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From the
Received for publication, August 30, 2002, and in revised form, September 13, 2002
The anaerobic pathway for unsaturated fatty acid
synthesis was established in the 1960s in Escherichia coli.
The double bond is introduced into the growing acyl chain by FabA, an
enzyme capable of both the dehydration of Unsaturated fatty acid
(UFA)1 biosynthesis is
essential for the maintenance of membrane structure and function in
many groups of bacteria that embrace the anaerobic life style. In
eukaryotes, olefin formation requires molecular oxygen (1), and double bonds are introduced into the fatty acids following the completion of
their synthesis via the type I, multifunctional fatty acid synthase
(2). In contrast, bacteria synthesize fatty acids using the
dissociated, type II fatty acid synthase system in which each of the
steps is catalyzed by distinct enzymes that are encoded by separate
genes (3, 4). The key players in UFA synthesis were first defined by
the isolation and characterization of UFA-auxotrophs (5). In the type
II system, the double bond is introduced anaerobically into the growing
acyl chain at the 10-carbon intermediate by The availability of numerous bacterial genomes sequences allows the
reconstruction of type II fatty acid synthase in these organisms using
standard bioinformatics analysis tools. It is notable that
fabA and fabB genes occur together in most
bacteria that produce UFA (15). However, many anaerobes that synthesize UFA, such as the Streptococci and Clostridia, do not have a
recognizable fabA homolog in their genomes, and also have a
fabF rather than a fabB subtype of elongation
condensing enzyme. Clearly, UFA are synthesized by a distinct
biochemical mechanism in these organisms, and the goal of this study
was to identify the enzyme(s) responsible for olefin formation in
Streptococcus pneumoniae. Like E. coli, S. pneumoniae produces straight-chain saturated and monounsaturated fatty acids predominately of 16 and 18 carbon chain lengths (16). Our
experiments show that this organism does not utilize a FabA-like mechanism for introducing a double bond into the growing acyl chain,
but rather accomplishes this task using a previously unknown enzyme,
termed trans-2, cis-3-decenoyl-ACP isomerase
(FabM). Reconstitution of the S. pneumoniae UFA synthetic
pathway in vitro and in vivo lead to the
conclusion that the branch point for UFA synthesis occurs at the
enoyl-ACP intermediate, and the amount of UFA produced arises from the
competition of FabM and FabK for enoyl-ACP.
Materials--
Sources of supplies were: Amersham Biosciences,
[2-14C]malonyl-CoA (specific activity, 56 mCi/mmol);
Sigma, antibiotics, acyl-CoA, ACP; Difco, microbiological media;
Promega, molecular reagents and restriction enzymes; Invitrogen, T4
ligase; Novagen, pET vectors and expression strains; Qiagen,
Ni2+-agarose resin. Protein was quantitated by the Bradford
method (17). The Mycobacterium tuberculosis mtFabH, E. coli ecFabD, ecFabG, ecFabA, S. pneumoniae spFabZ, and
spFabF proteins were purified as described previously (18-22). All
other chemicals were reagent grade or better.
Cloning and Purification of FabM--
The fabM gene
was amplified from genomic DNA from S. pneumoniae R6.
The spfabM PCR primer pair consisted of
5'-AAATAAAAAGGAGCCCATATG and
5'-GGATCCTCAAAGAATGATGCAAG. The primers introduced
novel restriction sites for NdeI at the initiator methionine
codon of the predicted coding sequence and BamHI downstream
of the stop codon. The PCR products were ligated into the plasmid
pCR2.1 and sequenced to verify the absence of PCR mutations. The
plasmid was isolated and digested with NdeI and
BamHI, and the gene fragment was isolated and ligated into
plasmid pET-15b digested with the same enzymes. The resulting plasmid
was used to transform strain BL21(DE3) codonplus-RIL strain, and the
protein was overexpressed and purified as described previously (20).
Affinity chromatography was followed by gel filtration on Superdex-200
HR 26/60. The enzyme was homogeneous as judged by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. The apparent molecular
weight of FabM was estimated by gel filtration chromatography using a
Superdex-200 HR10/30 column calibrated with globular protein standards.
Measurement of FabM Isomerase Activity in Vitro--
Cycles of
fatty acid elongation were reconstituted in vitro to detect
isomerase activity using the purified individual enzymes that catalyze
the fatty acid biosynthesis cycle essentially as described previously
(20, 23). The reaction mixtures contained 100 µM ACP, 10 mM dithiothreitol, 0.1 M sodium phosphate
buffer, pH 7.0, 100 µM NADPH, 100 µM NADH,
50 µM octanoyl-CoA, 100 µM [14C]malonyl-CoA (specific activity, 56 mCi/mmol), mtFabH
(1.0 µg/reaction), ecFabD (1.0 µg), ecFabG (1.0 µg), spFabZ (2.3 µg/reaction), ecFabA (2 µg/reaction), spFabF (3 µg) or spFabM (5 µg) in a final volume of 40 µl. The assay mixtures were incubated
at 37 °C for 20 min and analyzed by conformationally sensitive gel
electrophoresis in 15% polyacrylamide gels containing 2.5 M urea. Electrophoresis was performed at 25 °C and 32 mA/gel. The gels were dried, and the bands were quantitated using a
phosphorimager screen. Specific activities were calculated from the
slopes of the plot of product formation versus protein
concentration in the assay. Bands were identified based on the
generation of standards as described previously (20, 21). The substrate
specificity of FabM was addressed by substituting 100 µM
either decanoyl-CoA, lauroyl-CoA, or myristoyl-CoA for octanoyl-CoA in
the assay.
Direct Detection of FabM Isomerase Activity--
This assay
measures spectrophotometrically the conversion of
trans-2-octenoyl-NAC to cis-3-octenoyl-NAC. A
solution containing 100 µM
trans-2-octenoyl-NAC in 10 mM potassium
phosphate, pH 7.0, was placed in a cuvette, and the substrate
concentration was verified by determining the absorbance at 263 nm
( Mass Spectrometry--
The solutions of acyl-ACP derivatives
from the reconstituted assays described above were adjusted to 2%
acetic acid, and the protein mass determinations were performed by the
Hartwell Center for Bioinformatics and Biotechnology at St. Jude
Children's Research Hospital. Approximately 100 pmol of the protein
were diluted to 50 µl with 2% acetonitrile, 1% acetic acid. The
protein was loaded on a C8 reversed phase nano-extraction
cartridge (Western Analytical, Murrieta, CA) and washed extensively
with the same buffer to remove bound salts. The protein was eluted from
the column with 30 µl of 80% acetonitrile, 1% acetic acid and then
diluted with 1% acetic acid to a final acetonitrile concentration of
50%. Mass measurements were performed using an LCT electrospray-time
of flight spectrometer (Micromass Inc, Beverly, MA) equipped with a
Z-spray electrospray interface (Micromass Inc, Beverly, MA). A flow
rate of 10 µl/min was maintained using a VLP200 syringe pump (Harvard
Apparatus, Holliston, MA), and the desalted protein was introduced by
loop injection. Data were collected for an m/z
range of 500-2500 at a cone voltage of 35 V and a manual pusher time
of 70 µs. All other instrument settings are those typically used for
protein measurements on this instrument. Deconvolution of the protein spectrum was accomplished using the maximum entropy algorithm of the
MassLynx software (Micromass Inc, Beverly, MA) (25).
Complementation Assay using fabA(Ts) Strain--
Strain JT60
(fabA(Ts)) was unable to grow at the non-permissive
temperature (42 °C) unless unsaturated fatty acids (oleate) were
supplied or the fabA gene expressed (26). The ability of spFabM alone or in conjunction with caFabK to complement the
fabA(Ts) phenotype was tested after transformation of JT60
with plasmids carrying either spfabM, cafabK, or
both. The same vector (pBluescript KSII(+)) and construction method
were used to constitutively express the His-tagged versions of spFabM,
caFabK, and ecFabA (20). The Clostridium acetobutylicum
cafabK gene (CAC3576) was amplified from genomic DNA
from the ATCC strain 824. The cafabK PCR primers were:
5'-CATATGTTAAAAACTCAGTTTTGTG and
5'-GGATCCCCTATTTAATTCTATCTATAACT. The primers
introduced restriction sites for NdeI at the initiator methionine and BamHI downstream of the stop codon. The
caFabK protein was expressed from pBluescript and found to substitute for all of the spFabK functions. Like plasmids expressing
spfabK (27), cafabK expression restored growth of
an E. coli fabI(Ts) mutant at 42 °C,
illustrating its function as an enoyl reductase in type II fatty acid
synthesis in vivo. Expression of caFabK also conferred
triclosan resistance to E. coli. In order to select for the
presence of both FabK and FabM, pBluescript vector bearing the
cafabK was modified to replace the AmpR gene
with a KanR cassette using AvaII and
DraIII restriction sites. The pBluescript (empty vector),
pfabA (positive control), pfabM, pfabK, or a combination of
pfabK/pfabM, pfabK/pfabA were transformed into JT60 and selected with
the appropriate antibiotic combinations. Cells were grown at the
permissive temperature for the host strain (30 °C), and individual
colonies were spotted onto rich broth agar plates without or with 0.15 µg/ml triclosan and incubated at 42 °C. Plates were scored for
growth after 24 h at 42 °C.
Characteristics of FabM--
An analysis of the type II fatty acid
biosynthetic genes in S. pneumoniae show that they cluster
at a single location within the genome (Fig.
1). A comparison of the predicted protein
sequences of these open reading frames to the known enzymes of E. coli showed that the S. pneumoniae gene cluster
lacked both FabI and FabA homologs. Recently, the open reading
frame termed fabK (Fig. 1) was demonstrated to encode a
novel flavoprotein enoyl-ACP reductase that replaces FabI in the
S. pneumoniae type II system (27). There are two unknown
genes at the end of the cluster of known fatty acid biosynthetic genes.
One gene, SP0416, is predicted to encode a helix-turn-helix DNA binding
protein of the MarR family that may be a transcriptional regulator
involved in controlling the expression of this gene cluster. Adjacent
to the transcription factor is the SP0415 open reading frame that
is renamed in this work as fabM.
Further bioinformatics analysis of the gene cluster reveals a potential
connection between the fabM, HTH (SP0416), and
fabK genes. The MarR transcription factor is a dimer that
utilizes a winged-helix motif to bind a DNA palindrome (28, 29). Often bacterial transcription factors are autoregulated, and their DNA binding motifs are located within their own promoter regions. A DNA
palindrome was located in the promoter region of the putative SP0416
transcriptional regulator (Fig. 1). Significantly, this same sequence
palindrome is found in the promoters of the fabM and
fabK genes (Fig. 1). Unraveling the transcriptional
regulation in this large cluster is beyond the scope of this study. The
significance of the bioinfomatic analysis is that it ties
fabM to the fatty acid biosynthetic gene cluster and
suggests that the fabM and fabK genes may be
coordinately regulated.
The fabM open reading frame specifies a protein that is a
member of the hydratase/isomerase superfamily (Pfam 000378, Ref. 30). A
comparison of the predicted protein sequence to three members of this
superfamily and to the family consensus sequence is illustrated in Fig.
2. FabM has a strong similarity to the consensus sequence of Pfam 000378 (Fig. 2) exhibiting 28% identity over the 169-amino acid sequence. This family of enzymes catalyze a
wide variety of reactions centered on double bond isomerizations and
water addition and elimination at the Expression and Purification of FabM--
The FabM open reading
frame was cloned into pET-15b, and the His-tagged fusion protein
purified by affinity chromatography and gel filtration as described
under "Experimental Procedures." The purified protein has a
monomeric molecular size of 31 kDa (Fig.
3). Members of the hydratase/isomerase
protein family are uniformly multimeric proteins with hexamers of
identical subunits being a common configuration, although some members
are dimers and tetramers. We therefore examined the size of native FabM
by gel filtration chromatography to estimate its aggregation state (Fig. 3). These results are consistent with FabM existing as a tetramer
of identical subunits in solution.
Enzymatic Activity of FabM--
The ability of FabM to act as an
isomerase was tested in a reconstituted fatty acid biosynthetic system
designed to detect isomerase activity (Fig.
4). Cycles of fatty acid elongation were reconstituted in vitro using purified enzymes as described
under "Experimental Procedures." The assay employed the FabH enzyme from M. tuberculosis to generate
The reconstituted fatty acid synthase assay is a crude tool for
detailed biochemical characterization of an individual enzyme specificity, but we employed this assay to evaluate the substrate specificity of FabM isomerase (Fig. 4, panel B). The
mtFabH/FabG/spFabZ system was used to present FabM with different chain
length enoyl-ACP substrates. The formation of an elongation product
arising from FabM isomerase activity as a function of FabM protein in
the assay was used to estimate the activity of FabM. FabM was most
active when the 10-carbon enoyl-ACP was presented as the substrate
exhibiting a specific activity under these defined conditions of
1.6 ± 0.09 pmol/min/µg. The 12-carbon enoyl-ACP was also
utilized in vitro, albeit at a much lower rate (0.4 ± 0.08 pmol/min/µg). We examined 14- and 16-carbon enoyl-ACPs as
substrate; however, there was no evidence for isomerization of these
longer substrates (not shown). The low activity of mtFabH with
hexanoyl-CoA (18) did not permit the analysis of FabM activity on
trans-2-octenoyl-ACP using this assay. The specific activity
of ecFabA for the 10-carbon substrate under these same assay conditions
was 33 ± 1 pmol/min/µg. Thus, FabM was 20-fold less efficient
than ecFabA in the formation of cis-double bonds in the
in vitro fatty acid synthase assay reconstituted with the
indicated constellation of enzymes and the E. coli ACP
cofactor. These data are consistent with FabM, like ecFabA (20, 37),
being most active on 10-carbon enoyl-ACP, but capable of isomerizing
longer chain substrates at a lower rate.
Mass Spectrometry Analysis of FabM Products--
The ACP thioester
intermediates in the reconstitution assays (Fig. 4A) were
analyzed by electrospray ionization mass spectrometry (ESI-MS) to
confirm the identities of the products (Fig.
5). The major mass peak for the ACP
starting material occurred at 8849 with a minor peak at mass 8980 corresponding to the ACP molecules that retained the amino-terminal
methionine residue. A new mass peak of 9019 with the expected mass
increase of 170 appeared in the Direct Demonstration of FabM Isomerase Activity--
We employed a
direct assay for isomerase activity using substrate analogs to
complement reconstituted fatty acid synthase assay. Although the
enzymes of E. coli fatty acid synthesis are highly specific
for ACP thioester substrates, in all cases these enzymes utilize
substrate analogs (either CoA or NAC thioesters) presented in high
concentrations in biochemical assays (38, 39). Thus, we developed an
assay employing the substrate analog trans-2-octenoyl-NAC
that was modeled on the spectrophotometric assay using NAC substrate
analogs to detect the isomerization activity of ecFabA (40).
Enoyl-thioesters absorb at 263 nm (24), and this absorption is lost
upon conversion to the cis-3-thioester. FabM was capable of
isomerizing trans-2-octenoyl-NAC (Fig.
6). FabM activity was dependent on time
and protein concentration (Fig. 6, panel A) and increased
with increasing substrate concentration (Fig. 6, panel B).
FabM activity was linear for the first minute, and although relatively
high concentrations of the substrate analog were employed, it was clear
from panel B that the experiments were performed at
octenoyl-NAC concentrations that were below saturation. The specific
activity calculated using 200 µM
trans-2-octenoyl-NAC was 87 ± 2.8 pmol/min/µg. The
importance of these studies with a substrate analog was that they
provided a direct demonstration that FabM alone is capable of
catalyzing the isomerization of enoyl-thioesters.
Activity of FabM in E. coli--
We cloned fabM,
spfabZ, and ecfabA into pBluescript vectors and
used these constructs to test whether the gene could complement the
temperature-sensitive growth phenotype of strain JT60
(fabA(Ts)). As expected, the fabA construct
permitted growth at 42 °C; however, the constructs engineered to
express fabM or spfabZ did not. All strains grew
at 42 °C on plates supplemented with oleate. We also cloned
fabM into a plasmid that places it under arabinose
regulation. However, complementation was not observed at any
concentration of arabinose tested (data not shown). These data
illustrate that fabM isomerase activity alone cannot
substitute for fabA within the context of the E. coli fatty acid synthase system.
We attributed this result to the differences in the overall
organization of the UFA biosynthetic pathway in E. coli
and S. pneumoniae (Fig. 7).
The idea was that the complementation experiments described above
failed because of the inability of FabM to successfully compete with
FabI for the available enoyl-ACP within the context of the E. coli fatty acid synthase system. In S. pneumoniae, FabI is absent and FabM competes with FabK. We hypothesized that FabM would
be capable of complementing fabA(Ts) mutants if the cells expressed FabK, and the endogenous FabI activity was eliminated. This
was accomplished by introducing FabM and FabK expression using plasmids
and ablating FabI activity with the potent inhibitor triclosan. FabI is
exquisitely sensitive to triclosan (38, 41), whereas FabK is not
affected by this drug (27). If this hypothesis was correct, then
fabM would only be able to complement the
fabA(Ts) temperature-sensitive growth defect in the presence
of fabK and triclosan.
The results from this complementation experiment are summarized
in Table I. At 42 °C in the
absence of triclosan, only strains expressing the fabA gene
were able to grow. In the presence of triclosan at 42 °C, the only
strains that grew were those harboring the pfabK plasmid in
conjunction with either the pfabA or pfabM plasmids. Thus, FabM was capable of complementing fabA(Ts)
mutants only when FabI activity was abolished and in the presence of
FabK. These data corroborate the conclusion that FabM functions as an isomerase in type II fatty acid synthase in vivo.
The study of the mechanism of oxygen-independent double bond
formation was brought to the forefront by Konrad Bloch's research group (42) whose interest in the anaerobic bacteria in the clostridium family was piqued by the presence of unsaturated fatty acids in these
organisms. Bloch pursued his investigation of the unsaturated fatty
acid pathway in E. coli culminating in the discovery of FabA, and in the 40 years since this discovery, FabA in combination with FabB, became the paradigm for anaerobic olefin formation (see
Introduction). The advent of genome sequencing and bioinformatics genomic analysis reveals the surprising result that several groups of
bacteria contain neither FabA nor FabB, although they have an anaerobic
metabolism and synthesize unsaturated fatty acids. Specifically,
S. pneumoniae possess only the FabZ dehydratase, and lacks
both FabA and FabB enzymes (Fig. 1). Thus, this bacterium must employ a
different mechanism for the formation of unsaturated fatty acids. The
first possibility was that the FabZ-related protein expressed in this
organism (Fig. 1) not only dehydrates The findings in this study, coupled with the recent discovery of
enoyl-ACP reductase II, FabK, reveals the fundamental differences between type II fatty acid synthesis in S. pneumoniae and
the model organism, E. coli (Fig. 7). In E. coli,
the branch point in unsaturated fatty acid synthesis occurs at the
level of the FabM homologs are only found in the Streptococcus species
and thus the discovery of FabM does not explain how all anaerobic bacteria synthesize UFA. Other genera, like the Clostridia, contain UFA, but do not possess either a FabA or a FabM homolog in their genomes. Thus, it is likely that organisms in this group utilize yet
another mechanism to introduce a double bond into the growing acyl
chain of type II fatty acid synthase.
We thank Amy Sullivan for technical
assistance, the St. Jude Protein Production Facility for preparing the
FabM used in this study, and William Lewis in the St. Jude Hartwell
Center for the mass spectra.
*
This work was supported by National Institutes of Health
Grant GM 34496, Cancer Center (CORE) Support Grant CA 21765, and the
American Lebanese Syrian Associated Charities.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.
§
Present address: Division of Environmental Life Sciences, College
of Natural Science, Seoul Women's University, Seoul, Korea 139-774.
Published, JBC Papers in Press, September 16, 2002, DOI 10.1074/jbc.M208920200
2
Fatty acid abbreviations: 10:1( The abbreviations used are:
UFA, unsaturated
fatty acid;
ACP, acyl carrier protein;
SFA, saturated fatty acids;
FabA,
A New Mechanism for Anaerobic Unsaturated Fatty Acid Formation in
Streptococcus pneumoniae*
,§, and¶
Department of Infectious Diseases, Protein
Science Division, St Jude Children's Research Hospital, Memphis,
Tennessee 38105 and the ¶ Department of Molecular Biosciences,
University of Tennessee Health Science Center, Memphis, Tennessee
38163
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxydecanoyl-acyl
carrier protein (ACP) to trans-2-decenoyl-ACP, and the
isomerization of trans-2 to cis-3-decenoyl-ACP.
However, there are a number of anaerobic bacteria whose genomes do not
contain a fabA homolog, although these organisms
nonetheless produce unsaturated fatty acids. We cloned and
biochemically characterized a new enzyme in type II fatty acid
synthesis from Streptococcus pneumoniae that carries out
the isomerization of trans-2-decenoyl-ACP to
cis-3-decenoyl-ACP, but is not capable of catalyzing the
dehydration of -hydroxy intermediates. This tetrameric enzyme,
designated FabM, has no similarity to FabA, but rather is a member of
the hydratase/isomerase superfamily. Thus, the branch point in
the biosynthesis of unsaturated fatty acids in S. pneumoniae occurs following the formation of trans-2-decenoyl-ACP, in contrast to E. coli
where the branch point takes place after the formation of
-hydroxydecanoyl-ACP.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxydecanoyl-ACP dehydratase, FabA (6). FabA is capable of both the removal of water to
generate trans-2-decenoyl-ACP and the isomerization of this
intermediate to the cis-3-decenoyl-ACP (3, 7). However, FabA
is not the only protein that is required for introduction of the double
bond and does not catalyze the rate-limiting step in UFA formation (8).
A second unsaturated fatty acid auxotroph was isolated that corresponds
to the fabB gene, which encodes -ketoacyl-ACP synthase I. In fabB mutants, saturated fatty acid synthesis persists due
to the presence of the other elongation condensing enzyme in
Escherichia coli, FabF (9, 10). Although FabF readily
elongates 16:1 to 18:1 (10), the inability to support UFA synthesis in
fabB mutants leads to the conclusion that FabF cannot
elongate a key intermediate in UFA biosynthesis (3, 4). The analysis of
fabB and fabF mutants, coupled with the catalytic
properties of FabB and FabF in vitro supports a function for
FabB in UFA synthesis and a role for FabF in the thermal modulation of
membrane fatty acid composition (11-14).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
= 6700 M
1cm1) (24).
The reaction was started by the addition of the enzyme to 150-µl
final volume, and the decrease in absorbance at 263 nm was followed on
a Shimadzu UV-visible spectrophotometer UV-1601. Each reaction was run
in triplicate. One unit is defined as the disappearance of 1 pmol of
trans-2-octenoyl-NAC per min under the defined conditions.
Specific activity was expressed as units per microgram of protein.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
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Fig. 1.
Organization of the S. pneumoniae
fatty acid biosynthetic gene cluster. All of the genes
required for type II fatty acid biosynthesis are located in a single
cluster in the S. pneumoniae genome. The thick
arrows indicate the relative sizes of the genes. The numbers
above the arrows indicate the gene designations
in the S. pneumoniae TIGR 4 data base, and the gene names
below the arrows indicate the E. coli
genes that correspond to the open reading frames in the S. pneumoniae cluster. fabM, 10:1( 
2t)-ACP isomerase and
the topic of this paper; HTH, a helix-turn-helix DNA-binding
protein of unknown function; fabH, -ketoacyl-ACP synthase
III; acpP, ACP; fabK, enoyl-ACP reductase II;
fabD, malonyl-CoA:ACP transacylase; fabG,
-ketoacyl-ACP reductase; fabF, -ketoacyl-ACP synthase
II; accB, acetyl-CoA carboxylase subunit; fabZ,
-hydroxyacyl-ACP dehydratase; accC, accD, and
accA, subunits of acetyl-CoA carboxylase. The
asterisks indicate the location of the palindromes
identified in the promoter regions of the fabM,
HTH, and fabK genes. The alignment of these DNA
sequences is shown under the gene organization chart, and a consensus
sequence is indicated.
, carbons of thioester substrates. The structures of family members show a common active site
design that provides for CoA binding, an expandable acyl chain binding
pocket, an oxyanion hole for polarizing the thioester carbonyl, and
multiple active site stations for the positioning of acidic and basic
amino acid side chains to facilitate proton shuffling (31). The SP0415
(fabM) open reading frame is annotated in the data base as
an enoyl-CoA hydratase/isomerase. The hydratase/isomerase activity that
the data base entry is referring to is associated with the enzymes
responsible for either hydrating enoyl-CoA to -hydroxyacyl-CoA or
isomerizing cis-3 to trans-2-enoyl-CoA
intermediates in fatty acid -oxidation. However, S. pneumoniae lacks cytochromes and does not possess enzymes of the
-oxidation pathway making it highly unlikely that FabM functions in
this context (32). PhaB (PaaB) is an enzyme essential for the
catabolism of phenylacetic acid in Pseudomonas putida and is
thought to carry out either the hydroxylation or isomerization of the
double bonds once the aromatic ring has been opened (33). This protein
is part of a multifunctional phenylacetic acid degradation complex and
shows 35% identity and 51% similarity to FabM. ChcB is a novel
3,2-enoyl-CoA isomerase involved in the biosynthesis of the
cyclohexanecarboxylic acid moiety of the polyketide ansatrienin A (34).
ChcB has 27% identity and 41% similarity to FabM. FadB is a
multifunctional protein involved in fatty acid -oxidation in
E. coli, and contains as one of its activities a
cis-3-trans-2-enoyl-CoA isomerase (35). The
section of this protein that aligns with FabM is the component of the
complex thought to be responsible for the enoyl-CoA isomerase activity
required in the degradation of unsaturated fatty acids and over this
segment of the protein has a 30% identity and 46% similarity to FabM.
These strong similarities to enzymes known to catalyze isomerizations
of enoyl thioesters led us to test the hypothesis that FabM encodes a
trans-2 to cis-3-decenoyl-ACP isomerase.
View larger version (90K):
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Fig. 2.
Comparison of the FabM protein sequence to
members of the hydratase/isomerase superfamily. The predicted
protein sequence of FabM from S. pneumoniae (NCBI
accession number NC_003028) is compared with the predicted sequences of
PhaB (PaaB), the enoyl-CoA isomerase from P. putida
(accession number AAC24330) involved in the catabolism of phenylacetic
acid (33, 46), ChcB, the 3,2-enoyl-CoA isomerase (accession
number AAF73478) involved in the synthesis of ansatrienin A (34), the
3-cis, 2-trans-enoyl-CoA isomerase module
(accession number P21177) of the FadB multifunctional fatty acid
-oxidation complex from E. coli (35), and the consensus
sequence for the enoyl-CoA hydratase/isomerase superfamily, pfam000378
(30). Residues identical between FabM and the other sequences are
highlighted.
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Fig. 3.
Purification and apparent molecular weight of
FabM. Analysis of the purified FabM protein by SDS gel
electrophoresis using a 12% polyacrylamide gel is shown in the
left inset. His-tagged FabM was expressed and purified as
described under "Experimental Procedures" and the purified 31-kDa
product visualized by staining with Coomassie Blue. The protein was
applied to a Superdex-200 HR 10/30 column (Amersham Biosciences)
equilibrated with 50 mM Tris-HCl pH 7.5, 1 mM
dithiothreitol, and 1 mM EDTA and was eluted at a flow rate
of 0.5 ml/min. FabM was monitored at 280 nm and eluted at 11.8 ml. The
molecular mass was estimated to be 129 kDa by graphic analysis of a
standard curve based on the elution volumes of protein molecular mass
markers (Sigma) (right inset). Ferritin (440 kDa); catalase
(232 kDa); aldolase (158 kDa), bovine serum albumin (66 kDa); ovalbumin
(43 kDa); chymotrypsinogen (25 kDa); RNaseA (13.7 kDa) were used to
calibrate the column.
-keto[14C]decanoyl-ACP starting with
octanoyl-CoA and [2-14C]malonyl-ACP (via
ecFabD) as substrates. The NADPH-dependent ecFabG reduced
the intermediate to the initial substrate for the assays,
-hydroxy[14C]decanoyl-ACP (Fig.
4A, lane 1). The addition of ecFabA (lane 2) results in the conversion of the -hydroxy intermediate to a
mixture of trans-2- and cis-3-decenoyl-ACPs.
These isomeric forms are not distinguished on the gel, but previous
work suggests that the trans intermediate would predominate
(36). The enoyl-ACP cannot be elongated by a condensing enzyme, but the
cis-3 intermediate can. Accordingly, the addition of spFabF,
the elongation condensing enzyme of S. pneumoniae,
condenses the cis-3-decenoyl-ACP with malonyl-ACP, and
following reduction by ecFabG, gives rise to the accumulation of a new
band on the gel corresponding to
-hydroxy-cis-5-dodecanoyl-ACP (Fig. 4A,
lane 3). Since this reaction mixture did not contain an enoyl-ACP
reductase, additional rounds of elongation cannot occur, and the
product accumulates at the 12-carbon stage. Furthermore, ecFabA is
characteristically inactive with unsaturated -hydroxy intermediates
(20), so there is little conversion of the
-hydroxy-cis-5-dodecenoyl-ACP to the trans-2
intermediate. The addition of spFabZ also converts the
-hydroxydecanoyl-ACP to the enoyl-ACP (Fig. 4A,
lane 4); however, the addition of spFabF to this reaction did not
lead to the appearance of the elongated 12-carbon unsaturated
intermediate (Fig. 4, lane 5). These data illustrate that
spFabZ is a dehydratase and that it is not capable of isomerizing the
trans-2-enoyl-ACP to the cis-3 intermediate. The
addition of FabM to the base reaction did not lead to the formation of
enoyl-ACP (Fig. 4A, lane 6), and in combination
with spFabF did not lead to the appearance of any additional products
(lane 7). Thus, FabM lacked -hydroxyacyl-ACP dehydratase
activity. The combination of spFabZ and FabM led to the formation of
enoyl-ACP (lane 8), but it was not possible to discern if
the cis intermediate was formed in this reaction mixture. In
the presence of spFabZ, spFabF, and FabM, a new product appeared indicating that cycles of elongation occurred (Fig.
4A, lane 9). This product(s) arises from the
elongation of the cis-3 intermediate by spFabF and the
dehydration by spFabZ. In contrast to FabA and like ecFabZ (20), spFabZ
was capable of utilizing unsaturated -hydroxy intermediates to form
enoyl-ACPs. These experiments with the reconstituted fatty acid
synthase enzymes establish that FabM is capable of isomerizing
trans-2-enoyl-ACP to cis-3-acyl-ACP, but cannot
dehydrate -hydroxyacyl-ACP. In addition, spFabF is capable of
elongating cis-3-acyl-ACP intermediates.
View larger version (26K):
[in a new window]
Fig. 4.
FabM catalyzes the formation of
cis unsaturated acyl-ACP intermediates in
vitro. Panel A, a reconstitution assay
designed to demonstrate the catalytic properties of FabM, spFabF, and
spFabZ within the context of the other enzymes of the type II fatty
acid synthase cycle. The assays contained the indicated Fab enzymes and
were initiated with octanoyl-CoA and [2-14C]malonyl-CoA.
The reaction products were separated by conformationally-sensitive gel
electrophoresis as described under "Experimental Procedures." The
appearance of either -hydroxy-12:1(5c)-ACP or 12:2(5c,2t)-ACP
indicated the ability of the system to isomerize 10:1(2t)-ACP to
10:1(3c)-ACP, which can be elongated by spFabF. Panel B,
the specific activity of FabM using either 10:1(2t)-ACP or
12:1(2t)-ACP as the substrate in the reconstitution assay. The FabM
isomerase assay was performed using different concentrations of FabM
protein and the collection of Fab enzymes to generate either
10:1(2t)-ACP (
) (initiated with 8:0-CoA) or
12:1(2t)-ACP (
) (initiated with 10:0-CoA) as the
substrate, and the formation of the respective
cis-5,trans-2-acyl-ACPs was quantitated using the
phosphorimager. A standard curve was used to determine the amount of
labeled product, either 12:1(5c,2t)-ACP () or 14:1(7c,2t)-ACP
(), which was plotted as a function of protein concentration.
-hydroxydecanoyl-ACP starting
material (lane 1). The trans-2-and cis-3-decenoyl-ACP mixture (lane 2) formed after
dehydration by ecFabA led to the appearance of a new peak at mass 9001 (ACP+152). The elongated product of cis-3-decenoyl-ACP by
spFabF was predicted to be the
-hydroxy-cis-5-dodecenoyl-ACP (lane 3), and
accordingly the mass spectrum of the mixture contained a new peak at
9044 corresponding to ACP+196. Samples from the reactions in
lanes 4 and 5 contained
trans-2-decenoyl-ACP and displayed the expected mass peak at
9001. The -hydroxydecanoyl-ACP precursor (lanes 6 and
7) was not converted to other products by FabM, and the predominant acyl-ACP peak in these reactions mixtures was 9019, diagnostic for -hydroxydecanoyl-ACP (Fig. 5A). In
lane 8, trans-2- and/or
cis-3-decenoyl-ACPs were revealed by the appearance of a
characteristic mass peak at 9001. The FabM-catalyzed isomerization of
the trans-2 to cis-3-decenoyl-ACP was revealed by
the appearance of acyl-ACP products elongated by spFabF (lane
9). Two products were detected as illustrated by the appearance of
a peak for cis-5, trans-2-C12:2-ACP (ACP+178) as
well as cis-7,cis-5,trans-2-C14:3-ACP (ACP+204) with predicted and observed molecular masses of 9027 and
9053, respectively (Fig.
5B).2 These data
confirm the identities of the labeled intermediates indicated in
Fig. 4A and substantiate the conclusion that FabM was
capable of isomerizing both C10 and C12 enoyl-ACPs in
vitro.
View larger version (17K):
[in a new window]
Fig. 5.
Electrospray ionization-mass spectrometry of
precursor and products of the FabM-catalyzed reaction. Panel
A, mass spectrum of the reaction mixture in lane 7 showing the -hydroxydecanoyl-ACP substrate. Panel B, mass
spectrum of the products of the isomerization of
2-trans-decenoyl-ACP by FabM to
3-cis-decenoyl-ACP (C10:1, 3c-ACP) (9001),
5c,3c/2t-dodecadienoyl-ACP (C12:2,5c,3c/2t-ACP)
(9027), and to
7-cis,5-cis,2-trans(3-cis)-tetradecatrienoyl-ACP
(C14:3, 7c,5c,2t/3c-ACP) (9053). In both spectra, the M+131 ions
represent the molecular ions of ACP molecules that retained the
amino-terminal methionine residue.
View larger version (16K):
[in a new window]
Fig. 6.
Direct detection of FabM isomerase
activity. Incubation mixtures contained
trans-2-octenoyl-NAC (C8:1-NAC) in 150 µl of 10 mM potassium phosphate, pH 7.0. Reactions were started by
the addition of the indicated amounts of FabM and followed at 263 nm
for 3 min. Panel A, time course of the isomerization of
trans-2-octenoyl-NAC by purified FabM. Reactions were
started by the addition of 10 or 40 µg of FabM, and the conversion of
trans-2-octenoyl-NAC (100 µM) to
cis-3-octenoyl-NAC was monitored by the decrease in
absorbance at 263 nm. The controls without substrate and without enzyme
showed no significant change in absorbance. The curves have been
adjusted to the same zero time absorbance in the figure. Panel
B, initial rate of conversion of trans-2-octenoyl-NAC
by FabM. Reactions containing trans-2-octenoyl-NAC (100 or
200 µM) were started by the addition of increasing
amounts of FabM. The FabM specific activity calculated was 87 ± 2.8 pmol/min/µg.
View larger version (19K):
[in a new window]
Fig. 7.
Comparison of the fatty acid biosynthetic
pathway in E. coli and S. pneumoniae.
Left panel, the branch point in saturated/unsaturated
fatty acid synthesis in E. coli occurs at the dehydratase
step. This bacterium contains two dehydratases: FabZ, which functions
on all chain-lengths (20, 47), and FabA, which not only dehydrates the
-hydroxy intermediates, but also isomerizes the double bond to
produce a mixture of C10:1(2t)-ACP and C10:1(3c)-ACP (6, 7, 48).
SFA biosynthesis proceeds by the action of FabI on the
trans-2 intermediate followed by further elongation cycles
initiated by either FabB or FabF condensing enzymes. C10:1(3c)-ACP
is the least abundant product of FabA at equilibrium (49), thus UFA
requires FabB to efficiently utilize of the cis-3
intermediate and initiate the elongation cycles that form the major
long-chain unsaturated fatty acids. Accordingly, the FabR transcription
factor controls the cellular content of UFA primarily by altering the
expression of fabB (44). Right Panel, in S. pneumoniae there is only a single dehydratase, FabZ, that forms
the C10:1(2t)-ACP intermediate. SFA are formed by the action of the
FabK enoyl-ACP reductase followed by further elongation cycles
initiated by FabF. UFA arise from the isomerization of the
trans-2 intermediate by FabM followed by further elongation
cycles initiated by FabF. The branch point in E. coli is
based on the competition of the two dehydratases for the
-hydroxyacyl-ACP intermediate, and the activity of FabB, which pulls
the FabA product down the UFA branch of the pathway. In S. pneumoniae the branch point is at the enoyl-ACP level and the
proportion of products is determined by competition between enoyl-ACP
reductase II, FabK, and FabM, the trans-2,
cis-3-decenoyl-ACP isomerase.
Complementation of the fabA(Ts) phenotype with fabM and fabK
, no growth; +, growth).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxyacyl-ACP, but like
FabA, also isomerizes enoyl-ACP to 3-cis-acyl-ACP. However, spFabZ clearly is only capable of catalyzing the dehydration reaction of the type II synthase (Fig. 4). Thus, a previously undiscovered enzyme must be present to carry out an isomerization reaction and
genomic analysis reveals a candidate gene associated with the fatty
acid biosynthetic gene cluster termed FabM (Fig. 1). Indeed,
reconstitution of fatty acid synthesis in vitro with FabM in
combination with spFabZ and spFabF resulted in the formation of
unsaturated acyl-ACP intermediates (Figs. 4 and 5) and FabM catalyzes
the isomerization of enoyl substrate analogs (Fig. 6). Thus, S. pneumoniae uses the combination of FabZ and FabM to replace FabA
and introduce a double bond into the growing acyl chain. In the absence
of FabB, spFabF carries out the elongation of both saturated and
unsaturated acyl-ACPs in this organism.
-hydroxydecanoyl-ACP intermediate. The FabA or FabZ
dehydratases compete for utilization of this substrate. The FabZ
isozyme only forms the trans-2-enoyl-ACP, whereas the FabA
enzyme possesses the unique property of being able to isomerize the
trans-2 dehydration product to
cis-3-decenoyl-ACP. Also, FabA competes with FabI for trans-2-enoyl-ACP. The FabB condensing enzyme directly
elongates cis-3-decenoyl-ACP to initiate unsaturated fatty
acid formation. The saturated branch of the pathway begins with the
reduction of trans-2-decenoyl-ACP by FabI followed by
subsequent cycles of elongation catalyzed by either the FabB or FabF
condensing enzyme. Thus, the ratio of saturated to unsaturated fatty
acid is determined not only by the action of FabA, but also by the activity of FabB (8, 43). Indeed, the level of fabB
expression is controlled by the FabR (44) and FadR (15) transcription factors, and higher intracellular levels of this enzyme lead to increased UFA production. In contrast, the branch point in the S. pneumoniae pathway is one step downstream at the enoyl-ACP step
(Fig. 7). The FabK enoyl-ACP reductase II competes with the FabM
isomerase for the trans-2-decenoyl-ACP intermediate. Both of
these reactions produce intermediates that are subsequently elongated
to either saturated or unsaturated fatty acids by the single FabF
elongation condensing enzyme in S. pneumoniae. These differences offer an explanation for the inability of fabM
alone to complement E. coli fabA(Ts) mutants. In
the E. coli type II system, FabA competes with FabI and
FabZ, whereas UFA production in S. pneumoniae arises from
FabM competition with FabK. Our restoration of UFA synthesis in
E. coli (Table I) with FabM and FabK illustrates the
importance of kinetic competition. FabM alone is not sufficiently active to effectively compete with FabI for the trans-2
intermediates in this heterologous system, and for this protein to
function in UFA biosynthesis, FabI must be eliminated and FabM must be co-expressed with its competitive partner, FabK. In this context, the
finding of similar transcription factor binding sites within the
promoters of the fabM and fabK gene in S. pneumoniae (Fig. 1) suggests that these two partners in UFA
biosynthesis are coordinately regulated, reminiscent of the coordinate
regulation of fabA and fabB in E. coli
(15, 44, 45).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Infectious Diseases, Protein Science Division, St Jude Children's
Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. Tel.:
901-495-3491; Fax: 901-495-3099; E-mail:
charles.rock@stjude.org.
2t), number of
carbon atoms:number of double bonds. is double bond, number is the
location of the double bond, and c or t is the cis or
trans configuration.
ABBREVIATIONS
-hydroxydecanoyl-ACP dehydratase/isomerase;
FabB, -ketoacyl-ACP synthase I;
FabF, -ketoacyl-ACP synthase II;
FabH, -ketoacyl-ACP synthase III;
FabI, enoyl-ACP reductase I;
FabK, enoyl-ACP reductase II;
FabZ, -hydroxyacyl-ACP dehydratase;
FabD, malonyl-CoA:ACP transacylase;
NAC, N-acetylcysteamine;
ec, E.
coli;
sp, S. pneumoniae;
mt, M.
tuberculosis;
ca, C. acetobutylicum.
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M. L. Mohedano, K. Overweg, A. de la Fuente, M. Reuter, S. Altabe, F. Mulholland, D. de Mendoza, P. Lopez, and J. M. Wells Evidence that the Essential Response Regulator YycF in Streptococcus pneumoniae Modulates Expression of Fatty Acid Biosynthesis Genes and Alters Membrane Composition J. Bacteriol., April 1, 2005; 187(7): 2357 - 2367. [Abstract] [Full Text] [PDF]
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 K. Takayama, C. Wang, and G. S. Besra Pathway to Synthesis and Processing of Mycolic Acids in Mycobacterium tuberculosis Clin. Microbiol. Rev., January 1, 2005; 18(1): 81 - 101. [Abstract] [Full Text] [PDF]
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M. S. Kimber, F. Martin, Y. Lu, S. Houston, M. Vedadi, A. Dharamsi, K. M. Fiebig, M. Schmid, and C. O. Rock The Structure of (3R)-Hydroxyacyl-Acyl Carrier Protein Dehydratase (FabZ) from Pseudomonas aeruginosa J. Biol. Chem., December 10, 2004; 279(50): 52593 - 52602. [Abstract] [Full Text] [PDF]
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M. C. Mansilla, L. E. Cybulski, D. Albanesi, and D. de Mendoza Control of Membrane Lipid Fluidity by Molecular Thermosensors J. Bacteriol., October 15, 2004; 186(20): 6681 - 6688. [Full Text] [PDF]
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 C. J. Orihuela, J. N. Radin, J. E. Sublett, G. Gao, D. Kaushal, and E. I. Tuomanen Microarray Analysis of Pneumococcal Gene Expression during Invasive Disease Infect. Immun., October 1, 2004; 72(10): 5582 - 5596. [Abstract] [Full Text] [PDF]
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H. Wang and J. E. Cronan Functional Replacement of the FabA and FabB Proteins of Escherichia coli Fatty Acid Synthesis by Enterococcus faecalis FabZ and FabF Homologues J. Biol. Chem., August 13, 2004; 279(33): 34489 - 34495. [Abstract] [Full Text] [PDF]
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E. M. Fozo and R. G. Quivey Jr. The fabM Gene Product of Streptococcus mutans Is Responsible for the Synthesis of Monounsaturated Fatty Acids and Is Necessary for Survival at Low pH J. Bacteriol., July 1, 2004; 186(13): 4152 - 4158. [Abstract] [Full Text] [PDF]
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S. K. Sharma, M. Kapoor, T. N. C. Ramya, S. Kumar, G. Kumar, R. Modak, S. Sharma, N. Surolia, and A. Surolia Identification, Characterization, and Inhibition of Plasmodium falciparum {beta}-Hydroxyacyl-Acyl Carrier Protein Dehydratase (FabZ) J. Biol. Chem., November 14, 2003; 278(46): 45661 - 45671. [Abstract] [Full Text] [PDF]
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