©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Comparison of the Reactivity of Tetradecenoic Acids, a Triacsin, and Unsaturated Oximes with Four Purified Saccharomyces cerevisiae Fatty Acid Activation Proteins (*)

(Received for publication, April 14, 1995)

Laura J. Knoll Otto F. Schall Iwao Suzuki George W. Gokel Jeffrey I. Gordon (§)

From the Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Saccharomyces cerevisiae contains at least five acyl-CoA synthetases (fatty acid activation proteins, or Faaps). Four FAA genes have been recovered to date. Recent genetic studies indicate that Faa1p and Faa4p are involved in the activation of imported fatty acids, while Faa2p activates endogenous pools of fatty acids. We have now purified Faa4p from S. cerevisiae and compared its fatty acid substrate specificity in vitro with the specificities of purified Faa1p, Faa2p, and Faa3p. Among C8-C18 saturated fatty acids, Faa4p and Faa1p both prefer C14:0. Surveys of C14 fatty acids with single cis-double bonds at C2-C12 indicated that Faa4p and Faa1p prefer Z9-tetradecenoic acid, although Faa4p's preference is much greater and also evident in C16 and C18 fatty acids. Faa4p's selectivity for fatty acids with a C9-C10 cis-double bond is a feature it shares with Faa3p and is notable since in yeast Ole1p, a microsomal cis-Delta^9 desaturase, accounts for de novo production of monoenoic acyl-CoAs from saturated acyl-CoA substrates. Faa4p has no detectable acyl-CoA synthetase activity when incubated with tetradecenoic acids having a trans-double bond at C2-3, C4-5, C5-6, C6-7, C7-8, or C9-10. Faa3p can only use E9-tetradecenoic acid as a substrate, while E4-, E6- and E9-tetradecenoic acids can be used by Faa1p and Faa2p. E2-tetradecenoic acid is an Faap inhibitor, with Faa2p exhibiting the greatest sensitivity (IC = 2.6 ± 0.2 µM). Triacsin C (1-hydroxy3-(E,E,E,2`,4`,7`-undecatrienylidine)-1,2,3-triazene) has trans-double bonds at positions that correspond to those in E2-, E5-, and E7-tetradecenoic acids. This compound is a potent inhibitor of Faa2p (K = 15 ± 1 nM; competitive with fatty acid), less potent against Faa4p (K = 2 µM), and not active against Faa1p or Faa3p (IC > 500 µM). Analysis of an n-tetradecanal plus a series of oximes (tridecanal oxime, 1-azadeca-1,3,5-trienol, and 1-azaundeca-1,3,5-trienol) indicated that the combination of an azenol moiety (R-CH&cjs0808;N-OH) plus adjacent unsaturation are critical for triacsin C's selective inhibition of Faa2p. Triacsin C and oxime derivatives appear to be very useful for defining differences in molecular recognition among S. cerevisiae acyl-CoA synthetases. The >25,000-fold range in the inhibitory effects of triacsin C on these four Faaps suggests that it may be possible to develop other selective inhibitors of eukaryotic acyl-CoA synthetases.


INTRODUCTION

Saccharomyces cerevisiae is an excellent, genetically manipulatable model system to study the regulation of acyl-CoA metabolism. De novo production of saturated acyl-CoAs from acetyl-CoA and malonyl-CoA is catalyzed by the fatty acid synthetase complex (Fas; (1) ). Palmitoyl-CoA and stearoyl-CoA are the principal products of Fas. Ole1p, a microsomal cis-Delta^9 desaturase, accounts for all de novo production of monoenoic acyl-CoAs from saturated acyl-CoA substrates(2, 3, 4, 5) . Exogenously derived and endogenous free fatty acids are activated to their acyl-CoA derivatives by at least five long chain acyl-CoA synthetases encoded by fatty acid activation (FAA) (^1)genes(6, 7, 8) .

The functions of four unlinked S. cerevisiae FAA genes have been characterized by noting the phenotypes of isogenic strains of yeast with all possible combinations of faa1, faa2, faa3, and/or faa4Delta (null) alleles(8) . The results indicate that Faa1p and Faa4p are the principal acyl-CoA synthetases responsible for activating imported fatty acids, while Faa2p appears to be involved in activation of endogenous fatty acids to their CoA derivatives. The physiologic role of Faa3p has not been defined. The existence of FAA5 was inferred from the observation that an faa1Deltafaa2Deltafaa3Deltafaa4Delta strain is still able to survive on media containing myristate, palmitate, or oleate as the sole carbon source, presumably because it is able to import these fatty acids, activate them, and direct the resulting acyl-CoAs to peroxisome-based beta-oxidation pathways(8) .

The overall reaction catalyzed by an acyl-CoA synthetase is as follows:

Berg (9, 10) hypothesized that activation of acetate (and presumably other fatty acids) involved at least two steps:

assumes the formation of a mixed phosphoryl-carboxyl anhydride (R-CO-O-PO(2)-O-ribose-adenine). Bar-Tana and co-workers used partially purified rat liver microsomal palmitoyl-CoA synthetase to present three lines of evidence that the catalytic mechanism involves formation of an enzyme-bound intermediate(11, 12, 13, 14, 15) . First, the initial velocity pattern of the forward reaction suggested a Bi Uni Uni Bi Ping Pong or a Bi Bi Uni Uni Ping Pong mechanism where ATP and palmitate must bind to enzyme before any product is released (14) . Second, ^18O present in [carboxy-^18O]palmitate is incorporated into AMP and palmitoyl-CoA but not into pyrophosphate(14) . Third, an enzyme intermediate was recovered by gel filtration chromatography after incubation of the acyl-CoA synthetase with [^14C]ATP and [^3H]palmitate. The molar ratio of fatty acid to nucleotide in the intermediate was 1:1(15) .

S. cerevisiae's four Faaps provide an opportunity to define common as well as divergent features in the structure/activity relationships of acyl-CoA synthetases. Faa1p, Faa2p, and Faa3p have been expressed in and purified from Escherichia coli strains that do not produce the single endogenous bacterial acyl-CoA synthetase encoded by the fadD gene(16, 17) . A survey (17) of their in vitro chain length selectivity using 4-24-carbon saturated fatty acids (C4:0-C24:0) indicated that Faa1p prefers C12:0 to C16:0 fatty acids, with C14:0 and C15:0 having the highest activities. C4:0-C9:0 and C20:0-C24:0 are inactive. Faa2p tolerates a broader range of fatty acyl chain lengths; C9:0-C13:0 are the most active substrates but C7:0-C8:0 and C14:0-C17:0 can be accommodated with less than a 2-fold reduction in acyl-CoA production. C22:0 and C24:0 are inactive. Faa3p has a 2-160-fold lower acyl-CoA synthetase activity with C9:0-C18:0 substrates compared with Faa1p and Faa2p. However, Faa3p is able to activate C22:0 and C24:0 (lignoceric acid). Surveys of C14, C16, and C18 fatty acids with and without cis-double bonds at C9-C10 indicate that Faa3p prefers palmitoleic and oleic acids as substrates. These are the two most abundant fatty acids in S. cerevisiae when it is grown on rich media (e.g. see (18) ). Faa4p has not been purified, nor has its acyl chain selectivity been defined. A recent genetic study indicated that a cDNA encoding rat liver acyl-CoA synthetase can complement at least some of the functions served by FAA4, namely activation of two exogenous fatty acids (myristate and palmitate), which are then utilized by intracellular phospholipid biosynthetic pathways(19) .

We have now isolated Faa4p from S. cerevisiae and compared its substrate selectivity with the selectivities of the other three known yeast Faaps using a series of saturated C8-C18 fatty acids, isomerically pure cis- and trans-tetradecenoic acids, a previously characterized triacsin inhibitor of mammalian acyl-CoA synthetases, and several aldoximes that are similar in some ways to the triacsin.


EXPERIMENTAL PROCEDURES

Production of Faaps in E. coli

Faap-6xHis Expression Vectors

A tag of six histidine residues (6xHis) was added to the carboxyl terminus of Faa1p, Faa2p, Faa3p, and Faa4p to allow for rapid and efficient selection of the full-length proteins by nickel-nitrilotriacetic acid (Ni-NTA)-agarose affinity chromatography. Details of the construction of Faa1p-6xHis (pBB319), Faa2p-6xHis (pBB321), Faa3p-6xHis (pBB341) are provided in Knoll et al.(17) . Each plasmid contains a kanamycin resistance gene and a p15 origin of replication (20) . Faap production is controlled by the isopropyl-beta-D-thiogalactopyranoside-inducible Ptac promoter(21) . An analogous Faa4p-6xHis expression vector (pBB371) was generated in three steps. (i) A 2.3-kilobase pair XbaI fragment was recovered from pBB364 (8) and ligated to XbaI-digested pMON5839(22) , yielding pBB370. (ii) A 280-bp KpnI-SalI fragment encompassing Gly-Thr of Faa4p, six histidine codons, a stop codon (TGA), and a SalI restriction site was produced using the polymerase chain reaction, 5`-CGCTGAATGGACCCCCAAGGG-3` and 5`-CCCCGTCGACTCAGTGATGGTGATGGTGATGAGTGTTTTCTTTATAAACTCTTTCCACATCTGGC-3` as primers, and pBB348 as template DNA. (iii) The PCR fragment was then subcloned into KpnI-SalI-digested pBB370, producing pBB371.

Purification of Faa1p-6xHis, Faa2p-6xHis, and Faa3p-6xHis from E. coli

E. coli strain LS6928 (fadRfadD27 zea::Tn10)(23) , containing pBB319, pBB321, or pBB341, was grown at 24 °C in Luria broth plus kanamycin (100 µg/ml) to an A approx 0.8. Faap-6xHis production was induced for 1 h with isopropyl-beta-D-thiogalactopyranoside (1 mM). Cells were harvested by centrifugation, resuspended in volume of buffer A (50 mM sodium phosphate, pH 7, 300 mM NaCl, 40 mM imidazole, 5 mM beta-mercaptoethanol, 1 mM Pefabloc SC (Boehringer Mannheim), 2 mM ATP), and burst open with a French press at 2000 p.s.i. Lysates were spun at 10,000 g for 20 min, and the Faap-6xHis was recovered by batch binding the supernatant to Ni-NTA-agarose (Quiagen; 10 ml of resin/5 liters of culture). The column was washed with 10 volumes of buffer A, and each Faap-6xHis was eluted with a linear gradient between buffer A and buffer B (buffer B is identical to buffer A except that it contains 200 mM imidazole). Column fractions were assayed for acyl-CoA synthetase activity using the enzyme-coupled assay described below, and protein concentration was determined(24) . CoA (2 mM) was added to the fractions with the highest specific activity. Faa1p-6xHis and Faa2p-6xHis can be stored up to 1 year at -80 °C without loss of activity. However, it is important to note that 2 mM ATP and CoA need to be included in the storage buffer to preserve enzyme activity. In contrast, Faa3p-6xHis loses >80% of its activity with a single freeze thaw cycle. Its activity also diminishes to a comparable extent if incubated for more than 6 h at 4 °C. Therefore, all assays employing this enzyme were performed within 0.5-2 h after isolation.

Metabolic Labeling of Cellular Phospholipids Produced by a fadDStrain of E. coli Containing Faap Expression Vectors

E. coli strain LS6928 was transformed with one of four vectors, each of which contained a p15 origin of replication, a kanamycin resistance gene, and an open reading frame specifying an Faap without a COOH-terminal 6xHis tag, under the control of the Ptac promoter. Cells with pBB277 (Faa1p; (16) ), pBB320 (Faa2p; (17) ), pBB323 (Faa3p; (17) ), or pBB370 (Faa4p) were labeled during exponential growth at 24 and 37 °C in Luria broth/kanamycin with [^3H]myristate, [^3H]palmitate, or [^3H]oleate (final specific activity of each fatty acid, 10 Ci/mmol). Cellular phospholipids were extracted and fractioned by high performance thin layer chromatography(16) . The radiolabeled lipids were visualized by fluorography. Experiments were repeated three times.

Expression of Faa4p-6xHis in S. cerevisiae

pBB373 (GPD-Faa4p-6xHis) was constructed by subcloning a 2.2-kilobase pair XbaI/SalI fragment derived from the bacterial expression plasmid, pBB371 (see above), into XbaI/SalI-digested pBB358(8) . pBB358 is a low copy YCp-based episome containing URA3. The FAA4-6xHis DNA insert in pBB373 is under the control of the constitutively expressed glyceraldehyde 3-phosphate dehydrogenase (GPD) promoter.

The functional effects of adding a 6xHis tag to Faa4p were defined by comparing the growth of isogenic S. cerevisiae strains under a variety of conditions. These strains, YB332 (MATa NMT1 ura3 his3Delta200 ade2 lys2-801 leu2 FAA1 FAA2 FAA3 FAA4) and YB525 (MATa NMT1 ura3 his3Delta200 ade2 lys2-801 leu2 faa1Delta::1.9::HIS3 FAA2 FAA3 faa4Delta0.3::LYS2), are described in (7) and (8) , respectively.

YB332 (FAA1FAA4) was transformed with the parental vector without FAA insert (pBB358). Strain YB525 (faa1Deltafaa4Delta) was transformed with pBB358 (GPD-vector), pBB364 (GPD-Faa4p without a COOH-terminal 6xHis tag; (8) ), or pBB373 (GPD-Faa4p-6xHis). The strains were grown at 30 °C to an A = 1 in supplemented minimal medium without uracil (SMM-URA; 0.67% yeast nitrogen base with ammonium sulfate (Bio101), 0.08% complete supplement minus uracil (Bio 101), 2% dextrose). Aliquots (50 µl) were removed from the cultures and added to 150 µl of sorbitol (1 M) and equivalent numbers of cells were stamped with a multiprong applicator (Replaclone) onto agar plates containing 1% yeast extract, 2% peptone, 2% dextrose (YPD) with or without 25 µM cerulenin, 500 µM myristate, 500 µM palmitate, or 500 µM oleate. (Brij-58 (1% w/v) was added to media containing fatty acids.) Plates were incubated at 24, 30, or 37 °C for 3-4 days. Experiments were performed in quadruplicate on two separate occasions.

Purification of Faa4p-6xHis from S. cerevisiae

S. cerevisiae strain YB525 (faa1Deltafaa4Delta) containing pBB373 (GPD-Faa4p-6xHis) was grown at 24 °C to an A approx 1 in 2 liters of SMM-URA. Cells were harvested by centrifugation, resuspended in 10 ml of ice cold homogenization buffer (0.2 M Tris-HCl, pH 8.1, 2 mM ATP, 4 mM EDTA, 5 mM beta-mercaptoethanol, 8 µM leupeptin, 1 mM Pefabloc SC, 10% glycerol, 0.1% Brij-35) and lysed by vortexing in an equal volume of 425-600-µm glass beads (Sigma; 4 cycles of 1 min each with cooling on ice between each cycle of vortexing). Lysates were spun at 10,000 g for 5 min at 4 °C. The cleared supernatant was poured over a 10-ml Poly-Prep spin column (Bio-Rad) containing 1.5 ml of Ni-NTA-agarose. The column was immediately spun at 300 g at 4 °C and then washed with 10 ml of ice cold buffer A (see above). Faa4p-6xHis was eluted with 4 ml of buffer B (see above) supplemented with 2 mM CoA. Acyl-CoA synthetase activity was measured using the HPLC assay described below. Faa4p-6xHis was used within 0.5-1 h after purification because of its instability when maintained for longer periods of time at 4 °C. The enzyme is not stable when subjected to a single cycle of freezing at -20 or -80 °C and thawing.

Organic Syntheses

Tridecanal Oxime (1)

Tridecanal (0.79 g, 4.0 mmol; Aldrich), hydroxylamine hydrochloride (0.35 g, 5.0 mmol), pyridine (3.0 ml), and ethanol (anhydrous, 3.0 ml) were stirred at ambient temperature for 1 h. The solvent was removed in vacuo, water (20 ml) and ether (20 ml) were added, the layers were separated, the aqueous layer was washed again with ether (20 ml), and the combined organic phase was dried (MgSO(4)) and evaporated. The product was obtained (0.32 g, 38%) after several recrystallizations (hexanes) as a white solid, melting point 82.5-83 °C; ^1H-NMR: 0.881 (t, J = 6.9 Hz, ^3H, CH(3)), 1.2-1.4 (m, 20H, methylenes), 1.488 (quintet, J = 7.1 Hz, 2H, C(3)H), 2.195 (quartet, J = 7.0 Hz, 1.2H, C(2)H), 2.364 (br quartet, J = 7.0 Hz, 0.8H, C(2)H), 6.7-6.8 (br s, 1.2H, C(1)H), 7.427 (t, J = 6.2 Hz, 0.8H, C(1)H).

1-Azadeca-1,3,5-trienol (2)

A mixture of nona-2,4-dienal (0.55 g, 4.0 mmol; Aldrich), hydroxylamine hydrochloride (0.35 g, 5.0 mmol), pyridine (3.0 ml), and EtOH (3.0 ml) was stirred at room temperature for 1 h, and the solvent was removed in vacuo. Water (20 ml) and ether (20 ml) were added to the syrupy residue, the ether layer was separated, the aqueous layer was extracted again with ether (20 ml), the ether layers were combined and dried over MgSO(4), and the solvent evaporated. The residue was purified by silica gel column chromatography (hexanes-ethyl acetate, 9:1). Fractions containing the desired product were combined, and the solvent was removed. Several crystallizations (hexanes) of the resulting solid afforded azatrienol (2) (0.13 g, 21%) as a colorless solid, melting point 110-111 °C. ^1H-NMR: 0.905 (t, J = 7.1 Hz, ^3H); 1.24-1.46 (m, 4H); 2.16 (q, J = 7.5 Hz, 2H); 5.98 (dt J = 7.0 and 15.1 Hz, 1H); 6.19 (dd, J = 10.4 and 15.1 Hz, 1H); 6.49 (dd, J = 10.4 and 15.4 Hz, 1H); 6.72 (dd, J = 9.5 and 15.3 Hz, 1H); and 7.10 (d, J = 9.3 Hz, 1H). Anal. calcd for C(9)HNO: C, 70.55; H, 9.87; N, 9.14%. Found, C, 70.45; H, 9.84; N, 9.07%.

1-Azaundeca-1,3,5-trienol (3)

A mixture of deca-2,4-dienal (0.61 g, 4.0 mmol; Aldrich), hydroxylamine hydrochloride (0.35 g, 5.0 mmol), pyridine (3.0 ml), and EtOH (3.0 ml) was stirred at room temperature for 1 h, and the solvent was removed in vacuo. Water (20 ml) and ether (20 ml) were added to the syrupy residue, the ether layer was separated, the aqueous layer was extracted again with ether (20 ml), the ether layers were combined and dried over MgSO(4), and the solvent evaporated. The residue was chromatographed over silica gel (hexanes-ethyl acetate, 9:1). Fractions containing the desired product were combined, and the solvent was removed in vacuo. Several crystallizations (hexanes) of the resulting solid afforded azatrienol (3) (0.14 g, 21%) as a colorless solid, melting point 98-99 °C. ^1H-NMR: 0.891 (t, J = 6.8 Hz, ^3H, CH(3)), 1.22-1.36 (m, 4H, C(8)H and C(9)H), 1.420 (quintet, J = 7.6 Hz, 2H, C(7)H), 2.146 (quartet, J = 7.1 Hz, 2H, C(6)H), 5.983 (dt, J = 7.0 and 15.1 Hz, 1H, C(5)H), 6.189 (dd, J = 10.4 and 15.1Hz, 1H, C(4)H), 6.492 (dd, J = 10.4 and 15.5Hz, 1H, C(3)H), 6.721 (dd, J = 9.5 and 15.4Hz, 1H, C(2)H), 7.101 (d, 1H, J = 9.6 Hz, C(1)H), 8.45-8.75 (br s, 1H, OH). Anal. calcd for CHNO: C, 71.81; H, 10.24; N, 8.37%. Found, C, 72.00; H, 10.17; N, 8.44%.

Fatty Acids

The methods used for synthesis of unsaturated derivatives of tetradecanoic acid are described in Kishore et al.(25) . The isomeric purity of each fatty acid was established by gas phase chromatography(25) .

In Vitro Assays for Acyl-CoA Synthetase Activity

Enzyme-coupled Assay

This assay was adapted from Ichihara and Shibasaki(26) . Reaction mixtures (50 µl) contained 150 mM Mops, pH 7.1, 2 mM CoA, 2 mM ATP, 10 mM MgCl(2), 200 µM fatty acid, 1 mM dithiothreitol, 1% methanol, 0.1% Triton X-100, 2000 units/ml bovine liver catalase (Sigma), 33 units/ml acyl-CoA oxidase (Boehringer Mannheim), and either purified Faa1p-6xHis (300 µg/ml), Faa2p-6xHis (300 µg/ml), Faa3p-6xHis (60 µg/ml), or Faa4p-6xHis (30 µg/ml). Reactions were assembled in each well of a 96-well polystyrene plate (Costar). The microtiter plate was incubated at 25 °C for 20 min. The reaction was stopped with 50 µl of 2 N KOH. Fifty microliters of 0.6% 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (prepared in 0.5 M HCl; Sigma) were added to each well, and the plate was incubated for an additional 10 min at 37 °C. One hundred microliters of 0.375% KIO(4), 0.2 M KOH was placed in each well, and the plate was incubated at 25 °C for 10 min. Absorbance was determined at 550 nm with a Thermo-Max Reader (Molecular Devices). The molar absorption coefficient ( = 29,200 M cm) (23) of the resulting purple dye was used to calculate the amount of acyl-CoA produced.

The rate of product formation was assessed using these assay conditions, myristate or oleate as substrates, each of the purified enzyme preparations, and incubation times of 1, 2, 4, 8, 16, 24, 32, and 40 min. The results established that formation of the triazolotetrazine dye was linear up to 32 min. Therefore, the activity of fatty acid analogs was surveyed using a 20-min incubation period. Each tetradecenoic acid was assayed in triplicate. All experiments were repeated at least three times.

HPLC-based Assay ((17) )

To determine acyl chain specificity, 100-µl reactions contained 150 mM Mops, pH 7.1, 2 mM CoA, 2 mM ATP, 10 mM MgCl(2), 1 mM dithiothreitol, 0.05% Triton X-100, 100 µM EGTA, a purified Faap-6xHis (final concentration, 1 µg/ml), and one of the following fatty acids (all at a final concentration of 200 µM and a final specific activity of 55 mCi/mmol; all purchased from American Radiolabeled Chemicals): [1-^14C]octanoic acid, [1-^14C]decanoic acid, [11,12-^3H]lauric acid, [9,10-^3H]myristic acid, [9,10-^3H]palmitic acid, [9,10-^3H]stearic acid, [9,10-^3H]palmitoleic acid, and [9,10-^3H]oleic acid. Following a 15-min incubation at 25 °C, the reactions were terminated with an equal volume of 5% trichloroacetic acid/methanol. Labeled fatty acyl-CoA and fatty acid were resolved using a C4 reverse phase HPLC column (Vydac, 5-µm particle size, 4.6 150 mm) and the following isocratic gradients: (i) for C8:0 and C10:0, 95% 20 mM potassium phosphate buffer, pH 5.5, 5% acetonitrile for 10 min followed by 50% 20 mM potassium phosphate, pH 5.5, 50% acetonitrile for 12 min; (ii) for C12:0, 80% 20 mM potassium phosphate, 20% acetonitrile for 10 min followed by 50% 20 mM potassium phosphate, 50% acetonitrile for 12 min; (iii) for C14-C18 fatty acids, 60% 20 mM potassium phosphate, 40% acetonitrile for 10 min followed by 50% 20 mM potassium phosphate, 50% acetonitrile for 12 min. Radiolabeled acyl-CoAs and fatty acids were quantitated using an in-line scintillation counter (Radiomatic).

Product formation was assessed using these assay conditions, radiolabeled oleate as a substrate, and incubation times of 2, 5, 10, 15, and 30 min. With each purified Faap-6xHis, acyl-CoA production was linear up to 30 min. At 15 min, less that 20% of the fatty acid had been converted to its acyl-CoA derivative. We selected this time point for assaying other radiolabeled fatty acids. IC values were determined using similar assay conditions except that the reactions contained 5 µM [9,10-^3H]oleate (10 Ci/mmol), 0-1.5 mM of the test compound, 0.2 µg of a purified Faap-6xHis, and a 15-min incubation at 24 °C. (We confirmed that < 20% of [^3H]oleic acid is converted to its CoA derivative under these conditions in the absence of test compound.) All assays were done in triplicate on two or three occasions.

The K of purified Faa1p-6xHis and Faa2p-6xHis for oleate was defined by Eadie Hofstee plots(27, 28) . These plots were also used to calculate the K for the inhibition of Faa2p-6xHis and Faa4p-6xHis by triacsin C (Kameda Co.).


RESULTS

Faa4p Is Not Active in E. coli

Three observations indicated that, unlike the other known Faaps, Faa4p is unstable in E. coli or missing a heretofore unidentified co-factor required for its activity. (i) The same vector that directs efficient expression Faa1p, Faa2p, or Faa3p in a fadD strain of E. coli(16, 17) was used in an attempt to produce Faa4p. Lysates were prepared from cells grown at 24 °C and assayed for acyl-CoA synthetase activity with three tritiated fatty acids. Myristoyl-CoA-, palmitoyl-CoA-, or oleoyl-CoA synthetase activity was not detectable in lysates prepared from bacteria with the Faa4p plasmid, in contrast to lysates prepared from strains containing the Faa1p, Faa2p, or Faa3p expression plasmids (data not shown). (ii) Because Faa4p might have been degraded during lysate preparation, we attempted to measure its activity in vivo. Tritiated myristate, palmitate, or oleate was added to exponentially growing cultures of the fadD strain containing each of the expression plasmids and incorporation of label into newly synthesized cellular phospholipids measured by high performance thin layer chromatography. Phosphatidylethanolamine, phosphatidylglycerol, and phosphatidic acid were labeled when cells contained either the Faa1p, Faa2p, or Faa3p vectors(16) . Only phosphatidylethanolamine was labeled in cells containing the expression vector without insert or with the FAA4 open reading frame (data not shown; note that PE labeling occurs in bacteria lacking fadD through a pathway that involves activation of exogenous fatty acids by the bacteria's inner membrane acyl-acyl carrier protein synthetase/2-acyl-glycerophosphoethanolamine acyltransferase; Refs. 16, 29, 30). (iii) Plasmids encoding the Faaps with a COOH-terminal tag of 6 histidine residues were introduced in the fadD strain. Cultures were grown to mid-log phase at 24 or 30 °C, Faap-6xHis production was induced for 1 h, and cell lysates were subjected to Ni-NTA-agarose affinity chromatography. This procedure permits rapid and efficient recovery of homogeneous preparations of Faa1p, Faa2p, and Faa3p(17) . In contrast, neither protein of the expected size nor acyl-CoA synthetase activity was recovered from bacteria containing the Faa4p-6xHis plasmid (data not shown).

Purification and Initial Characterization of Faa4p from S. cerevisiae

Previous studies of cell lysates prepared from isogenic FAA1FAA4 and faa1Deltafaa4Delta strains indicated that Faa1p and Faa4p together account for 99% of endogenous cellular myristoyl-CoA and palmitoyl-CoA synthetase activities(19) . Therefore, we attempted to recover Faa4p-6xHis from faa1Deltafaa4Delta cell lysates using Ni-NTA-agarose affinity chromatography with the expectation that there would be virtually no contaminating acyl-CoA synthetase activities from other endogenous FAA gene products. Prior to this attempt at enzyme purification, we compared the biological properties of Faa4p with and without a COOH-terminal histidine tag. This was done by noting the ability of a low copy, centromeric GPD-FAA4 or GPD-FAA4-6xHis episome to rescue growth of a faa1DeltafaaDelta strain in YPD/agarose containing 25 µM cerulenin with or without 500 µM myristate, 500 µM palmitate, or 500 µM oleate. Cells with vector alone failed to grow at 24, 30, or 37 °C when de novo acyl-CoA production by Fas was blocked with cerulenin even if the medium was supplemented with fatty acids. (Fig. 1). The GPD-FAA4 and the GPD-FAA4-6xHis episomes were able to rescue growth in YPD/cerulenin plus C14:0, C16:0 or C18:1, at each of the three temperatures surveyed (Fig. 1). Moreover, the extent of the rescue was equivalent (except at 37 °C where oleate was less effective with both GPD-FAA4 and GPD-FAA4-6xHis). These findings demonstrate that (i) FAA4 can substitute for FAA1 and activate sufficient quantities of imported fatty acids to overcome the acyl-CoA deficiency produced by Fas' inhibition; (ii) addition of a COOH-terminal tag of six histidines does not interfere with the enzyme's ability to function in vivo; (iii) like Faa1p, Faa2p, and Faa3p(19) , Faa4p and its His-tagged derivative are not inhibited by cerulenin; and (iv) Faa4p and Faa4p-6xHis can use myristic, palmitic, and oleic acids as substrates.


Figure 1: A centromeric plasmid encoding Faa4p, with or without a COOH-terminal histidine tag, can rescue growth of an faa1Deltafaa4Delta strain of S. cerevisiae when fatty acid synthetase is inhibited and the medium is supplemented with fatty acids. An equal number of cells from YB332 (FAA1FAA4), transformed with the parental YCp-GPD vector (pBB358), or YB525 (faa1Deltafaa4Delta), transformed with either the parental vector, GPD-FAA4, or GPD-FAA4-6xHis, were plated onto YPD media supplemented with 25 µM cerulenin (CER), with or without 500 µM myristate (C14:0), palmitate (C16:0), or oleate (C18:1). Plates were incubated for 3-4 days at 24, 30, or 37 °C.



Ni-NTA-agarose affinity chromatography of faa1Deltafaa4Delta GPD-FAA4-6xHis cell lysates resulted in an average 120-fold enrichment in oleoyl-CoA synthetase activity (n = 6 experiments). In contrast, no oleoyl-CoA synthetase activity was recovered from faa1Deltafaa4Delta cells containing the GPD-vector without insert. The affinity purification protocol is simple and rapid (20 min from lysis of cells to assay). This rapidity is important; Faa4p-6xHis is only stable for 60 min after purification is completed.

Assay conditions were identified where [^3H]oleoyl-CoA production by purified Faa4p-6xHis was linear over a 30-min period (see ``Experimental Procedures''). The conditions were then used to identify the enzyme's pH and temperature optima. There is little change in oleoyl-CoA synthetase activity at 24 °C from pH 6.5 to 7.9 (Fig. 2A). Moreover, enzyme activity varies <2-fold between 24, 30 and 37 °C at pH 7.1 (Fig. 2B), in general agreement with the in vivo studies shown in Fig. 1.


Figure 2: The pH, temperature, and acyl chain selectivity of purified Faa4p-6xHis. pH and temperature optima were determined using [^3H]oleic acid as a substrate and the HPLC assay described under ``Experimental Procedures.'' The effects of changing pH were assessed at 24 °C. The temperature optimum was defined at pH 7.1. A panel of radiolabeled C8-C18 saturated fatty acids plus palmitoleic (C16:1) and oleic (C18:1) acids were used to generate the data shown in panelC (24 °C, pH 7.1). Mean values ± 1 S.D. are plotted (n = 2 experiments, each done in duplicate). An asterisk indicates that a radiolabeled acyl-CoA product was not detected.



The Acyl Chain Length Selectivity of Purified Faa4p-6xHis

Faa4p-6xHis was incubated with members of a panel of radiolabeled C8-C18 saturated fatty acids (Fig. 2C). C14:0 was the most active substrate. Removal of 2 methylenes (lauric acid; C12:0) results in a 20-fold reduction in acyl-CoA production, while addition of 4 methylenes (stearic acid; C18:0) produces a 4-fold reduction. These findings are consistent with results obtained when unfractionated cell lysates, prepared from the faa1Deltafaa4Delta strain containing GPD-Faa4p (without 6xHis tag), were incubated with tritiated myristate and palmitate(19) .

Faa4p-6xHis Prefers a cis-Double Bond at C9-C10

Introduction of a cis-double bond at C9-C10 of stearic acid (yielding C18:1) produces a 7-fold more active substrate compared with C18:0 (Fig. 2C). An increase in product formation is also observed when a cis-double bond is placed at this position in palmitic acid (compare C16:0 and C16:1 in Fig. 2C).

Radiolabeled myristoleic acid (C14:1) was not commercially available. However, we had previously synthesized a series of myristic acid analogs with single cis-double bonds at all possible positions in the alkyl chain (Z2-Z13-tetradecenoic acids; (25) ). This family of compounds allowed us to explore the enzyme's cis-double bond selectivity. Because they were unlabeled, we determined their relative activities as Faa4p-6xHis substrates in a system that detects acyl-CoA products through a series of secondary reactions: (i) Candida acyl-CoA oxidase is used to generate hydrogen peroxide from acyl-CoA; (ii) the hydrogen peroxide is used, together with catalase, to oxidize methanol to formaldehyde; (iii) formaldehyde reacts with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole to form a purple triazolotetrazine dye in the presence of basic periodate, and (iv) the concentration of the purple dye is determined colorimetrically. Failure to detect product in this enzyme-coupled assay may either reflect the fact that the tetradecenoic acid is not a substrate for the purified acyl-CoA synthetase or that the resulting analog-CoA may not be a substrate for the Candida acyl-CoA oxidase. We could conclude that an analog was not a substrate for Faa4p only if acyl-CoA production was detected with another Faap.

When the coupled assay system contained purified Faa4p-6xHis, Z9-tetradecenoic acid was the most active compound. The Z10 and Z11 isomers were 3-4-fold less active, while Z12 or the Z8-Z2-tetradecenoic acids did not yield detectable levels of product (Fig. 3A).


Figure 3: Comparison of the activities of Z-tetradecenoic acids as Faap substrates. Each purified Faap was incubated with members of the panel of unlabeled tetradecenoic acids with single cis-double bonds at various positions along the alkyl chain (e.g.Z2 = cis-double bond between C2 and C3; C1 = carboxyl). Acyl-CoA production was measured using the coupled enzyme assay described under ``Experimental Procedures.'' The mean value obtained with each analog has been plotted (± S.D.) from three independent experiments, each done in triplicate. Product, triazolotetrazine dye. An asterisk indicates that acyl-CoA production was below the limits of detection of the assay (<0.02 pmol/min/mg Faap). Note the difference in scale in the plot of product formation with Faa3p.



Comparison of the Selectivity of Purified S. cerevisiae Faa1p, Faa2p, and Faa3p for Z-Tetradecenoic Acids

Purified Faa3p-6xHis can only accommodate a cis-double bond at C9-C10 although its acyl-CoA synthetase activity with this compound was 5-fold lower than with Faa4p-6xHis (Fig. 3B). Faa1p-6xHis shows a preference for a cis-double bond at C9-C10 or C10-C11 with Z-9-tetradecenoic acid favored (Fig. 3D). Nonetheless, the acyl-CoA synthetase can accept a cis-double bond at all other positions except C2-C5 with no more than a 4-fold reduction in product formation. Faa2p-6xHis is the least selective of the four Faaps: Z4 to Z12-tetradecenoic acids have similar activities as substrates (Fig. 3C). These activities are comparable to C14:0 (data not shown). Thus, it appears that with the exception of Faa2p, the yeast acyl-CoA synthetases prefer a (C14) fatty acid with a cis-double bond at C9-C10. This makes physiologic sense. As noted in the Introduction, Ole1p is the only known desaturase in this organism.

E-Tetradecenoic Acids

The coupled enzyme assay was used to assess whether any of the Faaps could accommodate unlabeled tetradecenoic acids having a trans-double bond at C2-3, C4-5, C5-6, C6-7, C7-8, or C9-10. Faa4p had no detectable activity with any of the six, isomerically pure, analogs (Fig. 4). Faa3p could only accommodate E9-tetradecenoic acid. Faa1p and Faa2p showed remarkable apparent selectivity. E2- and E5-tetradecenoic acids produced no detectable acyl-CoA product with Faa1p, while the E4- and E6-isomers were accommodated as well as, if not better than, the corresponding Z-tetradecenoic acids (compare Fig. 4with Fig. 3). A similar pattern was observed with Faa2p: E4-, E6-, and E9-tetradecenoic acids were as active as the Z-isomers, while introduction of a trans-double bond at C2, C5, or C7 resulted in geq10-fold less activity than with the cis-tetradecenoic acids ( Fig. 4and Fig. 3).


Figure 4: Comparison of the activities of E-tetradecenoic acids as Faap substrates. Unlabeled tetradecenoic acids with trans-double bonds at the positions indicated were each incubated with a purified Faap-6xHis, and acyl-CoA product formation was detected with the coupled enzyme assay. Asterisk, acyl-CoA production below the limits of detection of the assay. See the legend to Fig. 3for further details.



The ``inactive'' E2, E5, and E7 isomers were evaluated further with an HPLC assay that measures the ability of these compounds to block conversion of [^3H]oleic acid to its CoA derivative. Only E2-tetradecenoic acid showed detectable inhibitory activity (at 10-100-fold molar excess compared with substrate). The degree of inhibition by E2-tetradecenoic acid was enzyme-specific: Faa2p was most sensitive (IC = 2.6 ± 0.2 µM). Faa1p-6xHis was less affected (IC = 148 ± 10 µM).

Faa4p's preference for a cis- but not a trans-double bond at C9-C10 highlights its stereoselectivity. The panel of E-tetradecenoic acids also revealed a stereoselectivity in Faa2p that was not apparent with the corresponding cis-isomers.

Triacsin C Is a Selective Inhibitor of S. cerevisiae Faaps

The triacsins were originally isolated from Streptomyces sp.(31, 32) . Triacsins A-D consist of an unbranched chain of 15 non-hydrogen atoms that terminates with a triazenol group (R-CH&cjs0808;N-N&cjs0808;N-OH). All triacsins contain multiple trans-double bonds. All triacsins are identical from the nitrogen at position 1 through position 8 (Fig. 5). Triacsins A and C differ from triacsins B and D in that the former have a saturated carbon at position 10 while the latter are unsaturated (Fig. 5). In vitro studies by Tomoda et al.(33) , using rat liver microsomal or partially purified Pseudomonas aeruginosa acyl-CoA synthetases and [^3H]oleic acid, indicated that triacsins A and C are more potent long chain acyl-CoA synthetase inhibitors (IC = 4-18 µM) than triacsins B and D (IC > 200 µM). Kinetic studies with the bacterial enzyme revealed that triacsin A was competitive with oleic acid (K of C18:1 = 100 µM; K for triacsin A = 8 µM) and noncompetitive with respect to ATP or CoA(33) . Two hydrolysis products of triacsins A and C (E,E-2,4-undecadienal and E,E,E-2`,4`,7`-undecatrienal, respectively) were not inhibitors, leading these workers to suggest that the N-hydroxytriazene domain is required for the molecules' inhibitory effects on long chain acyl-CoA synthetases(33) .


Figure 5: Comparison of the structures of triacsins A-D with tetradecanoic acid, n-tetradecanol, tridecanal oxime, 1-azadeca-1,3,5-trienol, and 1-azaundeca-1,3,5-trienol. The triazenol group in the triacsins has been boxed and aligned with comparable regions of the other compounds.



Triacsin C has trans-double bonds at positions that correspond to those in E2-, E5-, and E7-tetradecenoic acids (Fig. 5). As noted above, one of these isomers, E2-tetradecenoic acid, is a selective inhibitor of Faa2p. This led us to ask whether triacsin C is also a selective Faap inhibitor. Since oleic acid is a substrate for each Faap, we measured the effects of triacsin C on their oleoyl-CoA synthetase activities. The triacsin is not active against purified Faa1p-6xHis or Faa3p-6xHis (IC > 500 µM). It is a potent inhibitor of Faa2p, with an IC of 80 ± 10 nM and a K of 15 ± 1 nM (the enzyme's K for C18:1 is 25 ± 1 µM; Fig. 6). Eadie-Hofstee plots indicated that triacsin C is competitive with respect to oleate (Fig. 6). We were not able to determine whether triacsin C is noncompetitive with ATP or CoA, as is the case with the bacterial acyl-CoA synthetase, because Faa2p-6xHis has to be stored with these compounds (concentration of 2 mM) to remain stable. Triacsin C also inhibits purified Faa4p-6xHis but is less potent (IC = 4.5 ± 0.5 µM; K = 2 µM) than is the case with Faa2p.


Figure 6: Triacsin C is a potent competitive inhibitor of Faa2p. A, Eadie-Hofstee plots of the inhibition of Faa2p with varying concentrations of triacsin C using tritiated oleic acid as the substrate. Note that with increasing concentrations of triacsin C, the K for oleate (slope = -K) becomes greater while the V(max) (the y intercept) remains unaffected. This finding indicates that triacsin C is competitive with respect to fatty acid. B, plot of the concentration of triacsin C versus the K values determined from panelA. The K of triacsin C is 15 ± 1 nM. n = 3 independent experiments, each done in duplicate.



Oxime Analogs Reveal Structural Elements in Triacsin C That Are Important for Its Inhibitory Activity

It was unclear which domain or domains of triacsin C contributed to its potent inhibition of Faa2p. n-Tetradecanol has a chain length similar to triacsin C but does not share any of its other structural features except that it has a terminal hydroxyl group. n-Tetradecanol is not an effective inhibitor (IC with Faa2p = 215 ± 11 µM). Thus, the terminal -OH is not the unique determinant of triacsin C's inhibition. Like triacsin C, the oxime derivative of tridecanal has a terminal hydroxylamino group (Fig. 5). Corey-Pauling-Koltun models indicate that their overall chain lengths are equivalent. Tridecanal oxime lacks double bonds in the carbon chain and the nitrogen atoms at positions 2 and 3. Thus, this compound is an azenol (R-CH&cjs0808;N-OH) rather than a triazenol (R-CH&cjs0808;N-N&cjs0808;N-OH). We synthesized tridecanal oxime and obtained the E-isomer as the predominant product. The Z- and E-isomers were then separated chromatographically. NMR spectroscopy of the purified aldoxime isomers revealed the expected nuclear Overhauser effect signals. Tridecanal oxime has minimal effects on the oleoyl-CoA synthetase activity of Faa2p (IC = 245 ± 17 µM), nor does it produce detectable inhibition of Faa4p, indicating that the azenol fragment is not itself sufficient to inhibit these two enzymes.

The oxime derivatives of 2,4-nonadienal and 2,4-decadienal were also synthesized, yielding 1-azadeca-1,3,5-trienol and 1-azaundeca-1,3,5-trienol, respectively (Fig. 5). They were obtained as E-isomers. These compounds may be considered to be bis(desaza)triazenols (i.e. they contain a domain that resembles the triazenol functional group including two conjugated trans-double bonds (triazenol: R-CH&cjs0808;N-N&cjs0808;N-OH; bis(desaza)triazenol: R-CH&cjs0808;CH-CH&cjs0808;N-OH)). The compounds were obtained as E-isomers. 1-Azadeca-1,3,5-trienol and 1-azaundeca-1,3,5-trienol both inhibit Faa2p (IC = 6 ± 0.5 and 16 ± 1 µM, respectively). As with triacsin C itself, they show selectivity between the Faaps, i.e. IC > 1500 µM for Faa1p (Faa1p's C18:1K = 62 ± 3 µM).


DISCUSSION

Faa4p: Why and Where?

Faa4p is a 694-residue protein that has 78% identity with Faa3p, 61% identity with Faa1p, and 23% identity with Faa2p(8) . Physical mapping studies suggest that FAA4 and FAA1 may have arisen from a gene duplication event(8) . Faa4p and Faa1p also appear to share a common function, activation of imported fatty acids. This is based on the observation that either a GPD-FAA1 or a GPD-FAA4 episome can overcome the growth arrest of an faa1Deltafaa4Delta strain on rich media containing cerulenin and 500 µM C14:0, C16:0, or C18:1. A GPD-FAA2 episome produces no detectable rescue, while only minimal rescue is achieved with a GPD-FAA3 episome(8) . The difference between FAA1/FAA4's and FAA2's ability to rescue growth cannot be correlated with differences in the in vitro fatty acid substrate specificities of their purified protein products, leading to the notion that acyl-CoA metabolism is highly compartmentalized in S. cerevisiae and that part of this compartmentalization is achieved by compartmentalization of the organism's multiple acyl-CoA synthetases. Unfortunately, neither the location nor the relative steady state levels of the four Faaps are known. Definition of the intracellular distributions of the Faaps may explain the physiologic significance of their overlapping yet distinct in vitro fatty acid selectivities. For example, Faa2p accommodates the broadest range of fatty acids in vitro ( (17) and this report). If this enzyme functions to activate endogenous pools of fatty acids, a broad tolerance for chain length variations in saturated and unsaturated fatty acids may be needed. If Faa3p is associated with peroxisomes, its observed ability to activate very long chain fatty acids in vitro(17) makes physiologic sense.

Activation of imported fatty acids during growth at 24-37 °C on rich media is principally the function of Faa1p since deletion of FAA1 but not FAA4 impairs the ability of yeast to grow on YPD/cerulenin/fatty acid media(8) . The impairment is temperature- and fatty acid-dependent; growth retardation worsens with higher temperatures and when C16:0 or C18:1 is substituted for C14:0(8) . The combination of faa1 and faa4 null alleles produces inviable cells on YPD/cerulenin/fatty acid media when fatty acid = 14:0, 16:0, or C18:1. However, no other combination of two faa null alleles results in this lethal phenotype, and faa2Deltafaa4Delta and faa3Deltafaa4Delta cells demonstrate no diminution of growth on YPD/cerulenin/fatty acid media (8) .

These observations beg the obvious question, ``Why is there an Faa4p?'' Faa4p does not have to form a heterodimer with Faa1p for either enzyme to function since GPD-FAA1 or GPD-FAA4 episomes can rescue growth of faa1Deltafaa4Delta cells on YPD/cerulenin/fatty acid media ( Fig. 1and (8) ). The remarkable instability of purified Faa4p raises the possibility that it may normally reside in a cellular membrane. However, the enzyme does not appear to have a distinctive role in fatty acid transport across the yeast's plasma membrane. Measurement of the rate and extent of import of C14:0 or C16:0 into isogenic FAA1,FAA4 and faa1Deltafaa4Delta strains harvested during exponential growth indicated that (i) S. cerevisiae contains a saturable fatty acid transport system (Kfor the two fatty acids approx 50 µM, where K represents the substrate concentration at which the transport rate is half its maximal value) and (ii) fatty acid uptake is not coupled to activation by either Faa1p or Faa4p (i.e. there is no difference in the ability of FAA1FAA4 and faa1Deltafaa4Delta strains to take up fatty acids, nor does overexpression of Faa1p or Faa4p produce a detectable increase in C14:0 or C16:0 import(19) ). There is no dramatic difference in the fatty acid selectivities of the two purified enzymes in vitro; both prefer C14:0 among C8-C18 saturated fatty acids, and both favor a cis-double bond at C9-C10. It is likely that the physiologic function of Faa4p has not been determined because the effects of genetic manipulations have only been assessed under conditions that allow exponential growth (e.g. analysis of isogenic strains containing single faa null alleles indicates that during exponential growth when Fas is active, none of the FAA genes is essential). However, there is at least one circumstance when both FAA1 and FAA4 are needed, even when Fas is active. Wild type strains of S. cerevisiae can grow using glycerol as the sole carbon source if glycerol can be efficiently converted to glyceraldehyde-3-phosphate for entry into the glycolytic pathway. This requires adequate mitochondrial respiration so that the NADH produced by glycolysis can be removed. Isogenic strains with single faaDelta alleles grow on YP/glycerol at rates comparable to wild type cells(8) . Analysis of cells with all possible combinations of two faaDelta alleles revealed that only faa1Deltafaa4Delta strains exhibit diminished growth. This diminution is temperature-dependent; at 30 °C there is mild growth retardation, while at 37 °C no growth is observed(8) . These findings raise the possibility that FAA1 and FAA4 influence mitochondrial function, either directly or indirectly.

Additional studies of isogenic strains with various combinations of faa null alleles are clearly required, including an analysis of their viability/longevity during stationary phase when marked changes in lipid metabolism are known to occur(34) . Given the predilection of Faa1p and Faa4p for C14:0, an additional parameter to monitor in stationary phase cells with faa1Delta and/or faa4Delta is the effect of introducing mutations in the essential myristoyl-CoA:protein N-myristoyltransferase gene (NMT1), specifically mutations that reduce Nmt1p's affinity for its acyl-CoA substrate and hence produce a requirement for augmentation of cellular myristoyl-CoA pools to maintain survival (35, 36) .

Structure/Activity Relationships in Triacsin C

The triacsins are the only reported naturally occurring compounds known to contain the triazenol group. (^2)The structural features in the triacsins that are responsible for their effects on eukaryotic acyl-CoA synthetases have not been defined previously. The studies described above with purified Faaps illustrate one important point that must be considered before beginning an analysis of triacsin structure/activity relationships; there are multiple long chain acyl-CoA synthetases with overlapping yet distinct substrate specificities, even in a simple eukaryote such as S. cerevisiae, and each purified Faap may have remarkable differences in its sensitivity to a given triacsin. Among the Faaps, Faa2p has the greatest degree of sequence similarity to known mammalian acyl-CoA synthetases(8) . The potent inhibitory effects of triacsin C on Faa2p make this acyl-CoA synthetase a good model for identifying structural fragments in this compound that are responsible for its activity. The triacsins all possess four obvious chemical features: (i) identical overall length; (ii) a terminal hydroxyl group; (iii) nitrogen atoms; and (iv) trans-double bonds. Our studies of a series of oximes indicate that a combination of features is critical for triacsin C's inhibitory effects on Faa2p: the azenol moiety (R-CH&cjs0808;N-OH) plus the adjacent unsaturation. Studies with n-tetradecanol revealed that the inhibition is not due exclusively to the hydroxyl group. The azenol appears to be necessary but not sufficient since the tridecanal oxime, which possesses this functional group but no additional unsaturation, is not an active inhibitor. Addition of two conjugated trans-double bonds in a 9- or 10-carbon fragment ``allows'' expression of inhibitory activity. This latter finding is interesting because these compounds contain a trienol (-CH&cjs0808;CH-CH&cjs0808;CH-CH&cjs0808;N-OH) analogous to the double bond arrangement in triacsin C but lacking the triacsin's two internal nitrogens (-CH&cjs0808;CH-CH&cjs0808;N-N&cjs0808;N-OH). Thus, we can conclude that the internal nitrogens of the triazenol group in triacsin C may enhance but are not critical for inhibition of Faa2p (i.e. it is not the triazenol but the presence of a conjugated oxime (-CH&cjs0808;CH-CH&cjs0808;Y-Y&cjs0808;N-OH, where Y represents N or CH) that is required). It may be that the presence of a double bond adjacent to the hydroxyl (i.e. an enol) is critical for inhibition. If so, a carbon-based enol (C&cjs0808;C-OH) would be ineffective because it is unstable relative to the aldehyde tautomer. A nitrogen-based enol represents a simple and practical solution to this problem of stability because it does not tautomerize.

The azatrienols lack two of the five double bonds present in triacsin C. Thus, the fourth conjugated double bond found in triacsins A-D (Fig. 5) is not absolutely required for its inhibitory effect on Faa2p.

It is remarkable that the azatrienols ``preserve'' the Faap selectivity found with triacsin C. It is difficult to imagine that the chemical mechanism of catalysis is different between the Faaps. Our data with the unsaturated tetradecenoic acid analogs reveal distinct differences in acyl chain recognition (e.g. Faa2p is the most sensitive to the presence of a trans-double bond at C2-C3 while being least selective for double bonds at the other positions tested (C4-C12)). Differences in the positioning of the conjugated oxime within Faa2p (and Faa4p) compared with the other Faaps may determine its ability to prevent proper positioning of a fatty acid substrate for reaction with ATP and thus prevent formation the mixed anhydride (R-CO-O-AMP). Alternatively, the conjugated oxime may itself react with ATP. In this sense, triacsin and oxime derivatives may help define differences in molecular recognition among members of this family of four yeast acyl-CoA synthetases. The >25,000-fold range in the inhibitory effects of triacsin C on the four yeast Faaps suggests that it may be possible to develop other selective inhibitors of eukaryotic acyl-CoA synthetases. These may be useful therapeutically (38) .


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AI27179 and AI38200 and a grant from Monsanto. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Biology and Pharmacology, Box 8103, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-7243; Fax: 314-362-7058; jgordon{at}pharmdec.wustl.edu.

(^1)
The abbreviations used are: FAA, S. cerevisiae fatty acid activation gene; Faap, S. cerevisiae fatty acid activation protein (acyl-CoA synthetase); Z, cis-double bond; E, trans-double bond; Mops, 3-(N-morpholino)propanesulfonic acid; NTA, nitrilotriacetic acid; HPLC, high pressure liquid chromatography; dd, doublet of doublets; dt, doublet of triplets.

(^2)
A number of metal complexes derived from R-NH-N&cjs0808;N(O)R` or R-N(O)&cjs0808;N-NR`(2) are known (37), but these are distinct from the triazenols described here.


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