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Originally published In Press as doi:10.1074/jbc.M405233200 on June 22, 2004

J. Biol. Chem., Vol. 279, Issue 36, 37324-37333, September 3, 2004
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The Escherichia coli fadK (ydiD) Gene Encodes an Anerobically Regulated Short Chain Acyl-CoA Synthetase*

Rachael M. Morgan-Kiss{ddagger}§ and John E. Cronan{ddagger}||

From the Departments of {ddagger}Microbiology and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received for publication, May 11, 2004 , and in revised form, June 21, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We recently reported a new metabolic competency for Escherichia coli, the ability to degrade and utilize fatty acids of various chain lengths as sole carbon and energy sources (Campbell, J. W., Morgan-Kiss, R. M., and Cronan J. E. (2003) Mol. Microbiol. 47, 793–805). This {beta}-oxidation pathway is distinct from the previously described aerobic fatty acid degradation pathway and requires enzymes encoded by two operons, yfcYX and ydiQRSTD. The yfcYX operon (renamed fadIJ) encodes enzymes required for hydration, oxidation, and thiolytic cleavage of the acyl chain. The ydiQRSTD operon encodes a putative acyl-CoA synthetase, ydiD (renamed fadK), as well as putative electron transport chain components. We report that FadK is as an acyl-CoA synthetase that has a preference for short chain length fatty acid substrates (<10 C atoms). The enzymatic mechanism of FadK is similar to other acyl-CoA synthetases in that it forms an acyl-AMP intermediate prior to the formation of the final acyl-CoA product. Expression of FadK is repressed during aerobic growth and is maximally expressed under anaerobic conditions in the presence of the terminal electron acceptor, fumarate.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Acyl-CoA synthetases (more properly called fatty acid:CoA ligases or acyl-CoA ligases) perform key roles in metabolic and regulatory processes by activating free fatty acids to their CoA thioesters. The enzymatic mechanism is a two-step reaction that proceeds via the intermediate formation of an acyl-adenylate (acyl-AMP) intermediate: step 1, fatty acid + ATP -> fatty acyl-AMP + PPi and step 2, fatty acyl-AMP + CoA -> fatty acyl-CoA + AMP. Acyl-CoAs serve as important intermediates in diverse metabolic functions, such as fatty acid transport, {beta}-oxidative degradation of fatty acids, and phospholipid biosynthesis, as well as enzyme activation, cell signaling, and transcriptional regulation (1). Consistent with the diverse roles of acyl-CoA synthetases (ACSs)1 in cell metabolism, many eukaryotic organisms encode several different ACSs that specifically activate short (C6–C8), medium (C10–C12), long (C14–C20), or very long (>C22) chain length fatty acids (1, 2). Moreover, some organisms possess multiple enzymes for each set of acyl chain lengths. In contrast, only a single ACS called FadD has been reported in Escherichia coli, although, multiple ACS isoforms of varying molecular masses recently shown to be artifacts of proteolysis (3) had been reported (4, 5). E. coli FadD has been purified to homogeneity (6, 7) and has a broad substrate specificity for fatty acids of medium and long chain lengths. Activation of long chain fatty acids to their acyl-CoA thioesters is required not only for aerobic degradation of the acids, but also for induction of the fad (fatty acid degradation) genes that catalyze the degradation process. In the absence of long chain acyl-CoAs, the FadR regulatory protein binds to its operator sequences, where it functions as a repressor of transcription of the fad genes (and as a transcriptional activator of the fabA and fabB fatty acid biosynthetic genes) (8–10). However, in the presence of long chain acyl-CoAs, FadR is released from the fad operators and transcription of these genes is induced. Acyl-CoAs of 10 or fewer acyl carbon atoms cannot release FadR from the operator sites and thus the aerobic degradative pathway is not induced and the acids cannot be utilized for growth. However, when FadR is inactivated by mutation these strains can utilize medium chain length fatty acids as the sole carbon source (8–10).

ACSs belong to a larger family of AMP-forming enzymes, which include firefly luciferase, acetyl-CoA synthetases, and nonribosomal peptide synthetases (11–14), all of which form an acyl-adenylate intermediates in their reaction cycles. The sequences of members of this family share 20 to 40% homology, and possess a number of conserved motifs (11). ACSs contain an additional motif, the fatty acid CoA-ligase (FACS) signature (15, 16). Two putative AMP binding motifs (AMP1 and AMP2) as well as the FACS motif have been assigned to FadD (15, 16). All of these cofactor binding motifs are found in a 43-kDa C-terminal cleavage product that results from OmpT-catalyzed proteolysis consistent with retention of enzymatic activity by this fragment (3).

Recently, we reported that that E. coli can utilize fatty acids as sole carbon sources during growth under anaerobic conditions provided that an electron acceptor is present in the medium (8). This anaerobic {beta}-oxidation pathway is distinct from the previously studied aerobic pathway in a number of properties. These are: (i) mutational studies demonstrated that several of the fad enzymes (FadD, FadBA, and FadE) required for the aerobic pathway are not required for anaerobic growth on fatty acids, (ii) the anaerobic fatty acid oxidation pathway is not under the control of FadR, the major regulator of the aerobic fad regulon, and (iii) fatty acids of different chain lengths are utilized by the two pathways (8). We identified two gene clusters encoding putative homologues of several fad enzymes, yfcYX and ydiQRSTD. The yfcYX operon encodes putative homologues for fadBA, and was renamed fadIJ (8). The ydiQRSTD operon contains several open reading frames encoding proteins that could also play a role in anaerobic {beta}-oxidation. Proceeding upstream from ydiD, the genes encode a putative ferrodoxin (YdiT), a flavoprotein (YdiS) that could accept electrons and use these to reduce a quinone, and two proteins (YdiR and YdiQ) that seem likely to form a heterodimeric electron transport flavoprotein complex that might transfer electrons to YdiS. There is also a putative acyl-CoA dehydrogenase, ydiO, located immediately upstream of the ydiP gene (a possible AraC-like regulator of the ydiQRSTD operon). The proteins encoded by the ydiQRST genes have high sequence homologies to those encoded by the fixABCX operon of anaerobic carnitine metabolism (17), and may function as the electron transport chain linking the {beta}-oxidation of fatty acids with the respiratory chain. The last gene of this putative operon is ydiD, which we renamed fadK and argued was likely to encode an ACS (8). This argument was based on the fact that FadK can be aligned with the FadD over its entire length (albeit with many gaps) and contains plausible AMP and FACS motifs. Furthermore, the double fadD fadK mutant failed to grow on fatty acids under either aerobic or anaerobic conditions (8), although fadD mutants grew on fatty acids under anaerobic conditions. These results indicated that FadK could functionally replace FadD under anaerobic conditions. However, fadD mutants are unable to grow on long chain fatty acids aerobically (8, 18, 19) and thus the failure of FadK to replace FadD under aerobic conditions was attributed to insufficient expression of FadK or to impairment of enzyme function.

We report that FadK is indeed an ACS that is primarily active on short chain fatty acids. FadK is not expressed under aerobic growth conditions. The enzyme is expressed under anaerobic conditions, but the level of expression depends on the terminal electron acceptor available.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Labeled fatty acids were purchased from American Radiolabeled Chemicals (St. Louis, MO). Acyl-CoA standards, fatty acids, antibiotics, and most other chemicals were purchased from Sigma (Sigma). Octanoyl-AMP and oleoyl-AMP standards were synthesized according to Reed et al. (20) and Borgstrom (21), respectively. Timentin was from GlaxoSmithKline. The EnzChek® Pyrophosphate assay kit was purchased from Molecular Probes (Eugene, OR).

Growth Conditions and Media—Rich broth (RB) contained (g/liter) tryptone, 10; NaCl, 5; and yeast extract, 1. RB/MOPS medium was supplemented with 0.1 M sodium MOPS (pH 7.8). 2x YT medium contained (g/liter), tryptone, 16; NaCl, 5; and yeast extract, 10. Minimal medium was M9 medium (22) supplemented with 1 mM MgSO4, 0.1 mM CaCl2, 0.01% casamino acids (Difco), and 0.5 mg/ml thiamine (23). For aerobic growth glucose and glycerol were used at concentrations of 0.4 and 0.2%, respectively. These concentrations were double when growth was anaerobic. Fatty acids were neutralized with KOH and solubilized with Tergitol Nonidet P-40 and were used at final concentrations of 1 g/liter (w/v) in aerobic or 2 g/liter in anaerobic cultures. Anaerobic cultures were supplemented with either 25 mM NaNO3 or 40 mM sodium fumarate. Growth in a strictly anaerobic environment was performed in a Coy anaerobic chamber containing an atmosphere of 5% H2, 75% N2, and 20% CO2 as previously described (8). Solid media contained 1.5% (w/v) Bactoagar (Difco, Milwaukee, WI).

Construction of Plasmids and Bacterial Strains—All bacterial strains were derivatives of E. coli K12 (Table I). Strains RMK73 and RMK76 were constructed from strain MC1061 (24) by the {lambda} Red-mediated recombination method of Datsenko and Wanner (25) using helper plasmid pKD46. The chloramphenicol acetyltransferase gene from plasmid pKD3 and the kanamycin resistance gene from pKD4 were amplified via PCR using primers pKD-fadK-L plus pKD-fadK-R or pKD-fadD-L plus pKD-fadD-R (Table II). The resulting PCR products were then used to replace the entire coding sequence of fadK or fadD with the chloramphenicol acetyltransferase or kanamycin resistance genes. Several antibiotic-resistant transformants were verified by colony PCR using primers fadK-C1 plus fadK-C2 or fadD-C1 plus fadD-C2 (Table II) for strains RMK73 or RMK76, respectively. The antibiotic resistance cassettes were removed using the FLP recombinase encoded on the temperature-sensitive helper plasmid, pCP20 (26). Strain RMK92 was produced by P1 transduction of the fadD::kan phenotype from RMK89 into strain BL21DE3/pLysS. The fadD mutation was verified by the inability of strain RMK92 to grow on oleate as a sole carbon source. Transduction with phage P1vir and other basic genetic techniques were carried out as described previously (8).


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  		TABLE I
Bacterial strains/plasmids

 


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  		TABLE II
Oligonucleotide primers used in this work

 
Construction of Genes Encoding His6-tagged FadK and FadD—The fadD and fadK genes of strain MG1655 were amplified via PCR using PfuTURBO polymerase (Stratagene) under standard PCR conditions using primer set pET16b-fadD-L plus pET16b-fadD-R for fadD and primer set pET16b-fadKLM1 plus pET16b-fadK-R for fadK (Table II). The resultant 1.6-kb PCR products were purified via Qiagen spin columns and cloned into pCR2.1 (Invitrogen). The inserts of the resultant plasmids were sequenced to verify the fidelity of the cloned PCR products. Two of the sequenced plasmids were digested with NdeI and BamHI (sites within the primers) and ligated to pET16b cut with the same enzymes to give in-frame fusions of the sequence encoding the His6 tag of pET16b with the 5' end of fadK or fadD in plasmids pRK34 and pRK41, respectively (Table I).

Tagging of the Chromosomal fadD and fadK Genes—To facilitate monitoring of the protein expression levels of FadD and FadK during growth, the chromosomal genes were directly tagged with a sequence encoding a C-terminal hexameric histidine tag based on the Datsenko and Wanner method (25) for constructing gene deletions. A linear PCR product was synthesized from the CAT gene of pKD3 using modified primers. The upstream primer contained a sequence encoding six histidine codons followed by a stop codon. This sequence was flanked by 40 to 45 bp of homology to the 3' end of either fadD or fadK (with the natural stop codon removed) as well as homology to the P1 region of the pKD3 plasmid (25). The downstream primer was designed to contain 40 to 45 bp of homology to the region about 50 bases downstream of the 3' end of the gene plus the P2 region of the pKD3 plasmid (25). The primer set FadK-HIS-L plus FadK-HIS-R was used to construct the fadK-his6 fusion, and primer set FadD-HIS-L plus FadD-HIS-R was used to construct the fadD-his6 fusion (Table II). Correct insertion of the hexameric histidine tag was verified by PCR, sequencing (data not shown), and Western blot analyses (see below).

Expression and Purification of His6-FadD and His6-FadK—Strain RMK92/pLysS was transformed with plasmids pRK34 or pRK41 encoding either His6-FadD or His6-FadK, respectively. Cultures (50–250 ml) were grown in LB broth supplemented with 12 µg/ml Timentin at 30 °C until an optical density of about 1 was reached and then induced with 1 mM isopropyl-{beta}-D-thiogalactopyranoside for 2.5 h. The cells were harvested by centrifugation at 4,000 x g for 10 min. The pellets were resuspended (4-fold concentration) in ice-cold 20 mM Tris-HCl (pH 8.0) and centrifuged for 5 min at 4,000 x g. The pellets were stored at –80 °C until further use. All further purification steps were performed at 5 °C.

For purification of the His-tagged enzymes, frozen pellets were thawed on ice for 15 min prior to the addition of 5–10 ml of buffer A (50 mM NaH2PO4, pH 8.0, 300 mM NaCl) containing 10 mM imidazole and 2 mM phenylmethylsulfonyl fluoride. Lysozyme (1 mg/ml) was added and after 1 h of incubation on ice the cells were disrupted via sonication (three 20-s pulses). The lysates were centrifuged at 10,000 x g for 30 min and 1–3 ml of Ni2+-nitrilotriacetic acid resin (Qiagen, Inc., Valencia, CA) was added to the resultant supernatant. The slurry was rotated slowly at 5 °C for 90 min and the resin was loaded into a 5-ml column and washed with 5 volumes of Buffer A containing 20 mM imidazole. The His6-tagged protein was eluted by 1 ml of buffer B (50 mM sodium TES, pH 8.0, containing 300 mM NaCl) and 250 mM imidazole. The elution was repeated 2 or 3 times. The purity of the samples was monitored via SDS-PAGE (27). To remove trace amounts of contaminating phosphate, the purified enzyme preparations were dialyzed in a Pierce Slide-A-Lyzer cassette (Pierce) against two changes of buffer B for 12 h at 5 °C. Following dialysis, 10 mM 2-mercaptoethanol and 10% glycerol were added prior to storage at –80 °C.

Coupled Spectrophotometric Assay for Acyl-CoA Synthetase Activity—Acyl-CoA synthetase activity was assayed by monitoring the inorganic pyrophosphate (PPi) produced in the first half-reaction (28). Pyrophosphate production was assayed as phosphate using a commercial spectrophotometric assay (EnzChek® Pyrophosphate Assay Kit, Molecular Probes) in the presence of pyrophosphatase. The assay was very sensitive to the presence of contaminating inorganic phosphate; therefore, to remove contaminant Pi all enzyme preparations were dialyzed in Buffer B (see above) prior to use, and the presence of exogenous Pi was assayed in all enzyme preparations as well as all other reaction components by performing the assay in the absence of pyrophosphatase. The standard 1-ml ACS reaction contained: 20 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 0.4 mM ATP, 0.4 mM CoA, 1 mM fatty acid, 0.2 mM 2-amino-6-mercapto-7-methylpurine ribonucleoside, 1 unit of purine nucleoside phosphorylase, and 0.1 unit of pyrophosphatase. Because the unreacted 2-amino-6-mercapto-7-methylpurine ribonucleoside substrate contributed to a significant background absorbance (A360 > 0.3), the spectrophotometer was calibrated in the presence of a solution containing 20 mM Tris-HCl and 0.2 mM 2-amino-6-mercapto-7-methylpurine ribonucleoside prior to assay measurements. For continuous assays, reactions were incubated for 10 min at room temperature prior to enzyme addition to allow for traces of contaminating Pi to be consumed. For end point assays, the reaction was started by the addition of variable amounts of enzyme, and the reaction was incubated at room temperature for 30 min. The change in absorbance at 360 nm was measured on a Beckman DU640 spectrophotometer (Beckman-Coulter). In all experiments, the amount of PPi produced during the reaction was determined by subtracting the background absorbance in a control reaction containing buffer B in place of the enzyme preparation.

Radioactive Assay for Acyl-CoA Synthetase Activity—The radioactive assay used to measure the formation of 14C-fatty acyl-CoAs from 14C-fatty acids was based on the assay of Kameda and Nunn (7). Unless otherwise stated a standard 200-µl reaction mixture (29) contained 200 mM Tris-HCl (pH 7.5), 50 µM ATP, 8 mM MgCl2, 2 mM EDTA, 20 mM NaF, 0.1% Triton X-100, 0.5 mM CoA, 10 µM fatty acid, and enzyme (2–5 and 0.2–5 µg for FadK and FadD, respectively). The reactions were incubated at room temperature for 15 min and then spun at top speed of a microcentrifuge for 1 min. For HPLC analysis (see below), 0.5 µM of the appropriate acyl-CoA standard(s) (Sigma) was added immediately prior to injecting the sample. For analysis by scintillation counting, the reaction was terminated by the addition of 1 ml of isopropyl alcohol, n-heptane, 1 M H2SO4 (40:10:1, by volume). The residual radioactive free fatty acid was removed by extraction with n-heptane. The aqueous fraction was then counted in a Beckman-Coulter LS6500 scintillation counter to measure synthesis of fatty acyl-CoA (or fatty acyl-AMP when CoA was omitted from the assay).

HPLC analysis was performed on a Waters C18 column (30). The starting condition was 98.8% 50 mM ammonium acetate (pH 5.0) and 1.2% acetonitrile, followed by a gradient of 55% ammonium acetate, 45% acetonitrile over 5 min and then undiluted acetonitrile for 40 min. Radioactivity was monitored by an in-line Beckman 110B Radioisotope Detector with a flow-through scintillation counter. ACS activity was also monitored via thin layer chromatography. The reaction mixture was essentially as described above except that the reaction volume was scaled down to 15 µl. Following incubation of the reaction mixture for 15 min at room temperature, reactions were quenched with 5% acetic acid and loaded on a silica gel thin layer chromatography plate. The products were separated in 1-butanol, acetic acid, water (80:25:40) at 4 °C for about 5 h according to Trivedi et al. (31). Radiolabeled products were visualized by exposing the TLC plate to Kodak X-AR film for 2 to 3 days.

SDS-PAGE and Western Blotting—Samples from whole cell extracts, or the soluble and insoluble fractions were prepared for SDS-PAGE analysis by solubilization by boiling for 5 min in cracking buffer (60 mM Tris-HCl, pH 6.8, 1% 2-mercaptoethanol, 10% glycerol, 0.01% bromphenol blue, 1% SDS). The extracted proteins were loaded on an equal protein basis and separated on an 8% resolving, 4% stacking polyacrylamide gel using a Mini-Protean II apparatus (Bio-Rad). Proteins separated by PAGE were then electrophoretically transferred to Immobilon-P membranes (Millipore) for 45 min at 100 mV. The membranes were pre-blocked in TBS buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.05% Tween 20 and 5% nonfat milk powder. The membranes were probed overnight with an anti-pentameric histidine antibody (Qiagen) diluted 1:2000 in the above buffer. Following incubation with a secondary antibody conjugated with horseradish peroxidase (Amersham, 1:5000), the labeled His-tagged proteins were visualized by incubation of the blots in ECL plus chemiluminescent detection reagents (Amersham Biosciences) and developed on ECL Hyperfilm (Amersham Biosciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
FadK Has Strong Sequence Similarities to ACS Proteins— The crystal structure of Salmonella enterica acetyl-CoA synthetase (32) reported after submission of our prior paper (8) provided structural explanations for the conservation of two ACS motifs called A5 (which lies within a motif called AMP2) and A8 that are found in FadK (Fig. 1A). The A5 sequence is involved in binding the acyl-adenylate intermediate and A8 is involved in binding of both acyl-adenylate and CoA. These data strongly indicated that FadK is an ACS, as we had proposed (8). A third motif called AMP1 that includes a motif called A3 is also present in FadK. However, rather than binding the acyl-adenylate as previously assumed, the AMP1 motif forms a surface loop in acetyl-CoA synthetase that probably binds the pyrophosphate formed in the first partial reaction (32). Although FadK contains only 15 of the 25 residues of the FACS signature motif (15) (DGWLHTGDIGXWXPXGXLKIIDRKK), that overlaps the N-terminal end of the A8 motif (see Fig. 1), 8 of the 10 highly conserved residues of this motif are found in FadK, including the three underlined invariant glycine residues. Finally, the A10 motif containing the conserved Gly-Lys dipeptide is located near the FadK C terminus as seen in other ACSs (data not shown). Given these data plus our prior in vivo studies (8), it seemed clear that FadK was an ACS. However, the activity and specificity of the enzyme remained to be directly demonstrated. Therefore, we purified FadK to homogeneity and compared the enzyme with E. coli FadD, a well studied and more typical member of the ACS family.



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  		FIG. 1.
The ACS motifs of FadK and FadD. A, alignment of the AMP-binding motifs (labeled I and II) and FACS signature of FadD (16) with FadK and two yeast ACS proteins, Faa1p and Faa4p. The conserved ACS motifs (32), A3 and A5, are shown as boxes. The FadD site cleaved by the inner membrane protease, OmpT, is also shown by an arrow. B, the two possible ATG initiation codons (single underlines) of the 5'-sequence of fadK. The termination codon of the upstream gene ydiT is also shown (double underline).

 
Purification and Properties of His-tagged FadK and FadD Proteins—To test FadK for ACS activity we constructed two genes encoding N-terminal hexameric histidine (His6)-tagged forms of the protein (His6-FadKM1 and His6-FadKM2). Two constructs were made because two potential fadK translational initiation sites are located downstream of ydiT (Fig. 1B). Both methionine codons are conserved in a number of related bacteria (data not shown) and thus the functional initiation codon was determined empirically by constructing genes that encoded proteins beginning at either the upstream ATG codon (protein His6-FadKM1) or at the downstream ATG codon (protein His6-FadKM2). For comparison a gene encoding FadD with an N-terminal His6 tag (His6-FadD) was also made. It was found that expression of the tagged forms of FadK and FadD was optimal in cultures grown at 30 °C. The His6-tagged proteins were purified to over 95% homogeneity using Ni2+-chelate chromatography (Fig. 2). Both of the His6-FadK proteins had apparent masses of 63 kDa on SDS-PAGE, a value slightly smaller than His6-FadD (68 kDa). Because the predicted molecular masses of the His6-tagged proteins were about 64 kDa, it seems that these proteins have somewhat atypical SDS-PAGE migration rates, possibly because of the hexahistidine tag. The preliminary characterization of the ACS activities of the purified His6-tagged proteins used an enzyme-coupled assay that monitored the production of pyrophosphate dependent on oleate, CoA, and ATP. The specific activity of His6-FadKM1 was 2.5-fold greater than that of His6-FadKM2, although both proteins had much lower levels of activity than did His6-FadD. Moreover, in the spectrophotometric assay the activity of His6-FadKM2 decreased with increasing protein concentration when protein concentrations exceeded 2 µg/ml (data not shown). This together with the finding that reaction mixtures containing >5 µg/ml of His6-FadDM2 became cloudy during the reaction indicated aggregation of the protein (data not shown) suggesting that the upstream start codon was the start site used in vivo. Finally, storage of His6-FadKM2 at either –20 or –80 °C resulted in losses of activity that exceeded 80%, whereas the longer FadK form was stable under these conditions (data not shown). Therefore, we believe that the upstream ATG codon is more likely to be the initiation codon used in vivo (if so, fadK would be translationally coupled with ydiT) and thus His6-FadKM1 (which will be henceforth referred to as His6-FadK) was utilized for the remainder of the experiments reported. The activity versus pH profiles of His6-FadK and His6-FadD were also determined (data not shown). His6-FadD had the highest oleoyl-CoA synthetase activity at pH 7.5 to 7.8, whereas somewhat higher pH values (pH 7.7 to 8.2) gave the highest levels of His6-FadK activity.



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  		FIG. 2.
SDS-PAGE showing stages of purification of the N-terminal hexameric histidine-tagged proteins. Panel A shows the purification of FadK and panel B shows the purification of FadD. Molecular mass markers (kDa) are indicated to the left. Lane 1, un-induced culture; lane 2, induced culture; lane 3, soluble fraction; lane 4, insoluble fraction; lane 5, flow through; lane 6, wash; lane 7, first elution; lane 8, second elution.

 
Fatty Acid Chain Length Specificity of His6-FadK—To determine the cofactor requirements as well as fatty acid chain length preferences of FadK, the radioactive solvent partition assay of Kameda and Nunn (7) was used to monitor the overall reaction. The activity of His6-FadK decreased when either CoA or ATP were omitted from the assay, although His6-FadK retained significantly higher levels of activity in the absence of CoA than did His6-FadD (Table III). This activity is attributed to oleoyl-adenylate synthesis. (Oleoyl-adenylate like oleoyl-CoA partitions into the aqueous phase of the assay.) As expected omission of CoA, ATP, or Mg2+ (via addition of EDTA) decreased the activity of His6-FadD by >95% (Table III). His6-FadK had low activity on medium or long chain fatty acids and was maximally active on C6:0 and C8:0 substrates (Fig. 3A). In contrast, His6-FadD activity was the highest with fatty acid substrates of >10 carbon atoms (Fig. 3B) in agreement with previous reports (7).


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  		TABLE III
Requirements for the His6-FadK and His6-FadD ACS activities

Enzyme activity was assayed as formation of [1-14C]oleoyl-CoA formed in the presence of 0.2–5 µg of a purified His-tagged protein (n > 3) by the radioactive extraction assay ("Experimental Procedures"). The maximal activities were 1.95 and 153.2 nmol/min/mg protein for His6-FadK and His6-FadD, respectively.

 



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  		FIG. 3.
Fatty acyl chain length specificities of His -FadK (A) and His6-FadD (B). Enzyme activity (nanomole of product 6formed per min/mg of protein) was assayed by measuring formation of 1-14C-fatty acyl-CoAs (7) as described under "Experimental Procedures." Values represent the mean ± S.D. (n = 4). C18:1 is oleic acid.

 
Detection of Acyl-AMP and Acyl-CoA Products—To directly monitor the products of both half-reactions an HPLC-based assay was modified from the radioactive assay. We first tested the assay with the FadD protein. As expected in the presence of [1-14C]oleate and all required cofactors, His6-FadD converted more than 90% of the substrate to [1-14C]oleoyl-CoA (Fig. 4). In contrast, [1-14C]octanoate was a much less efficient substrate for His6-FadD in that only about 20% of the [1-14C]octanoate was converted to [1-14C]octanoyl-CoA (Fig. 4C, peak 2). In agreement with the spectrophotometric (data not shown) and radioactive extraction assays (Fig. 3), the HPLC-based enzyme assay indicated that His6-FadK was significantly less active than His6-FadD (Fig. 4). However, FadK clearly catalyzed acyl-CoA synthesis. In the presence of [1-14C]oleate and other required reaction components, a radiolabeled product was detected that had the same retention time as an oleoyl-CoA commercial standard (Fig. 4B, peak 2). Furthermore, in the presence of [1-14C]octanoate and CoA His6-FadK reactions accumulated octanoyl-CoA (Fig. 4D). We also performed the HPLC-based acyl-CoA synthetase assay in the absence of CoA to detect the acyl-AMP intermediate. Although with both FadK and FadD we were able to detect minor peaks having the retention times of acyl-AMP standards, these peaks were not well resolved probably because of hydrolysis during chromatography (data not shown). Therefore, we used thin layer chromatography done at 4 °C (35) to test for synthesis of the expected acyl-AMP intermediate. The reaction products formed from either [1-14C]octanoate or [1-14C]oleate in the presence or absence of CoA were determined (Fig. 5). In the absence of enzyme no products were formed. When [1-14C]octanoate was the substrate in the absence of CoA FadK formed a product having an RF of 0.67 as expected for an acyl-adenylate (35). However, when CoA was added to the reaction mixture, the RF of the product shifted to 0.51 (Fig. 5), the migration position of an acyl-CoA (31). Similarly, when [1-14C]oleate was provided as a fatty acid substrate, FadK catalyzed formation of products having RF values of 0.62 and 0.51 in the presence or absence of CoA, respectively (Fig. 5). These data confirm that the FadK proceeds via the expected acyl-AMP intermediate to the acyl-CoA product. In contrast, in the absence of CoA, FadD reactions failed to accumulate detectable levels of octanoyl-AMP, although formation of [1-14C]octanoyl-CoA was detected when CoA was added to the reaction mixture (Fig. 5). However, when oleate was the substrate both oleoyl-AMP and oleoyl-CoA products were detected (Fig. 5). Finally, we tested for His6-FadK- and His6-FadD-catalyzed conversion of exogeneously supplied octanoyl-AMP or oleoyl-AMP to the fatty acyl-CoAs using the HPLC assay and [3H]CoA and were unable to detect thioester formation (data not shown) suggesting that these enzymes are unable to acquire the acyl-AMP intermediate from solution.



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  		FIG. 4.
HPLC analysis of radiolabeled products formed by purified His6-FadD (A and C) or His6-FadK (B and D). Panels A and B show formation of [1-14C]oleoyl-CoA in the complete reaction mixture, whereas panels C and D show formation of [1-14C]octanoyl-CoA in the complete reaction mixture. The enzyme reactions were performed as given under "Experimental Procedures." The solid lines denote radioactivity, whereas the broken lines are internal standards detected by UV absorbance (30). The peak designations are: peak 1, CoA; peak 2, acyl-CoA (either octanoyl or oleoyl as appropriate); and peak 3, free fatty acid. Note that the elution volumes differed with the chain length of the substrate.

 



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  		FIG. 5.
Thin layer chromatographic analysis of the products of purified His6-FadK and His6-FadD. The fatty acid substrates were left panel, [1-14C]octanoate or right panel, [1-14C]oleate and assays were done in the presence or absence of CoA. Lane 1 of both panels lacked enzyme. Lanes 2, 3, 7, and 8 contained His6-FadK, whereas lanes 4, 5, 9, and 10 contained His6-FadD. Lanes 2, 4, 7, and 9 lacked CoA, whereas CoA was present in the other lanes. The area at the top of the plate where unreacted fatty acids migrate (31) was excised before autoradiography to prevent obscuring of the faint acyl-adenylate bands by the much greater radioactivity in the fatty acid spots.

 
Regulation of FadK and FadD—We previously reported that expression of FadK can complement a fadD mutant only during growth under anaerobic conditions (8). The failure of FadK to allow growth of fadD mutants under aerobic conditions could be either the result of poor enzymatic function in the presence of oxygen or insufficient expression of FadK during aerobic growth. Because it became clear that FadK has ACS activity under aerobic conditions in vitro (Figs. 4 and 5 and Table III), the former possibility was excluded and we turned to the measurement of FadK expression. To facilitate these measurements we constructed RMK96, a strain that encodes a C-terminal His6-tagged FadK expressed at the normal chromosomal location. A similar fadD-his6 fusion in a strain called RMK97 was also constructed. Neither strain had a growth phenotype when grown on the appropriate fatty acids indicating that the presence of the His6-encoding sequence did not have detrimental effects on enzyme function. Cultures of strain RMK96 were grown aerobically in RB/MOPS medium in the presence or absence of either glucose or short (C8) versus long (C18:1) chain fatty acids. The cells were broken by sonication and the resulting lysates were separated into soluble and insoluble fractions by centrifugation. These fractions were then analyzed by SDS-PAGE followed by Western blot analysis with an antibody that recognizes a His5 peptide sequence (Figs. 6 and 7). Densitometric quantitation was also performed on the Western blots (Fig. 8). Using this assay we detected only trace levels of FadK-His6 in aerobic cultures grown under any conditions tested (Figs. 6, A and B, and 8A). When cultures were grown under anaerobic conditions FadK-His6 was detected, but the levels of expression varied with the terminal electron acceptor supplied. In the presence of nitrate FadK-His6 protein levels were low under all growth conditions (Fig. 7, A and C), whereas in the presence of fumarate FadK-His6 was present in the insoluble fraction at relatively high levels under all growth conditions (Fig. 7D). In addition to the electron acceptor dependence, FadK-His6 expression was also dependent on the growth phase at which the cells were harvested. During log phase anaerobic growth expression of FadK-His6 was repressed in the presence of glucose and modestly induced in the presence of oleate or octanoate (Fig. 8C). However, in stationary phase cultures roughly comparable levels of FadK-His6 were seen regardless of the growth medium used (Fig. 8D). Last, we were able to detect only a 45-kDa C-terminal fragment of FadK-His6 irrespective of the growth conditions used. However, when the FadK-His6 protein was purified from the insoluble fraction of fumarate-grown cultures by nickel-nitrilotriacetic acid chromatography, low levels of a full-length protein were detected (data not shown). Therefore, it seems that under the experimental conditions used for the Western blot analyses, most of the FadK-His6 became cleaved.



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  		FIG. 6.
Differential expression of fadK and fadD during aerobic growth. Cultures of the FadK-His6 encoding strain RMK96 (A and B) or the FadD-His6 encoding strain RMK97 (C and D) were grown aerobically in RB-MOPS medium in the presence or absence of glucose (GLUC) or oleate (OLE). Samples were collected during the log (4 h) or stationary phases of growth (8 h). Cells were broken by sonication and protein expression (FadK-His6 and FadD-His6) in the supernatant (A and C) versus the pellet. B and D were assayed by immunoblotting with an antibody raised against His5. The medium was RB with the additions given. Lanes 1 and 4, no additions; lanes 2 and 5, glucose; lanes 3 and 6, oleate; lanes 7 and 8, FadD standards. The abbreviations used are: sup, supernatant; pel, pellet.

 



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  		FIG. 7.
Differential expression of fadK and fadD during anaerobic growth. Cultures expressing either RMK96 (A–D) or RMK97 (E–H) were grown under stringent anaerobic conditions in the presence of either nitrate (left panel) or fumarate (right panel) as terminal electron acceptors in RB/MOPS medium supplemented with glucose or oleate. Supernatant or pellet samples were separated via SDS-PAGE and probed with the anti-pentahistidine antibody to measure FadK-His6 and FadD-His6 levels. The designations are as described in the legend to Fig. 6.

 



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  		FIG. 8.
Quantitation of the effects of terminal electron acceptor and growth media on abundance of FadK-His6 (A and C) and FadD-His6 (B and D). Cultures of strains expressing the His-tagged proteins were grown either aerobically (A and B) or anaerobically in the presence of fumarate (C and D) in the presence or absence of glucose (GLUC), oleate (C18:1), or octanoate (C8). The values for the cell pellets are denoted by the solid bars, whereas the supernatant values are denoted by the open bars. The relative amounts of the polypeptides were estimated from densitometric analysis of Western blots. The values were normalized to 1 µg of the purified His6-tagged protein (n = 2).

 
It has been previously shown that FadD is cleaved by the OmpT outer membrane protease to give 19-kDa N-terminal and a 43-kDa C-terminal fragments (3), thus it seemed possible that FadK might also be specifically cleaved by OmpT. However, introduction of an ompT deletion mutation into the fadK-his6 fusion strain gave no change in the cleavage pattern of the His-tagged proteins of fumarate-grown cells (data not shown). Moreover, addition of OmpT-containing cell lysates to those from the ompT fadK-his6 strain (3) also failed to change the cleavage pattern (data not shown). These data indicated that cleavage of FadK is because of proteases other than OmpT.

We also examined fadD expression under the above growth conditions by use of a chromosomal fadD gene that encoded a His6 tag on the C terminus of the protein constructed as described above. It has been previously reported that fadD expression is derepressed during stationary phase (33) and thus cells were harvested from both log and stationary phase cultures. Under aerobic growth conditions, two His-tagged bands were detected in lysates of strain RMK97, which expresses the 78-kDa FadD-His6 fusion protein. One band was at the position expected for the full-length protein with the second band at 50 kDa (Fig. 6, C and D). However, the 50-kDa band was only present in the soluble fraction (Fig. 6C). Thus, the 50-kDa band seemed likely to be the 42-kDa C-terminal fragment previously reported to be the result of OmpT-mediated proteolysis (3). This was confirmed in vivo by the absence of the 50-kDa band in a strain (RMK 99) containing the fadD-his6 gene fusion as well as an ompT::kan null mutation (data not shown). Log phase growth in the presence of the long chain fatty acid, oleate, resulted in a 1.6-fold higher level of total FadD-His6 protein relative to cells grown in the absence of fatty acids (Figs. 6, C and D, and 8B). In contrast, stationary cultures had 1.2–2-fold increased levels of total FadD-His6 protein when grown in either the presence or absence of fatty acids (Fig. 8B). The addition of glucose strongly repressed FadD-His6 expression regardless of the growth phase (Figs. 6, C and D, and 8B) and addition of a short chain fatty acid (octanoate) also gave decreased expression for unknown reasons. Therefore, the data for aerobic growth we obtained by use of the chromosomal FadD-His6 construct agree well with those previously obtained with a fadD-lacZ construct (33). Because there are scant data regarding the regulation of the fad regulon enzymes during anaerobic growth, we also measured FadD levels in cells grown under anaerobic conditions. In marked contrast to aerobically grown cultures only low levels of FadD-His6 were detected in the insoluble fraction of cells grown under anaerobic conditions, regardless of the growth medium used (Figs. 7, E–H, and 8D). In addition, the pattern of expression was dependent on the source of terminal electron acceptor used to support growth. In nitrate-grown cultures FadD-His6 expression was repressed in the presence of glucose and failed to be induced by the presence of oleate. Anaerobic cultures grown in either the presence or absence of the fatty acids showed increased FadD expression in stationary phase in cultures grown in the presence of nitrate (Fig. 7E). The FadD-His6 protein levels of the fumarate-grown cultures of strain RMK97 were approximately uniform in growth media supplemented with either glucose or oleate during log phase, but were lower when grown in the presence of octanoate (Fig. 8D). Furthermore, the FadD C-terminal fragment was more prevalent in the fumarate-grown cells than in the nitrate-grown cells and a second His-tagged band of ~45 kDa was also detected (Fig. 6H). Similar bands were also detected by Yoo et al. (3) and hence it is likely that the 45-kDa band is an additional C-terminal fragment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We recently reported that E. coli has the ability to grow anaerobically on a wide range of fatty acid substrates as sole carbon and energy sources given the presence of an efficient terminal electron acceptor such as nitrate or fumarate (8). Although these results disagreed with previous assumptions that the {beta}-oxidative fatty acid degradation pathway does not function under anaerobic conditions (34), our findings were not surprising given that a natural environment of this organism, the mammalian bowel, is highly oxygen-limited and is often replete with fatty acids. We found that several E. coli fad mutants completely blocked in aerobic growth on fatty acids grew on fatty acids under anaerobic conditions and thus it was clear that an alternative pathway was involved in anaerobic fatty acid degradation. The minimum enzymatic requirements for a {beta}-oxidation pathway are: (i) enzymatic activation of free fatty acids to their CoA thioesters to facilitate chemistry at the {beta}-carbon of the acyl chain, (ii) several enzymatic activities targeting the {beta}-carbon that result in thiolytic cleavage of the chain, and (iii) redox protein(s) linking the reducing equivalents produced by {beta}-oxidation to the respiratory chain. As described in the Introduction two operons, yfcYX and ydiQRSTD, seemed likely to encode a full set of enzymes for the anaerobic {beta}-oxidative pathway. The last gene of the latter operon, ydiD (renamed fadK), encoded the putative ACS. Our prediction was based on the genetic and sequence analyses, but ACS activity was not demonstrated. We also argued that FadK should show a preference for short chain length fatty acids because strains lacking FadIJ activity fail to grow on short chain fatty acids (8) and the FabBA proteins have only very weak activity on short chain substrates (35). Therefore, the short chain preference of FadIJ would argue that the functionally associated ACS should have a similar specificity for short/medium chain length substrates, a prediction we have confirmed by in vitro assays of purified FadK. The substrate specificity of FadK argues that short and medium chain length fatty acids are abundant in environments where E. coli grows anaerobically. These acids could be obtained directly from the diet of the colonized animal or be the product of metabolism of longer chain acids by other intestinal flora.

FadK is active in both of the partial reactions characteristic of FACS proteins. In the presence of fatty acids, ATP, and CoA, the major product formed by either FadK or FadD is fatty acyl-CoA. However, when CoA was omitted from the reaction mixture, FadK catalyzed the formation of small amounts of acyl-AMP intermediate when either a short (C8) or a long chain acid (C18:1) was provided as the fatty acid substrate. The molar ratio of enzyme to acyl-AMP intermediate was roughly one, suggesting that FadK forms an enzyme-bound acyl-AMP intermediate, as has been previously proposed for FadD (15, 19). In contrast, in FadD reactions an acyl-AMP intermediate was only detectable when a long chain fatty acid was provided. This finding suggests that the short chain octanoyl-AMP is not efficiently shielded from solvent by the FadD active site, but rather is released into free solution where it hydrolyzes. FadK seems to have a higher affinity for the medium chain acyl-AMP intermediate and protects the intermediate from solvent. Note that FadK is a much poorer ACS than FadD. This low activity could be an intrinsic property of the enzyme or because of the fact that we have purified and studied FadK as a soluble protein while it is normally membrane-associated. However, FadD allows anaerobic growth of a strain carrying a fadK (ydiD) null mutation on octanoate in the presence of nitrate (8). Because FadD is poorly expressed under these conditions (Fig. 7) and octanoate is a poor substrate for this enzyme (7) (Fig. 3), it seems that only low levels of ACS activity are required for anaerobic fatty acid utilization.

FadK contains a region with high homology to a newly identified short/medium chain acyl-CoA synthetase motif (36) that includes part of the FACS motif plus the A8 motif that could be involved in substrate specificity (Fig. 9). Consistent with this notion, FadD aligns poorly with this motif. However, recent results argue that the FACS region has a more complex role than simple substrate binding. The recent acetyl-CoA synthetase crystal structure (32) shows this region to be the junction between the N- and C-terminal domains of the enzyme. Gulick and co-workers (32) propose that following acetyl-adenylate synthesis the C-terminal domain undergoes a large rotation relative to the N-terminal domain upon binding CoA. This rotation is thought to position the CoA thiol for nucleophilic attack on the acetyl group of the adenylate. Rotation would involve residues of the FACS and A8 motifs that could explain the sequence conservation within this region. However, very recently a family of mycobacterial enzymes thought to be FadD homologues by sequence comparisons were found unable to synthesize acyl-CoA thioesters (31). These enzymes, called the fatty acyl-AMP ligases, transfer the acyl group of the acyladenylate intermediate to proteins of polyketide synthesis for further elongation. Despite the inability of these proteins to catalyze ligation of CoA to fatty acids, their sequences contain reasonable and appropriately located FACS motif sequences. The exception is that the first strictly conserved glycine of this motif seems to have been deleted from the FACS motif of the fatty acyl-AMP ligases. It remains to be seen whether or not the lack of this glycine residue can distinguish fatty acyl-AMP ligases from fatty acyl-CoA ligases. However, it does seem clear that these two ligase classes differ in the structure of the A8 region because trypsin treatment of all tested fatty acyl-AMP ligases gave two discrete products (consistent with a single cleavage within the linker region) while the same treatment of several fatty acyl-CoA ligases from the same organism failed to produce distinct cleavage products (31).



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  		FIG. 9.
Comparison of predicted short/medium chain ACS domain (M-domain) (36) of FadK. FadK is aligned with the short/medium chain ACSs: Mig (Mycobacterium avium, putative), AlkK (Pseudomonas oleovorans), AlkK-5 (Archeoglobis fulgidus), and Dein (Deinococcus sp., putative), and the long chain ACS FadD (E. coli).

 
We now have shown that the failure of FadK to functionally replace FadD under aerobic conditions was because of insufficient expression. To monitor the expression levels of FadK and FadD a simple method for constructing C-terminal tagged proteins was designed and extended to FadD. The main advantage of this tagging system was that the C-terminal tag did not interfere with the function of the protein. Preservation of functionality was particularly critical for FadD, because the long chain acyl-CoAs required to induce the fad regulon is the product of FadD. In addition, the His6 tag epitope could be easily detected via Western blot analysis using a commercially available antibody. This avoided the need for the production of antibodies against both FadD and FadK and eliminated the potential problem of differential efficiencies of epitope recognition influencing detection of the proteins. As we had suspected (8) only trace levels of FadK-His6 are expressed under aerobic conditions regardless of the carbon source provided or the stage of growth sampled (Fig. 6). Furthermore, during anaerobic growth FadK was minimally expressed when nitrate was provided as a terminal electron acceptor, but was highly expressed in the presence of fumarate suggesting that the electron transport chain responsible for oxidation of the reducing equivalents produced during anaerobic {beta}-oxidative degradation of fatty acids may better utilize fumarate. (In our prior work (8) where cell growth was the indicator of gene expression the fact that fumarate is used as a carbon source as well as a electron acceptor obscured the greater expression on fumarate.) Because fadK is encoded in an operon containing several putative flavoproteins (ydiQ, ydiR, and ydiS) involved in an electron transport chain, it is reasonable to assume that transcriptional regulation of the operon would be governed by the presence of the preferred terminal electron acceptor. FadK was only detectable as a protease-cleaved form in the insoluble fraction suggesting that it may be a membrane-associated protein, like FadD. A model has been put forth in which FadD is transiently associated with the inner membrane (15). In this model the presence of long chain fatty acids results in FadD becoming membrane-associated in an active form, whereas in the absence of the inducer the enzyme has a cytoplasmic location and is inactive (15). However, in the present study, regardless of the presence or absence of the inducer, the intact FadD was found only in the insoluble (membrane) fraction of aerobically grown cells. The only soluble FadD was the C-terminal OmpT cleavage product (3). Because OmpT strains grow normally on fatty acids (data not shown) FadD cleavage by OmpT is not of physiological importance. Finally, our current models of fadD regulation (15) cannot explain the low levels of FadD present in anaerobically grown cells and the effects of glucose or octanoate addition suggesting that other regulatory circuits remain to be found.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant AI15650. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported in part by a National Science and Engineering Research Council of Canada Postdoctoral Fellowship. Back

|| To whom correspondence should be addressed: Dept. of Microbiology, University of Illinois, B103 Chemical and Life Sciences Laboratory, 601 S. Goodwin Ave., Urbana, IL 61801. Tel.: 217-333-7919; Fax: 217-244-6697; E-mail: j-cronan{at}life.uiuc.edu.

1 The abbreviations used are: ACS, acyl-CoA synthetase; MOPS, 3-(N-morpholino)propanesulfonic acid; fad, fatty acid degradation; FACS, fatty acid CoA-ligase; RB, rich broth; HPLC, high performance liquid chromatography; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-ethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank W. Metcalf and J. Imlay for use of their anaerobic chambers.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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