
synthases is the holo form of acyl carrier protein.
ACPs,1 as free proteins or as
domains of large multifunctional proteins, function in a variety of
synthases as hubs to which growing acyl intermediates and nascent
product molecules are covalently tethered during the elongation and
modification steps required to produce the final compound. The apo to
holo conversion of ACPs by post-translational modification with the
4'-phosphopantetheine (4'-PP) prosthetic group is essential to their
function, since all acyl chain molecules undergoing biosynthesis by
these systems are attached to the terminal cysteamine thiol of 4'-PP
via a thioester bond.
The E. coli fatty acid synthase has served as a model system
for understanding the 4'-PP post-translational modification, structure,
and function of ACPs. The E. coli ACP is a highly acidic protein of 77 residues. It is composed primarily of a 3-helix bundle
(3, 4), and recent structural studies reinforce the notion that many
ACPs from different synthases and/or organisms are of similar size and
structure to E. coli ACP (5-7).
E. coli ACP is post-translationally modified by holo-(acyl
carrier protein) synthase (EC 2.7.8.7) in a
Mg2+-dependent reaction, where the 4'-PP moiety
is transferred from CoA to the conserved serine 36 A previous study of in vivo ACP phosphopantetheinylation (9)
employed ethyl methanesulfonate mutagenesis of the pantothenate auxotrophic E. coli MP3 strain followed by screening for an
elevated pantothenate growth requirement to identify mutants with
altered AcpS activity. This approach was designed to yield mutants
having an increased Km for CoA, since the
intracellular concentration of CoA can be manipulated in strains
auxotrophic for pantothenate (a CoA precursor) (9). This work resulted
in the identification of the E. coli MP4 strain, which has a
deficiency in AcpS activity and elevated levels of intracellular apoACP
(9). Further study of this mutant by traditional genetic approaches
(such as cloning by complementation) was hindered by the pleiotropic
nature of the mutation and the rapid accumulation of revertants and/or suppressors.
Takiff et al. (10) first identified the E. coli
open reading frame containing the gene that encodes AcpS. At the time,
this gene was of unknown function and was named dpj. It was
identified as an essential gene through the use of the conditional
mutant E. coli HT253 strain, generated by a
mini-Tn10 transposable element deletion inserted upstream to
dpj. This transposon blocks transcription from the upstream
promoter, resulting in a polar effect on downstream genes (10).
Tetracycline induces the divergent promoters within the
mini-Tn10 transposon and partially restores transcription of
downstream genes (10). This analysis and that of Lam et al. (11) show dpj to be essential for the growth of E. coli.
Subsequent identification of dpj as the gene encoding the
E. coli AcpS activity resulted in the renaming of
dpj to acpS (12). This was the first gene known
to encode a phosphopantetheinyltransferase, and its discovery renewed
general interest in PPTases and the role of the 4'-PP
post-translational modification in activating the synthases that
produce a myriad of secondary metabolites (13). The E. coli
acpS sequence led to the identification and subsequent confirmation of other PPTases in a variety of organisms (14). This
includes two additional PPTases in E. coli: EntD, which
phosphopantetheinylates the EntB and EntF members of the Ent
biosynthetic gene cluster responsible for producing the enterobactin
siderophore (15), and YhhU, a previously uncharacterized open reading
frame of 195 amino acids (called o195) (14). Both the entD
and yhhU gene products were demonstrated to modify apoACP
in vitro at a very low rate (14). EntD has been shown to
efficiently modify EntF in vitro, which validates an
in vivo role of EntD in activating EntF consistent with the
co-induction of the two genes and their presence in the same operon.
The very low in vitro activity of the YhhU protein in
modifying EntF and ACP led to the conclusion that the physiological
substrate was an undiscovered acceptor protein (14).
Bacterial Strains and Plasmids--
Strains and plasmids that
were used are listed in Table I. All
bacterial strains are derivatives of E. coli K-12. Strains carrying plasmids were constructed by electroporation or by 50 mM CaCl2 treatment followed by heat shock with
the respective plasmids (16). Strain RF104 was constructed by
transduction of strain MP4 with a P1vir phage lysate grown
on strain HT210 with selection for kanamycin resistance.
Genetic Techniques--
P1 transduction was done according to
Miller (17). Arabinose-inducible plasmids were induced by additions of
0.4% arabinose, alternatively 0.4% glucose was added to decrease
basal expression from the arabinose promoter, or no sugars were added
to give basal level transcription.
Recombinant DNA Manipulations--
General DNA manipulations and
standard molecular biology procedures were carried out as described in
Sambrook et al. (16). Chromosomal DNA was prepared using the
Genome DNA Kit from Bio 101, Inc. Plasmid DNA was purified by Plasmid
Mini Kit from Qiagen, Inc. Southern blot analyses were performed with
the Genius System from Roche Molecular Biochemicals. Restriction
endonucleases were obtained from New England BioLabs, Inc. or Life
Technologies, Inc.
Identification of the Mutation in the acpS Gene of E. coli
MP4--
In the Boston laboratory, four independent PCR amplifications
of the acpS gene from the E. coli MP4 strain were
performed using the forward primer 5'-CGCCATCCCTGAGATGCATGA-3' and the
reverse primer 5'-ACACTGATCCATAATCGCTGT-3'. These primers anneal to the E. coli chromosome at approximately 100 bases upstream and
downstream from the acpS start and stop codons,
respectively. PCR products were purified by agarose gel
electrophoresis, and sequencing revealed a mutation in the
acpS of the E. coli MP4 strain (denoted
acpS1) containing a single G to A transition in the fifth
codon (underlined), 5'-ATGGCAATATTAGATXn. This mutation
corresponds to a G4D substitution in the expressed protein (denoted
AcpS1). This was mutation was confirmed in the Urbana laboratory upon
sequencing of the PCR product obtained using another set of primers.
Cloning of acpS1 from E. coli MP4 for in Vitro Kinetic
Studies--
acpS1 was PCR-amplified using a forward primer
that incorporated an NdeI restriction site (underlined) and
the start codon 5'-TGTACCTCAGACCATATGGCAATATTAGATTTAGGCACGG-3'. The
reverse primer incorporated a HindIII restriction site
(underlined) after the stop codon
5'-TGATGTCAGTCAAGCTTAACTTTCAATAATTACCGTGGCA-3'. The resulting PCR product was digested with NdeI and
HindIII, then subcloned into the corresponding restriction
sites in the pET22b expression plasmid from Novagen, Inc. This
construct was designated pRL102 and subsequently transformed into
BL21(DE3), creating the strain RL102 for overexpression of the AcpS1 protein.
Cloning of acpS for in Vivo Complementation Studies--
The
acpS plasmid pCY314 was made by ligating the
SspI-HindIII yhhU fragment of pTX290
(11) to the annealed product of two phosphorylated oligonucleotides,
5'-GTACCCATGGCTAT-3' and 5'-ATAGCCATGG-3'. The
ligation product was then ligated to pK18 (18) digested with
Acc65I and HindIII to give an acpS
gene having a NcoI site (underlined) overlapping the
acpS initiation codon. The
NcoI-HindIII fragment that encodes a wild type
AcpS protein was then ligated to NcoI plus the
HindIII digest of pBAD22. Finally the
ClaI-ScaI fragment of this construct was replaced
with the ClaI-HincII fragment of pACYC184 to give pCY314.
Cloning of acpS1 from E. coli MP4 for in Vivo Complementation
Studies--
The acpS1 gene was amplified by PCR from the
E. coli MP4 strain in the following manner. An
NcoI site (underlined) was engineered at the start codon
using primers 5'-CGTTTCCCATGGCAATATTAGAT-3' and
5'-GCATGCTGCACAAAATTAAAG-3'. The PCR product was blunt-ended with mung
bean nuclease from New England BioLabs, Inc. and subsequently digested
with NcoI. The NcoI/blunt product was ligated
into NcoI/SmaI double-digested pBAD22 (19) to
produce pYON107. To reduce the copy number, the
ClaI/ScaI fragment of pYON107 carrying
acpS1 was ligated into
HincII/ClaI-digested pACYC184. The resulting plasmid was named pYON108.
Cloning of yhhU from E. coli W3110 for in Vivo Complementation
Studies--
The YhhU overexpression plasmid pYON113 was made using a
PCR-amplified product from the wild type strain W3110 using an
amino-terminal primer containing an introduced AflIII site
(underlined) 5'-GGCCCTGACATGTATCGGATAGTTCTGGGG-3' and a
carboxyl-terminal primer containing an introduced HindIII site (underlined) 5'-GTCGGGTAAGCTTATCAGTTAACTGAATCG-3'. The
PCR products were digested with AflIII plus
HindIII and ligated into pBAD22 digested with
NcoI and HindIII.
Purification of Holo-(Acyl Carrier Protein) Synthases--
AcpS
was overexpressed from strain RL101 and purified according to a
previously developed method (12). An additional purification step for
the protein was appended to this method employing Sephacryl S-300 size
exclusion chromatography from Amersham Pharmacia Biotech to remove a
high molecular weight contaminant. AcpS1 from the E. coli
MP4 strain was overexpressed from strain RL102. Purification of the
AcpS1 was performed in a manner identical to wild type AcpS.
Amino-terminal protein sequencing of the purified AcpS1 confirmed the
presence of the G4D mutation.
Purification of Apoacyl Carrier Proteins--
The E. coli strains DK554, DK675, and DK700 were used to overexpress the
wild type, S36A, and S36T E. coli ACPs, respectively (20).
Purification of apoACP was performed using a scheme involving a
freeze/thaw osmotic shock release of ACP from cells followed by anion
exchange chromatography with Q Sepharose from Amersham Pharmacia
Biotech, Inc. The ACP containing eluant was passed through a Sephacryl
S-100 size exclusion chromatography column from Amersham Pharmacia
Biotech to remove a high molecular weight contaminant and to exchange
the sample into a nonreducing buffer. The ACP pool was then slowly
filtered through Thiopropyl Sepharose 6B from Amersham Pharmacia
Biotech to separate apoACP from the contaminant holoACP, which
covalently binds to the resin. The apoACP-containing flow-through was
then subjected to Sephacryl S-100 size exclusion chromatography a
second time to remove the trace disulfide-linked holoACP dimer, based
on its 2-fold molecular weight difference. Analysis of the relative apo
to holo content of the final purified protein using a previously
developed HPLC method (21, 22) indicated a >99% purity for
apoACP.2
The S36A and S36T apoACPs were purified in an analogous manner. Cells
overexpressing each protein were subjected to the freeze/thaw, Q
Sepharose, and Sephacryl S-100 purification steps. Subsequent filtration through Thiopropyl Sepharose 6B and Sephacryl S-100 were not
performed, since it was expected that overexpression of the S36A and
S36T ACPs would produce primarily the apo form, based on previous
studies (20). Characterization of the purified S36A and S36T apoACPs by
HPLC (21, 22) confirmed the absence of contaminant
holoACP.2
Preparation of [3H]CoA--
[3H]CoA
was obtained through tritium gas exposure by NEN Life Science Products.
Subsequent purification of [3H]CoA from degradation
products was performed using previously described methods (8, 12). The
resulting [3H]CoA had specific activities in the range of
50 to 150 Ci/mol. 70% of the 3H radiolabel resided in the
4'-PP moiety of the [3H]CoA (12).
4'-[3H]PP Transfer Assay--
Studies of AcpS
catalysis of apo to holo-ACP conversion were performed by monitoring
the incorporation of 4'-[3H]PP using an established assay
(8, 12, 14). Typical reaction mixtures contained 1 nM AcpS,
50 µM apoACP, 200 µM [3H]CoA,
10 mM MgCl2, 2.5 mM dithiotheitol,
and 75 mM Tris base, pH 8.8, in a 100 µl volume. After
incubation at 37 °C for 25 min, 900 µl of 10% trichloroacetic
acid was added to quench the reaction. The radiolabeled holo-ACP was
precipitated and isolated by adding 500 µg of bovine serum albumin as
a carrier and centrifugation at 10,000 × g for 5 min.
Unreacted [3H]CoA and 3',5'-[3H]ADP product
were removed by decanting the supernatant and rinsing the protein
pellet three times with 1 ml of 10% trichloroacetic acid. Pellets were
dissolved in 150 µl of 1.0 M Tris base (pH untitrated)
and transferred to scintillation vials containing 2.5 ml of
scintillation fluid from Packard, Inc. The 3H-labeled
holoACP was quantified with liquid scintillation counting. Reagents
incorporated in the reaction mixtures varied depending upon the study
being performed. Such exceptions to protocol are noted in relevant
results sections and figure legends.
Analysis of in Vivo ACP Pools--
ApoACP was analyzed by
urea-PAGE as described previously (23) with the following
modifications. Overnight cultures were diluted into fresh medium
subcultured several generations to mid-log phase and then treated with
5% trichloroacetic acid. After centrifugation, the protein pellets
were washed twice (at 4 °C) with 1% trichloroacetic acid and
resuspended in 1 M urea. These samples were then subjected to a 13% PAGE with 1 M urea. The separated proteins were
subsequently visualized by staining with Coomassie Blue.
Kinetic Characterization of AcpS--
Since the acpS1
mutation was suppressible by high intracellular CoA levels, the
phenotype of the E. coli MP4 mutant strain seemed likely to
be explained by kinetics of the mutant enzyme. However, the kinetic
parameters of the wild type enzyme had to first be determined. Studies
of the 4'-PP post-translational modification catalyzed by the wild type
AcpS were performed with the 4'-[3H]PP transfer assay.
This assay allows for direct measurement of apoACP to holoACP
conversion via trichloroacetic acid precipitation of the
3H-labeled holo product and subsequent quantification by
liquid scintillation counting. The predominant kinetic feature of AcpS was the severe substrate inhibition of the enzyme by apoACP at concentrations >5 µM (Fig.
1, panel A). AcpS had a
Km of 1.5 µM for apoACP and a peak
4'-PP transfer activity of kcat of 65 min NaCl Effects on AcpS Kinetics--
One explanation for the
observed substrate inhibition was an electrostatic interaction between
the basic AcpS enzyme (pI 9.6) and the acidic apoACP substrate (pI
4.1), which should be suppressed by high salt concentrations. Indeed,
at an NaCl concentration of 250 mM, a moderate relaxation
in the substrate inhibition of AcpS by apoACP was observed (Fig.
2). This relaxation was characterized by
a slight increase in the Km for apoACP and an
increase in the kcat of the enzyme at saturating
apoACP concentration. The maximum observed activity for AcpS in the
presence of 250 mM NaCl is 62 min Inhibitors of AcpS--
Prior in vivo work (20) showed
that a mutant ACP in which the target serine 36 had been mutated to a
threonine residue was an inactive substrate for
phosphopantetheinylation. This protein was tested to determine if is
was an inhibitor of AcpS as well as an inactive substrate. The in
vivo results were first confirmed in vitro. S36T apoACP
was overexpressed and purified to >99% homogeneity. In addition, S36A
apoACP was prepared in an identical manner as a
nonphosphopantetheinylatable control. Incubations of AcpS with [3H]CoA and either apoACP, S36A apoACP, or S36T apoACP
were conducted for periods up to 5 h. The results of these assays
clearly indicated the absence of any 4'-[3H]PP labeling
of S36T apoACP or the S36A apoACP control,2 although the
wild type ACP was readily labeled. The apparent inability of AcpS to
phosphopantetheinylate S36T apoACP was also confirmed through
separation of the apo and holo forms of ACPs after 1-h incubations with
AcpS and CoA,2 using a previously developed HPLC method
(21, 22).
S36A and S36T apoACPs were then characterized as inhibitors of AcpS
using the 4'-[3H]PP transfer assay. Several incubations
of AcpS with 10 µM apoACP were performed with
successively increased concentrations of either S36A apoACP or S36T
apoACP in the absence of NaCl2 as well as in the presence
of 500 mM NaCl (Fig. 3). An
IC50 of 10 µM was obtained for S36A apoACP
(Fig. 3, panel A). This value remained nearly the same,
regardless of the presence of NaCl in the reaction mixtures. An
IC50 of 7 µM was obtained for S36T apoACP in
the presence of 500 mM NaCl (Fig. 3, panel B).
An IC50 value for S36T apoACP in reactions conducted in the
absence of NaCl could not be obtained, because it was observed that
adding successively greater concentrations of S36T apoACP to these
incubations resulted in a slight increase in AcpS
activity.2
Ability of S36T apoACP to Relieve AcpS Substrate
Inhibition--
In response to the observation that successively
increasing amounts of S36T apoACP increased AcpS catalysis of 4'-PP
transfer to apoACP in the absence of NaCl, a kinetic study of the S36T apoACP activation of AcpS was performed. 100 µM S36T
apoACP was added to a series of 4'-[3H]PP transfer assays
investigating AcpS catalytic activity with respect to the apoACP
substrate concentration (Fig. 4).
Comparison of the results of this study with the previous
characterization of AcpS kinetics performed in the absence of S36T
apoACP clearly indicated that S36T apoACP activates AcpS through relief
of substrate inhibition by apoACP. In the presence of 100 µM S36T apoACP, AcpS had Michaelis-Menten kinetics with a
kcat of 80 min Identification of G4D Mutation in AcpS1--
The E. coli MP4 phenotype had been attributed to a deficiency of AcpS
activity due to expression of a mutant allele (denoted acpS1) encoding a protein having a postulated elevated
Km for CoA (9). PCR amplification and subsequent DNA
sequencing of acpS1 from E. coli MP4 revealed
that the fifth codon contained a single G to A transition. This base
change resulted in a glycine to aspartate substitution at the fourth
residue in the final acpS1 gene product. Recombinant
E. coli acpS1 was expressed and purified in a manner
identical to that used for AcpS. The G4D mutation in the purified AcpS1
protein was confirmed by amino-terminal protein sequencing.
Kinetic Characterization of AcpS1--
Kinetic characterization of
AcpS1 was conducted in a manner analogous to that of AcpS. The AcpS1
enzyme showed a ~5-fold reduction in its catalytic activity as a
result of the G4D mutation, with a kcat of 7.5 min acpS Mutants Accumulate ApoACP in Vivo--
Previous studies of
acpS activity in vivo utilized the E. coli MP4 acpS1 mutant strain, which has a complex
phenotype (9). Since a characterization of the AcpS1 protein in
vivo was desired, the more straightforward mutant E. coli HT253 strain of Takiff et al. (10) seemed ideal
because under nonpermissive conditions this strain should produce no
AcpS protein. This lack of protein production would preclude formation
of mixed AcpS dimers (having a mutant subunit and a wild type subunit)
which could complicate the phenotypic analysis. However, since strain
HT253 had not been examined biochemically, the ACP species present
in vivo were examined under both the permissive and
nonpermissive conditions. It was expected that if AcpS is the primary
or only enzyme capable of carrying out the 4'-PP modification of ACP in
E. coli, then a mutation that affects the transcription of
acpS should hinder modification of apoACP and result in
considerable accumulation of intracellular apoACP. The acpS
tetracycline-dependent, conditional mutant E. coli HT253 strain was grown overnight under permissive conditions
(the presence of tetracycline to induce the tet promoter), then subcultured under either permissive or nonpermissive (absence of
tetracycline) conditions. The ACP species present were subsequently identified by their migration patterns on urea-PAGE (Fig.
5). Strain HT253 accumulated detectable
quantities of apoACP even in the presence of tetracycline, indicating
that transcription off the mini-Tn10 element inserted
between acpS and its promoter did not result in normal
levels of AcpS production and holoACP synthesis (Fig. 5, lanes
1 and 2). In the absence of tetracycline the apoACP
level increased further, and the holoACP decreased (Fig. 5, lane
3). It should be noted that strain HT253 continued growth for a
few hours after tetracycline removal, an interval that probably
depleted the pool of ACP.
The acpS1 Allele of MP4 Is Responsible for the Mutant
Phenotype--
Experiments were undertaken to demonstrate that the
acpS1 of the E. coli MP4 strain is responsible
for its mutant phenotype, where high intracellular CoA concentrations
are required for growth. The acpS1 gene of the E. coli MP4 strain was cloned behind an inducible araBAD
promoter, and the ability to complement the
tetracycline-dependent acpS conditional mutant
E. coli HT253 strain was tested (Fig. 6). Unlike the wild type acpS
plasmid pCY314, which complemented the mutant without induction of the
arabinose promoter, the plasmid pYON108 carrying the acpS1
mutant allele complemented the mutation only upon induction by addition
of 0.4% arabinose (Fig. 6, plate 3). This experiment
indicated that the acpS1 allele encoded a less active enzyme
and that it is this mutant gene which is responsible for the phenotype
of the E. coli MP4 strain, requiring elevated CoA levels for
growth.
Additional studies of acpS1 examined phenotypes of strains
using the procedure of Polacco and Cronan (9), which was originally employed in the identification of the E. coli MP4 strain.
This assay examined the growth of strains carrying a panB6
lesion in minimal media supplemented with pantothenate in order to
distinguish among strains requiring different levels of intracellular
CoA for viability.
Strain RF103 was generated by transforming the E. coli MP4
strain with the complementing plasmid pDLC140, which carries the wild
type acpS under the control of its native promoter. Growth of this strain in minimal media supplemented with 0.25 µM
pantothenate and in the presence of ampicillin was monitored by light
absorbance at wavelength 600 nm. Comparison of the growth phenotype of
the strain RF103 with strains RF101 (MP3 carrying pBR322) and RF102 (MP4 carrying pBR322) indicates a partial recovery of the mutant phenotype through direct complementation of acpS1 (Fig.
7, panel A).
Strain RF104 was generated through P1 transduction, whereby the
chromosomal acpS1 of the E. coli MP4 strain was
replaced with wild type acpS. Phenotyping of this strain
using the Polacco and Cronan (9) assay also revealed partial recovery
of the MP4 phenotype when compared with strains MP3 and MP4 (Fig. 7,
panel B).
Overproduction of YhhU Suppressed the acpS Mutation of
HT253--
The exact role of the third E. coli PPTase YhhU
remains unclear (14). As with EntD, studies revealed that YhhU modified apoACP in vitro with very low efficiency (14). However,
there were two caveats to these findings. First, the protein was
produced as inclusion bodies, and refolding to the native structure was assumed but not demonstrated. Second, the protein produced contained an
extra glycine residue adjacent to the initiation methionine. Since the
mutation in acpS1 of the E. coli MP4 strain
indicated that the amino terminus played an important role in enyzme
activity, this extra residue might have compromised the activity of the protein.
The ability of the YhhU product to modify apoACP in vivo was
tested. The yhhU gene was placed behind the
araBAD promoter to give pYON113, in which the gene was
expressed from the plasmid promoter and ribosome binding site. When
transformed into the tetracycline conditional E. coli HT253
strain and induced with 0.4% arabinose, YhhU fully complemented the
tetracycline-dependent growth phenotype of the strain (Fig.
8, plate 3) and blocked the accumulation of apoACP (Fig. 9,
lane 4). However, when expression off the arabinose promoter
was not induced or was decreased by the addition of glucose to the
medium, growth of the strain was tetracycline-dependent
(Fig. 8, plate 2 and Fig. 9, lane 3). These results confirm the ability of the yhhU gene product to
functionally replace AcpS in vivo but only when expressed at
high levels. It is proposed that this gene be named acpT to
reflect this property.
Phosphopantetheinyl transfer by AcpS was studied in
vitro, establishing a context for understanding the physiology of
holo-ACP formation in E. coli. The predominant feature of
AcpS steady state kinetics is the unusually severe substrate inhibition
of the enzyme by apoACP (Fig. 1). Although this effect crippled AcpS
catalytic activity by nearly 50% at apoACP concentrations above 10 µM, there appeared to be no physiological basis for the
accumulation of apoACP and substrate inhibition of the enzyme in
vivo. Previous studies of in vivo ACP pools reveal that
the apo from of the protein is not detectable in wild type E. coli (25), implying a rapid and efficient conversion of the
protein to the holo form by AcpS. Indeed, the overexpression and
accumulation of apoACP in E. coli has been shown to be toxic
(20).
Factors that relieve AcpS substrate inhibition in vitro were
identified, providing clues about a possible biophysical mechanism(s) responsible for this effect. High concentrations of NaCl in the 250 to
750 mM range elicited a dramatic relief of the substrate inhibition of AcpS by apoACP (Fig. 2). This effect is attributed specifically to the Na+ cation rather than the
Cl The presence of S36T apoACP was also shown to relieve substrate
inhibition of AcpS by apoACP in the absence of NaCl (Fig. 4), even
though S36T apoACP itself cannot be
phosphopantetheinylated.2 The introduction of the
Having obtained a reasonable kinetic profile for phosphopantetheinyl
transfer by AcpS, the physiology of the E. coli MP4 strain and the basis for its deficiency in holoACP synthesis were
investigated. The E. coli MP4 strain was phenotypically
selected as a strain requiring growth medium supplemented with an
unusually high 25 µM pantothenate concentration (a CoA
precursor) for viability. At 0.25 µM pantothenate, a
level sufficient to support growth of strains having a wild type
acpS gene, E. coli MP4 cultures ceased growth and
had barely detectable holoACP levels. When supplemented with 25 µM pantothenate, E. coli MP4 cultures grew and
contained a mixture of both apo and holo forms of ACP. The rationale of this selection was that the addition of high levels of pantothenate to
the growth medium would increase the intracellular CoA concentration and allow function of a mutant AcpS activity having an elevated Km for CoA (9).
Sequencing of the acpS gene from the E. coli MP4
strain (denoted acpS1) identified a G4D mutation located
immediately amino-terminal to the (V/I)G(V/I)D motif conserved among
currently known PPTases (14). The kinetic profile of the AcpS1 protein
in vitro did not reveal a significantly elevated
Km for CoA. Instead, it is believed to be the
~5-fold reduction in catalytic efficiency kcat/Km of AcpS1 that results
in phenotypic 2- to 10-fold lower in vivo holoACP content of
the E. coli MP4 strain.
Although it had been shown that a point mutation existed in the
acpS1 gene isolated from the E. coli MP4 strain,
the possibility that a second mutation elsewhere in the chromosome
might be responsible for the elevated CoA growth phenotype of this
mutant had not been ruled out. Therefore, genetic analysis of the
acpS1 allele of the E. coli MP4 strain was
undertaken. Complementation of the tetracylcine-dependent
acpS strain HT253 by overproducing the acpS1
allele carrying the MP4 point mutation was successful (Fig. 6). In
addition, complementation and chromosomal replacement of the
acpS1 with wild type acpS in strains RF103 and
RF104, respectively, resulted in partial recovery of the MP4 growth
phenotype (Fig. 7). These results suggest that the mutation in the
acpS1 gene is largely responsible for the high in
vivo CoA concentrations required by the E. coli MP4
strain for growth. The original mapping of the MP4 mutation to a
different locus on the E. coli chromosome (9) could have
been due to a lack of genetic markers then available within the region,
resulting in inaccurate positioning of the mutation. Another
possibility may be that the pleiotropic nature of the mutation and the
rapid accumulation of revertants or suppressors led to mapping of a
suppressor locus.
Since these in vivo results indicate that the
acpS1 mutation is responsible for the phenotype of MP4, the
kinetic properties of the expressed AcpS1 protein must now be
reconciled with the physiology of the E. coli MP4 strain.
The ability of such a modest mutational alteration of AcpS catalytic
efficiency to produce such large physiological effects needs to be resolved.
E. coli AcpS is normally made in little functional excess
over the enzymatic level required to convert all of the apoACP to the
holo form. This was shown by Keating et al. (20), who report that even a 2-fold overproduction of apoACP resulted in detectable accumulation of the unmodified form in vivo. This unusually
close matching of the synthetic capacity of an enzyme to the level
needed for sufficient synthesis of the product means that only a small loss of catalytic efficiency is required to elicit a physiological effect. Therefore, the 5-fold reduction in catalytic efficiency of
AcpS1 when compared with wild type AcpS can readily explain the
accumulation of apoACP observed in the E. coli MP4 strain. However, the suppression of the physiological defect by the elevated CoA levels resulting from pantothenate supplementation remains to be explained.
It is believed that the mechanism of suppression of the effects of the
acpS1 mutation by elevated CoA levels also involves AcpS
synthetic capacity. It is clear that the wild type AcpS does not
function at its maximal synthetic capacity in vivo. Keating et al. (20) show that expansion of the intracellular CoA
pools in cells overproducing apoACP (through pantothenate
supplementation together with a mutation giving a feedback-insensitive
pantothenate kinase) markedly increased the conversion of apoACP to the
holo form. Therefore, in wild type E. coli the activity of
AcpS is limited by the CoA supply, and additional holoACP synthetic
capacity resulted from increased CoA pools. This view is consistent
with the Km for CoA that was obtained for AcpS and
the intracellular concentration of CoA (see below).
It is expected that CoA pool expansion similarly increases the
catalytic capacity of the AcpS1 protein, thus allowing sufficient holoACP synthesis for growth of the E. coli MP4 strain. The
magnitude of the increased synthetic capacity seems likely to be about
2- to 3-fold, since the intracellular concentration of CoA in
glucose-grown E. coli strains is about 50 µM,
whereas the Km for CoA of AcpS1 is 70 µM (28). Since intracellular CoA levels reach >500
µM upon the addition of 25 µM pantothenate
(9), the AcpS1 enzyme should operate at its maximal synthetic capacity
under these conditions to give a holoACP pool about 20-30% that of a wild type strain, a level that allows cell growth (9).
Previous studies also have demonstrated that disruptions of
acpS are inviable but could survive in the presence of
suppressor mutations (11). However, these disruptions were not
confirmed by Southern analysis and may have resulted in the formation
of tandem duplications on the chromosome that might have led to
residual AcpS activity. To rule out such trivial modes of suppression, a Southern analysis was performed on TX2004 (11), an
acpS::MudJ mutant. Using the entire
acpS gene as the probe, the MudJ insertion into
the acpS gene was
confirmed.3 Because
suppressor mutants of E. coli can be isolated despite the
lack of functional AcpS, alternative post-translational modification enzyme(s) for apoACP must exist in the organism.
There is evidence that the two other E. coli PPTases, EntD
and YhhU, are capable of inefficiently modifying apoACP in
vitro (14). Therefore, the ability of EntD and YhhU to modify
apoACP was tested in vivo. Expression of EntD and other
genes involved in enterobactin biosynthesis is under the regulation of
Fur, a transcriptional regulator that responds to intracellular iron concentrations (29). The presence of intracellular iron is sensed by
Fur, and Fur correspondingly acts as a transcriptional repressor for
iron-regulated genes. Regulation of AcpS has not been observed to be
repressed by Fur, and it is unlikely that residual expression of EntD
under normal, iron-rich conditions would provide sufficient activity to
modify apoACP in an acpS Up to this time, not much was known about YhhU other than the fact that
it has sequence homology with other PPTases. Similar to entD
above, the yhhU gene was cloned under an inducible promoter to test for the ability to suppress an acpS One possible explanation of acpS suppression by excess YhhU
is that E. coli evolved two enzymes that are capable of
modifying apoACP due to its crucial role in cell survival. In a normal
cell, AcpS could be the primary enzyme with more efficient activity. The secondary enzyme, YhhU, is less active and thus plays no major role
in 4'-PP modification of apoACP. Mutations in acpS could possibly allow selection for higher expression of the secondary enzyme
to provide apoACP modification for survival.
Previous physiological studies originally revealed the ability of the
E. coli AcpS to maintain complete holoACP pools in
vivo under normal conditions. The results presented in this work
indicate that AcpS also exists at levels minimally required and
operates with an efficiency minimally necessary to ensure the complete conversion of apoACP to holoACP. A disruption of AcpS production or a
small reduction of AcpS catalytic efficiency are capable of reducing
phosphopantetheinyl transfer to the point where significant (and
possibly toxic) levels of apoACP accumulate in vivo. Under circumstances where AcpS activity is compromised, possible mechanisms for physiological recovery by E. coli have been identified
and involve elevations in intracellular CoA pools or increased
expression of the complementing PPTase YhhU such that in
vivo holoACP is maintained at levels sufficient for cellular viability.
We thank David H. Keating for advice, Barry
T. Ballard for assistance in planning and executing the P1
transduction, and Vicki L. Healy for assistance in conducting the
kinetic studies.
*
This work was supported by National Institutes of Health
Grants GM20011 (to C. T. W.), AI15650 (to J. E. C.),
5T32-GM08313-07 (to R. S. F.), and GM16583-03 (to R. H. L.).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.
**
To whom correspondence should be addressed: Dept. of Biological
Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston MA 02115. Tel.: 617-432-1776; Fax: 617-432-0556; E-mail: walsh@walsh.med.harvard.edu.
2
R. S. Flugel, unpublished results.
3
Y. Hwangbo, unpublished results.
The abbreviations used are:
ACP, acyl carrier
protein;
AcpS, holo-(acyl carrier protein) synthase;
PPTase, phosphopantetheinyltransferase;
CoA, coenzyme A;
4'-PP, 4'-phosphopantetheine;
PAGE, polyacrylamide gel electrophoresis;
HPLC, high performance liquid chromatography;
PCR, polymerase chain
reaction.
Holo-(Acyl Carrier Protein) Synthase and Phosphopantetheinyl
Transfer in Escherichia coli*
§,
,, and§**
Committee on Higher Degrees in Biophysics,
Harvard University, Cambridge, Massachusetts 02138, § Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, and
¶ Departments of Microbiology and Biochemistry, University
of Illinois, Urbana, Illinois 61801
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-OH of apoACP. The resulting holo-acyl carrier
protein (holo-ACP) is then active as the central coenzyme of fatty acid
biosynthesis. The acpS gene has previously been identified
and shown to be essential for Escherichia coli growth.
Earlier mutagenic studies isolated the E. coli MP4 strain,
whose elevated growth requirement for CoA was ascribed to a deficiency
in holoACP synthesis. Sequencing of the acpS gene from the
E. coli MP4 strain (denoted acpS1) showed that
the AcpS1 protein contains a G4D mutation. AcpS1 exhibited a ~5-fold
reduction in its catalytic efficiency when compared with wild type
AcpS, accounting for the E. coli MP4 strain phenotype. It
is shown that a conditional acpS mutant accumulates apoACP in vivo under nonpermissive conditions in a manner similar
to the E. coli MP4 strain. In addition, it is demonstrated
that the gene product, YhhU, of a previously identified E. coli open reading frame can completely suppress the
acpS conditional, lethal phenotype upon overexpression of
the protein, suggesting that YhhU may be involved in an alternative
pathway for phosphopantetheinyl transfer and holoACP synthesis in
E. coli.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-OH of ACP (8).
AcpS therefore converts inactive apoACP to its activated holo form.
HoloACP is then capable of being acetylated or malonylated, initiating
the biosynthesis of a fatty acid (1, 2). Upon the completion of the
synthesis of the tethered fatty acyl chain, the acyl holoACP is a
substrate for acyltransferase activities, which link fatty acid
synthesis to phospholipid and lipid A synthesis (1, 2).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Bacterial strains and plasmids used
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1, occurring at an apoACP concentration of 5 µM. The kinetic profile of AcpS as a function of apoACP
concentration and all kinetic data displaying substrate inhibition have
been fit by linear regression with the general substrate inhibition
equation of Cleland (24). AcpS exhibits Michaelis-Menten kinetics and a
Km of 50 µM with respect to CoA as the
variable substrate (Fig. 1, panel B).
View larger version (13K):
[in a new window]
Fig. 1.
Kinetics of AcpS and AcpS1. Panel
A, [4'-3H]PP transfer assays were performed with
AcpS ( 
) and AcpS1 (
) under conditions where the
[3H]CoA concentration was fixed at 200 µM
and the apoACP concentration was varied over the range 0 to 50 µM. Panel B, [4'-3H]PP transfer
assays were performed with AcpS () and AcpS1 () under conditions
where the apoACP concentration was fixed at 50 µM and the
[3H]CoA concentration was varied over the range 0 to 200 µM. Each point represents the average result of three
reactions conducted at a particular substrate concentration.
1. At 500 and 750 mM NaCl, substrate inhibition of AcpS was
dramatically reduced, and the enzyme exhibited near Michaelis-Menten
behavior (Fig. 2). The enzyme continued to maintain a peak activity of ~62 min1. The apoACP concentration at which peak
activity occurred increased ~10-fold as a function of the NaCl
concentration, from 4 to 40 µM.
View larger version (23K):
[in a new window]
Fig. 2.
NaCl induced relief of substrate inhibition
of AcpS by apoACP. [4'-3H]PP transfer assays were
performed with the following NaCl concentrations: , 0 mM; , 250 mM; 
, 500 mM; 
,
750 mM.
View larger version (10K):
[in a new window]
Fig. 3.
Inhibition of AcpS with S36A and S36T
apoACPs. [4'-3H]PP transfer assays were performed
with 10 µM apoACP as substrate and either S36A or S36T
apoACP as a competitive inhibitor, in the presence of 500 mM NaCl to relieve the substrate inhibition effects of
apoACP. Panel A, S36A apoACP was added to assays in
concentrations ranging from 0 to 100 µM, resulting in an
IC50 = 7 µM for AcpS. Panel B,
S36T apoACP was added to assays in concentrations ranging from 0 to 100 µM, resulting in an IC50 = 10 µM for AcpS. Each point represents the average result of
two reactions conducted at a particular inhibitor concentration.
1 and a
Km of 18 µM for apoACP.
View larger version (18K):
[in a new window]
Fig. 4.
Relief of AcpS substrate inhibition with S36T
apoACP. [4'-3H]PP transfer assays were performed
under conditions identical to those outlined for Fig. 3A and
in the presence of 100 µM S36T apoACP ( ). The kinetic
data for AcpS-catalyzed 4'-PP transfer in the absence of S36T apoACP
() is shown as a reference. The presence of the
nonphosphopantetheinylatable S36T apoACP clearly activates AcpS through
relief of the substrate inhibition by apoACP.
1 at saturating apoACP concentration and a peak
activity of kcat = 10 min1 at an
apoACP concentration of 5 µM (Fig. 1, panel
A). Substrate inhibition of AcpS1 by apoACP was significantly less
severe than that which was observed for AcpS. The AcpS1
Km for apoACP was 2.5 µM, only
marginally greater than that observed for AcpS. AcpS1 had
Michaelis-Menten kinetics with respect to the CoA substrate with a
Km of 75 µM (Fig. 1, panel
B).
View larger version (81K):
[in a new window]
Fig. 5.
Accumulation of apoACP in strain HT253.
13% urea-PAGE of proteins stained with Coomassie Blue. Lane
1, proteins of the wild type strain W3110. Lane 2,
proteins of the tetracycline-dependent acpS
conditional strain HT253, grown overnight in tetracycline-containing
medium and subcultured onto the same medium and harvested at mid-log
phase after 2-3 cell doublings. Lane 3, same as lane
2 but subcultured in the absence of tetracycline. Lane
4, apoACP standard.
View larger version (182K):
[in a new window]
Fig. 6.
Complementation of the
tetracylcine-dependent acpS conditional
strain HT253 by the acpS1 allele of strain MP4.
Plate 1, medium containing tetracycline; plate 2,
medium lacking tetracycline; plate 3, medium lacking
tetracycline but supplemented with 0.4% arabinose for induction of
gene expression. Sections: a, strain containing pYON110
(vector); b, strain containing pCY314 (acpS);
c, strain containing pYON108 (acpS1).
View larger version (12K):
[in a new window]
Fig. 7.
Growth phenotypes demonstrating
complementation and necessity of acpS1 in MP4.
Panel A, growth of strains RF101 ( ), RF102 (), and
RF103 () in minimal media supplemented with 0.25 µM
pantothenate and ampicillin. Panel B, growth of strains MP3
(), MP4 (), and RF104 () in minimal media supplemented with
0.25 µM pantothenate.
View larger version (168K):
[in a new window]
Fig. 8.
Suppression of the
tetracycline-dependent acpS conditional
strain HT253 by yhhU expressed from the
araBAD promoter. Plate 1, medium
containing tetracycline. Plate 2, medium lacking
tetracycline. Plate 3, medium lacking tetracycline but
supplemented with 0.4% arabinose for induction of gene expression.
Section A, strain containing pBAD22 (vector). Section
B, strain carrying pYON113 (yhhU).
View larger version (74K):
[in a new window]
Fig. 9.
Modification of apoACP in strain HT253 upon
overexpression of yhhU. 13% urea-PAGE of
proteins stained with Coomassie Blue. Lane 1, strain
containing pBAD22 (vector) in medium containing tetracycline and
glucose. Lane 2, strain containing pBAD22 (vector) medium
containing tetracycline and arabinose. Lane 3, strain
containing pYON113 (yhhU) with medium containing
tetracycline and glucose. Lane 4, same as lane 3 except that the medium contained arabinose and lacked tetracycline.
Lane 5, strain containing pYON113 in medium containing
tetracycline and arabinose. Lane 6, apoACP standard.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
anion, since other monovalent and divalent chloride
salts have different impacts on AcpS kinetics.2 Monovalent
and divalent cations are known to activate specific component enzymes
in E. coli fatty acid biosynthesis (26) and can influence
ACP conformational stability and structure (27). It should be noted
that the severe substrate inhibition of AcpS observed in the absence of
NaCl is not likely to represent the true intracellular kinetic profile
of the enzyme, given the ionic content of cells.
-CH3 in S36T apoACP blocked the conversion of this
protein to the holo form, but the binding of S36T apoACP by AcpS may
induce an allosteric change in the homodimeric enzyme that relieves
substrate inhibition by apoACP. Based on these findings, it is believed
that AcpS substrate inhibition may arise from the presence of multiple
apoACP conformers, improper binding of apoACP to active site(s) on
AcpS, and/or negative cooperativity between apoACP binding sites on the
AcpS homodimer. Additional structural information about AcpS will be
required to fully understand the nature of this complex kinetic behavior.
strain. Nonetheless,
acpS suppression by derepression of entD by iron
starvation was examined, and no suppression of acpS was found under these conditions.3 Cloning of entD
under an inducible promoter and testing its ability to suppress an
acpS phenotype gave a negative
result,3 despite the fact that the EntD that was expressed
was capable of phosphopantetheinylating EntF in crude cell extracts.
This provides further evidence that entD is not able to
suppress acpS and modify apoACP in vivo.
phenotype. Induction of yhhU allowed the HT253 conditional
strain to grow in nonpermissive media lacking tetracycline, as well as completely modifying all of the apoACP pool (Figs. 8 and 9). The fact
that the in vitro analysis of YhhU demonstrated such low activity on apoACP (14) does not necessarily contradict this result.
One of the problems with the in vitro analysis of YhhU was
that the initial attempts in cloning and purification of YhhU led to an
additional glycine after the start site. Another problem was that the
purification process led to an inclusion-body form of YhhU that might
have not been properly refolded (14). Improper folding of YhhU could
have been responsible for the low in vitro activity. This
work demonstrated that YhhU is able to modify apoACP when overexpressed
in conjunction with an acpS mutation.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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R. Finking, J. Solsbacher, D. Konz, M. Schobert, A. Schafer, D. Jahn, and M. A. Marahiel Characterization of a New Type of Phosphopantetheinyl Transferase for Fatty Acid and Siderophore Synthesis in Pseudomonas aeruginosa J. Biol. Chem., December 20, 2002; 277(52): 50293 - 50302. [Abstract] [Full Text] [PDF]
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S. Y. Gerdes, M. D. Scholle, M. D'Souza, A. Bernal, M. V. Baev, M. Farrell, O. V. Kurnasov, M. D. Daugherty, F. Mseeh, B. M. Polanuyer, et al. From Genetic Footprinting to Antimicrobial Drug Targets: Examples in Cofactor Biosynthetic Pathways J. Bacteriol., August 15, 2002; 184(16): 4555 - 4572. [Abstract] [Full Text] [PDF]
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M. R. Mofid, R. Finking, and M. A. Marahiel Recognition of Hybrid Peptidyl Carrier Proteins/Acyl Carrier Proteins in Nonribosomal Peptide Synthetase Modules by the 4'-Phophopantetheinyl Transferases AcpS and Sfp J. Biol. Chem., May 3, 2002; 277(19): 17023 - 17031. [Abstract] [Full Text] [PDF]
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T. Yokochi and K. D. Robertson Preferential Methylation of Unmethylated DNA by Mammalian de Novo DNA Methyltransferase Dnmt3a J. Biol. Chem., March 29, 2002; 277(14): 11735 - 11745. [Abstract] [Full Text] [PDF]
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 Z. Suo, C. C. Tseng, and C. T. Walsh Purification, priming, and catalytic acylation of carrier protein domains in the polyketide synthase and nonribosomal peptidyl synthetase modules of the HMWP1 subunit of yersiniabactin synthetase PNAS, December 22, 2000; (2000) 21537498. [Abstract] [Full Text]
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K. A. McAllister, R. B. Peery, T. I. Meier, A. S. Fischl, and G. Zhao Biochemical and Molecular Analyses of the Streptococcus pneumoniae Acyl Carrier Protein Synthase, an Enzyme Essential for Fatty Acid Biosynthesis J. Biol. Chem., September 29, 2000; 275(40): 30864 - 30872. [Abstract] [Full Text] [PDF]
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H. D. Mootz, R. Finking, and M. A. Marahiel 4'-Phosphopantetheine Transfer in Primary and Secondary Metabolism of Bacillus subtilis J. Biol. Chem., September 28, 2001; 276(40): 37289 - 37298. [Abstract] [Full Text] [PDF]
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Z. Suo, C. C. Tseng, and C. T. Walsh Purification, priming, and catalytic acylation of carrier protein domains in the polyketide synthase and nonribosomal peptidyl synthetase modules of the HMWP1 subunit of yersiniabactin synthetase PNAS, January 2, 2001; 98(1): 99 - 104. [Abstract] [Full Text] [PDF]
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