Identification of lysine isobutyrylation as a new histone modification mark.

Short-chain acylations of lysine residues in eukaryotic proteins are recognized as essential posttranslational chemical modifications (PTMs) that regulate cellular processes from transcription, cell cycle, metabolism, to signal transduction. Lysine butyrylation was initially discovered as a normal straight chain butyrylation (Knbu). Here we report its structural isomer, branched chain butyrylation, i.e. lysine isobutyrylation (Kibu), existing as a new PTM on nuclear histones. Uniquely, isobutyryl-CoA is derived from valine catabolism and branched chain fatty acid oxidation which is distinct from the metabolism of n-butyryl-CoA. Several histone acetyltransferases were found to possess lysine isobutyryltransferase activity in vitro, especially p300 and HAT1. Transfection and western blot experiments showed that p300 regulated histone isobutyrylation levels in the cell. We resolved the X-ray crystal structures of HAT1 in complex with isobutyryl-CoA that gleaned an atomic level insight into HAT-catalyzed isobutyrylation. RNA-Seq profiling revealed that isobutyrate greatly affected the expression of genes associated with many pivotal biological pathways. Together, our findings identify Kibu as a novel chemical modification mark in histones and suggest its extensive role in regulating epigenetics and cellular physiology.

Biosynthesis of angelyl-CoA in Saccharomyces cerevisiae.

BACKGROUND: The angelic acid moiety represents an essential modification in many biologically active products. These products are commonly known as angelates and several studies have demonstrated their therapeutic benefits, including anti-inflammatory and anti-cancer effects. However, their availability for use in the development of therapeutics is limited due to poor extraction yields. Chemical synthesis has been achieved but its complexity prevents application, therefore microbial production may offer a promising alternative. Here, we engineered the budding yeast Saccharomyces cerevisiae to produce angelyl-CoA, the CoA-activated form of angelic acid. RESULTS: For yeast-based production of angelyl-CoA we first expressed genes recently identified in the biosynthetic cluster ssf of Streptomyces sp. SF2575 in S. cerevisiae. Exogenous feeding of propionate and heterologous expression of a propionyl-CoA synthase from Streptomyces sp. were initially employed to increase the intracellular propionyl-CoA level, resulting in production of angelyl-CoA in the order of 5 mg/L. Substituting the Streptomyces sp. propionyl-CoA carboxylase with a carboxylase derived from Streptomyces coelicolor resulted in angelyl-CoA levels up to 6.4 mg/L. In vivo analysis allowed identification of important intermediates in the pathway, including methyl-malonyl-CoA and 3-hydroxyl-2-methyl-butyryl-CoA. Furthermore, methyl-malonate supplementation and expression of matB CoA ligase from S. coelicolor allowed for methyl-malonyl-CoA synthesis and supported, together with parts of the ssf pathway, angelyl-CoA titres of approximately 1.5 mg/L. Finally, feeding of angelic acid to yeasts expressing acyl-CoA ligases from plant species led to angelyl-CoA production rates of approximately 40 mg/L. CONCLUSIONS: Our results demonstrate the biosynthesis of angelyl-CoA in yeast from exogenously supplied carboxylic acid precursors. This is the first report on the activity of the ssf genes. We envision that our approach will provide a platform for a more sustainable production of the pharmaceutically important compound class of angelates.

Structure-activity relationships of rationally designed AMACR 1A inhibitors.

α-Methylacyl-CoA racemase (AMACR; P504S) is a promising novel drug target for prostate and other cancers. Assaying enzyme activity is difficult due to the reversibility of the 'racemisation' reaction and the difficulties in the separation of epimeric products; consequently few inhibitors have been described and no structure-activity relationship study has been performed. This paper describes the first structure-activity relationship study, in which a series of 23 known and potential rational AMACR inhibitors were evaluated. AMACR was potently inhibited (IC50 = 400-750 nM) by ibuprofenoyl-CoA and derivatives. Potency was positively correlated with inhibitor lipophilicity. AMACR was also inhibited by straight-chain and branched-chain acyl-CoA esters, with potency positively correlating with inhibitor lipophilicity. 2-Methyldecanoyl-CoAs were ca. 3-fold more potent inhibitors than decanoyl-CoA, demonstrating the importance of the 2-methyl group for effective inhibition. Elimination substrates and compounds with modified acyl-CoA cores were also investigated, and shown to be potent inhibitors. These results are the first to demonstrate structure-activity relationships of rational AMACR inhibitors and that potency can be predicted by acyl-CoA lipophilicity. The study also demonstrates the utility of the colorimetric assay for thorough inhibitor characterisation.

Synthesis and Detection by HPLC of 3-Oxohexadecanoyl-CoA for the Study of Peroxisomal Bifunctional Proteins.

3-oxohexadecanoyl-CoA was synthesized for the study of D-bifunctional protein (EC 4. 2. 1. 107, EC 4. 2. 1. 119, EC 1. 1. 1. n12) and L-bifunctional protein (EC 4. 2. 1. 17, EC 5. 3. 3. 8, EC 1. 1. 1. 35). First, tetradecanal was subjected to the Reformatsky reaction with ethyl bromoacetate, and the product was then converted into ethyl 3-oxohexadecanoate. After acetalization of the 3-oxo ester with ethylene glycol, 3,3-ethlenedioxyhexadecanoic acid was obtained by alkaline hydrolysis. The acid was condensed with coenzyme A (CoA) by the mixed anhydride method, and the resulting CoA ester was deprotected with 4 M HCl to obtain 3-oxohexadecanoyl-CoA. In addition, the behavior of the CoA ester under several conditions of high-performance liquid chromatography (HPLC) was also investigated. We established separation detection of (R)-3-hydroxyhexadecanoyl-CoA, (S)-3-hydroxyhexadecaboyl-CoA, 3-oxohexadecanoyl-CoA, and trans-2-hexadecenoyl-CoA.

Effect of carbon chain length in acyl coenzyme A on the efficiency of enzymatic transformation of okadaic acid to 7-O-acyl okadaic acid.

Okadaic acid (OA), a product of dinoflagellate Prorocentrum spp., is transformed into 7-O-acyl OA in various bivalve species. The structural transformation proceeds enzymatically in vitro in the presence of the microsomal fraction from the digestive gland of bivalves. We have been using LC-MS/MS to identify OA-transforming enzymes by detecting 7-O-acyl OA, also known as dinophysistoxin 3 (DTX3). However, an alternative assay for DTX3 is required because the OA-transforming enzyme is a membrane protein, and surfactants for solubilizing membrane proteins decrease the sensitivity of LC-MS/MS. The present study examined saturated fatty acyl CoAs with a carbon chain length of 10 (decanoyl), 12 (dodecanoyl), 14 (tetradecanoyl), 16 (hexadecanoyl) and 18 (octadecanoyl) as the substrate for the in vitro acylation reaction. Saturated fatty acyl CoAs with a carbon chain length of 14, 16 and 18 exhibited higher yields than those with a carbon chain length of 10 or 12. Acyl CoAs with carbon chain lengths from 14 to 18 and containing either a diene unit, an alkyne unit, or an azide unit in the carbon chain were synthesized and shown to provide the corresponding DTX3 with a yield comparable to that of hexadecanoyl CoA. The three functional units can be conjugated with fluorescent reagents and are applicable to the development of a novel assay for DTX3.

A Chemo-Enzymatic Road Map to the Synthesis of CoA Esters.

Coenzyme A (CoA) is a ubiquitous cofactor present in every known organism. The thioesters of CoA are core intermediates in many metabolic processes, such as the citric acid cycle, fatty acid biosynthesis and secondary metabolism, including polyketide biosynthesis. Synthesis of CoA-thioesters is vital for the study of CoA-dependent enzymes and pathways, but also as standards for metabolomics studies. In this work we systematically tested five chemo-enzymatic methods for the synthesis of the three most abundant acyl-CoA thioester classes in biology; saturated acyl-CoAs, α,ß-unsaturated acyl-CoAs (i.e., enoyl-CoA derivatives), and α-carboxylated acyl-CoAs (i.e., malonyl-CoA derivatives). Additionally we report on the substrate promiscuity of three newly described acyl-CoA dehydrogenases that allow the simple conversion of acyl-CoAs into enoyl-CoAs. With these five methods, we synthesized 26 different CoA-thioesters with a yield of 40% or higher. The CoA esters produced range from short- to long-chain, include branched and α,ß-unsaturated representatives as well as other functional groups. Based on our results we provide a general guideline to the optimal synthesis method of a given CoA-thioester in respect to its functional group(s) and the commercial availability of the precursor molecule. The proposed synthetic routes can be performed in small scale and do not require special chemical equipment, making them convenient also for biological laboratories.

Study of Class I and Class III Polyhydroxyalkanoate (PHA) Synthases with Substrates Containing a Modified Side Chain.

Polyhydroxyalkanoates (PHAs) are carbon and energy storage polymers produced by a variety of microbial organisms under nutrient-limited conditions. They have been considered as an environmentally friendly alternative to oil-based plastics due to their renewability, versatility, and biodegradability. PHA synthase (PhaC) plays a central role in PHA biosynthesis, in which its activity and substrate specificity are major factors in determining the productivity and properties of the produced polymers. However, the effects of modifying the substrate side chain are not well understood because of the difficulty to accessing the desired analogues. In this report, a series of 3-(R)-hydroxyacyl coenzyme A (HACoA) analogues were synthesized and tested with class I synthases from Chromobacterium sp. USM2 (PhaCCs and A479S-PhaCCs) and Caulobacter crescentus (PhaCCc) as well as class III synthase from Allochromatium vinosum (PhaECAv). It was found that, while different PHA synthases displayed distinct preference with regard to the length of the alkyl side chains, they could withstand moderate side chain modifications such as terminal unsaturated bonds and the azide group. Specifically, the specific activity of PhaCCs toward propynyl analogue (HHxyCoA) was only 5-fold less than that toward the classical substrate HBCoA. The catalytic efficiency (kcat/Km) of PhaECAv toward azide analogue (HABCoA) was determined to be 2.86 × 10(5) M(-1) s(-1), which was 6.2% of the value of HBCoA (4.62 × 10(6) M(-1) s(-1)) measured in the presence of bovine serum albumin (BSA). These side chain modifications may be employed to introduce new material functions to PHAs as well as to study PHA biogenesis via click-chemistry, in which the latter remains unknown and is important for metabolic engineering to produce PHAs economically.

A study on the AMACR catalysed elimination reaction and its application to inhibitor testing.

α-Methylacyl-CoA racemase (AMACR; P504S) catalyses a key step in the degradation of branched-chain fatty acids and is important for the pharmacological activation of Ibuprofen and related drugs. Levels of AMACR are increased in prostate and other cancers, and it is a drug target. Development of AMACR as a drug target is hampered by lack of a convenient assay. AMACR irreversibly catalyses the elimination of HF from 3-fluoro-2-methylacyl-CoA substrates, and this reaction was investigated for use as an assay. Several known inhibitors and alternative substrates reduced conversion of 3-fluoro-2-methyldecanoyl-CoA by AMACR, as determined by (1)H NMR. The greatest reduction of activity was observed with known potent inhibitors. A series of novel acyl-CoA esters with aromatic side chains were synthesised for testing as chromophoric substrates. These acyl-CoA esters were converted to unsaturated products by AMACR, but their use was limited by non-enzymatic elimination. Fluoride sensors were also investigated as a method of quantifying released fluoride and thus AMACR activity. These sensors generally suffered from high background signal and lacked reproducibility under the assay conditions. In summary, the elimination reaction can be used to characterise inhibitors, but it was not possible to develop a convenient colorimetric or fluorescent assay using 3-fluoro-2-methylacyl-CoA substrates.

Prediction of novel synthetic pathways for the production of desired chemicals.

BACKGROUND: There have been several methods developed for the prediction of synthetic metabolic pathways leading to the production of desired chemicals. In these approaches, novel pathways were predicted based on chemical structure changes, enzymatic information, and/or reaction mechanisms, but the approaches generating a huge number of predicted results are difficult to be applied to real experiments. Also, some of these methods focus on specific pathways, and thus are limited to expansion to the whole metabolism. RESULTS: In the present study, we propose a system framework employing a retrosynthesis model with a prioritization scoring algorithm. This new strategy allows deducing the novel promising pathways for the synthesis of a desired chemical together with information on enzymes involved based on structural changes and reaction mechanisms present in the system database. The prioritization scoring algorithm employing Tanimoto coefficient and group contribution method allows examination of structurally qualified pathways to recognize which pathway is more appropriate. In addition, new concepts of binding site covalence, estimation of pathway distance and organism specificity were taken into account to identify the best synthetic pathway. Parameters of these factors can be evolutionarily optimized when a newly proven synthetic pathway is registered. As the proofs of concept, the novel synthetic pathways for the production of isobutanol, 3-hydroxypropionate, and butyryl-CoA were predicted. The prediction shows a high reliability, in which experimentally verified synthetic pathways were listed within the top 0.089% of the identified pathway candidates. CONCLUSIONS: It is expected that the system framework developed in this study would be useful for the in silico design of novel metabolic pathways to be employed for the efficient production of chemicals, fuels and materials.

Synthesis and characterization of cis-4-decenoyl-CoA, 3-phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA.

The measurement of acyl-CoA dehydrogenase activities is an essential part of the investigation of patients with suspected defects in fatty acid oxidation. Multiple methods are available for the synthesis of the substrates used for measuring acyl-CoA dehydrogenase activities; however, the yields are low and the products are used without purification. In addition, the reported characterization of acyl-CoAs focuses on the CoA moiety, not on the acyl group. Here we describe the synthesis of three medium-chain acyl-CoAs from mixed anhydrides of the fatty acids using an aqueous-organic solvent mixture optimized to obtain the highest yield. First, cis-4-decenoic acid and 2,6-dimethylheptanoic acid were prepared (3-phenylpropionic acid is commercially available). These were characterized by gas chromatography/mass spectrometry (GC/MS), (1)H nuclear magnetic resonance (NMR), and (13)C NMR. Then cis-4-decenoyl-CoA, 3-phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA were synthesized. These were then purified by ion exchange solid-phase extraction using 2-(2-pyridyl)ethyl-functionalized silica gel, followed by reversed-phase semipreparative high-performance liquid chromatography with ultraviolet detection (HPLC-UV). The purified acyl-CoAs were characterized by analytical HPLC-UV followed by data-dependent tandem mass spectrometry (MS/MS) analysis on the largest responding MS mass (HPLC-UV-MS-MS/MS) and (13)C NMR. The yields of the purified acyl-CoAs were between 75% and 78% based on coenzyme A trilithium salt (CoASH). Acyl-CoA dehydrogenase activities were measured in rat skeletal muscle mitochondria using, as substrates, the synthesized cis-4-decenoyl-CoA, 3-phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA. These results were compared with the results using our standard substrates butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA.

Non-radioactive detection of palmitoylated mitochondrial proteins using an azido-palmitate analogue.

While palmitoylation is typically thought of as a cytosolic process resulting in membrane attachment of the palmitoylated proteins, numerous mitochondrial proteins have been shown to be palmitoylated following in vitro labeling of mitochondria with radioactive or bioorthogonal analogues of fatty acids. The fatty acylation of two liver mitochondrial enzymes, methylmalonyl semialdehyde dehydrogenase and carbamoyl phosphate synthetase 1, has been studied in great detail. In both cases palmitoylation of an active site cysteine residue occurred spontaneously and resulted in inhibition of enzymatic activity, thus, suggesting that palmitoylation may be a direct means to regulate the activity of metabolic enzymes within the mitochondria. The progress of investigators working on protein fatty acylation has long been impeded by the long exposure time required to detect the incorporation of [(3)H]-fatty acids into protein by fluorography (often 1-3 months or more). Significant reduction in exposure times has been achieved by the use of [(125)I]-iodofatty acids but these analogues are also hazardous and not commercially available. Herein, we describe a sensitive chemical labeling method for the detection of palmitoylated mitochondrial proteins. The method uses azido-fatty acid analogues that can be attached to proteins and reacted with tagged phosphines via a modified Staudinger ligation. Recently, we used this labeling method, combined with mass spectrometry analysis of the labeled proteins, to identify 21 palmitoylated proteins from rat liver mitochondria.

An N-aroyltransferase of the BAHD superfamily has broad aroyl CoA specificity in vitro with analogues of N-dearoylpaclitaxel.

The native N-debenzoyl-2'-deoxypaclitaxel:N-benzoyltransferase (NDTBT), from Taxus plants, transfers a benzoyl group from the corresponding CoA thioester to the amino group of the beta-phenylalanine side chain of N-debenzoyl-2'-deoxypaclitaxel, which is purportedly on the paclitaxel (Taxol) biosynthetic pathway. To elucidate the substrate specificity of NDTBT overexpressed in Escherichia coli, the purified enzyme was incubated with semisynthetically derived N-debenzoyltaxoid substrates and aroyl CoA donors (benzoyl; ortho-, meta-, and para-substituted benzoyls; various heterole carbonyls; alkanoyls; and butenoyl), which were obtained from commercial sources or synthesized via a mixed anhydride method. Several unnatural N-aroyl-N-debenzoyl-2'-deoxypaclitaxel analogues were biocatalytically assembled with catalytic efficiencies (V(max)/K(M)) ranging between 0.15 and 1.74 nmol.min(-1).mM(-1). In addition, several N-acyl-N-debenzoylpaclitaxel variants were biosynthesized when N-debenzoylpaclitaxel and N-de(tert-butoxycarbonyl)docetaxel (i.e., 10-deacetyl-N-debenzoylpaclitaxel) were used as substrates. The relative velocity (v(rel)) for NDTBT with the latter two N-debenzoyl taxane substrates ranged between approximately 1% and 200% for the array of aroyl CoAs compared to benzoyl CoA. Interestingly, NDTBT transferred hexanoyl, acetyl, and butyryl more rapidly than butenoyl or benzoyl from the CoA donor to taxanes with isoserinoyl side chains, whereas N-debenzoyl-2'-deoxypaclitaxel was more rapidly converted to its N-benzoyl derivative than to its N-alkanoyl or N-butenoyl congeners. Biocatalytic N-acyl transfer of novel acyl groups to the amino functional group of N-debenzoylpaclitaxel and its 2'-deoxy precursor reveal the surprisingly indiscriminate specificity of this transferase. This feature of NDTBT potentially provides a tool for alternative biocatalytic N-aroylation/alkanoylation to construct next generation taxanes or other novel bioactive diterpene compounds.

Synthesis and use of isotope-labelled substrates for a mechanistic study on human alpha-methylacyl-CoA racemase 1A (AMACR; P504S).

Alpha-Methylacyl-CoA racemase (AMACR) is an important enzyme for the metabolism of branched-chain lipids and drugs. The enzyme is over-expressed in prostate and other cancers. AMACR 1A, the major splice variant, was purified from recombinant E. coli cells as a His-tag protein. Purified enzyme catalysed chiral inversion of both S- and R-2-methyldecanoyl-CoA, with an equilibrium constant of 1.09 +/- 0.14 (2S/2R). Reactions with (2)H-labelled substrate showed that loss of the alpha-proton was a prerequisite for chiral inversion. Reactions conducted in (2)H(2)O indicated that reprotonation was not stereospecific. These results are the first mechanistic study on any recombinant mammalian alpha-methylacyl-CoA racemase.

Oct-2-en-4-ynoyl-CoA as a specific inhibitor of acyl-CoA oxidase.

Oct-2-en-4-ynoyl-CoA was found to be a specific inhibitor of acyl-CoA oxidase in fatty acid oxidation in peroxisomes that has no inhibitory effect on acyl-CoA dehydrogenase in mitochondria. The inhibition reaction involves a nucleophilic attack of Glu421 to the delta carbon of the inhibitor. The result indicates that acyl-CoA oxidase and acyl-CoA dehydrogenase have certain differences in active-site structure, which makes it possible to control fatty acid oxidation selectively in either mitochondria or peroxisomes with different enzyme inhibitors.

Chemoenzymatic formation of novel aminocoumarin antibiotics by the enzymes CouN1 and CouN7.

The aminocoumarin antibiotics novobiocin, clorobiocin, and coumermycin A1 are highly potent inhibitors of the bacterial type II topoisomerase DNA gyrase. The key pharmacophore of both clorobiocin and coumermycin A1, the 5-methyl-2-pyrrolylcarbonyl moiety, targets the ATP-binding site of GyrB. The 5-methyl-2-pyrrolylcarbonyl group is transferred by the acyltransferases Clo/CouN7 from the carrier proteins Clo/CouN1 to the 3'-hydroxyl of the l-noviosyl scaffold during the late steps of clorobiocin and coumermycin A1 biosynthesis. We first examined the substrate specificity of the purified thiolation domain protein CouN1 in becoming primed by the phosphopantetheinyltransferase Sfp using a variety of synthetic CoA analogues of the 5-methyl-2-pyrrolylcarbonyl moiety. The acyl-S-CouN1 thioesters were then assayed as donors to the 3'-OH group of descarbamoylnovobiocin by the acyltransferase CouN7, resulting in 21 novel variants with heterocyclic acyl groups installed on the noviosyl moiety of the aminocoumarin scaffold. Scaleup of a 5-methylthiophene derivative yielded a compound with activity against both Gram-negative and Gram-positive bacteria. The minimal inhibitory concentration found for the Gram-positive bacteria was comparable to that of novobiocin.

Design, synthesis, and in vitro testing of alpha-methylacyl-CoA racemase inhibitors.

The enzyme alpha-methylacyl-CoA racemase (AMACR) is overexpressed in prostate, colon, and other cancers and has been partially validated as a potential therapeutic target by siRNA knockdown of the AMACR gene. Analogs of the natural substrate branched chain alpha-methylacyl coenzyme A esters, possessing one or more beta-fluorine atoms, have been synthesized using Wittig, conjugate addition, and asymmetric aldol reactions and found to be reversible competitive inhibitors. Each diastereomer of the previously reported inhibitor ibuprofenoyl-CoA was also tested. The compounds had Ki values of 0.9-20 microM and are the most potent inhibitors yet known. The presence of beta-fluorine on the alpha-methyl group or the acyl chain results in a significant lowering of the Ki value compared with nonfluorinated analogs, and this is attributed to a lowering of the pKa of the alpha-proton, facilitating enolization and binding. Several of the CoA ester inhibitors were formed by incubating the free carboxylic acid precursors with cell free extracts and CoA. alpha-Trifluoromethyltetradecanoic acid, the precursor to the most potent inhibitor, was shown to inhibit growth of cancer cell lines PC3, CWR22 Rv1, and Du145 in a dose-dependent manner and could be related to the expression level of AMACR.

Effects of ethanol on the remodeling of neutral lipids and phospholipids in brain mitochondria and microsomes.

We have analyzed the effects of ethanol in vitro on the remodeling of neutral lipids and phospholipids in mitochondria and microsomes isolated from chick brain. We used three different fatty acyl-CoAs of similar chain lengths but different degrees of unsaturation. Our results demonstrate the existence of active mechanisms for acyl-CoA transfer into neutral lipids and phospholipids in both mitochondria and microsomes. The profile of fatty acid incorporation was clearly different according to the membrane and lipid fraction in question. Thus, in mitochondrial lipids, the remodeling processes showed a clear preference for the saturated fatty acid whilst the polyunsaturated one was the preferred substrate for microsomal lipid acylation. With regard to the effects of ethanol in vitro, we were able to demonstrate that exposure of the membrane to ethanol led to an increase in the incorporation of polyunsaturated fatty acid into triacylglycerol (TG) in both mitochondria and microsomes, indicating that it directly stimulates the acylation of diacylglycerol (DG) to give TG. This effect may then contribute to the widely reported stimulation of TG biosynthesis in cases of both acute and chronic ethanol ingestion. It is noteworthy that the exposure of microsomes to ethanol in vitro also stimulated the incorporation of oleoyl-CoA into the aminophospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS). We also demonstrate that both mitochondria and microsomes synthesize fatty acid ethyl esters (FAEEs) from fatty acyl-CoA, although there is a clear difference in preference for the fatty acid used as substrate in the esterification of the alcohol. Thus, mitochondria were capable of forming FAEEs from the polyunsaturated fatty acid whilst in microsomes the saturated fatty acid was the preferred substrate. In both types of membrane, FAEE production was lowest with the monounsaturated fatty acyl-CoA.

In vitro reactivity of carboxylic acid-CoA thioesters with glutathione.

The chemical reactivity of acyl-CoA thioesters toward nucleophiles has been demonstrated in several recent studies. Thus, intracellularly formed acyl-CoAs of xenobiotic carboxylic acids may react covalently with endogenous proteins and potentially lead to adverse effects. The purpose of this study was to investigate whether a correlation could be found between the structure of acyl-CoA thioesters and their reactivities toward the tripeptide, glutathione (gamma-Glu-Cys-Gly). The acyl-CoA thioesters of eight carboxylic acids (ibuprofen, clofibric acid, indomethacin, fenbufen, tolmetin, salicylic acid, 2-phenoxypropionic acid, and (4-chloro-2-methyl-phenoxy)acetic acid (MCPA)) were synthesized, and each acyl-CoA (0.5 mM) was incubated with glutathione (5.0 mM) in 0.1 M potassium phosphate (pH 7.4, 37 degrees C). All of the acyl-CoAs reacted with glutathione to form the respective acyl-S-glutathione products, with MCPA-CoA having the highest rate of conjugate formation (120 +/- 10 microM/min) and ibuprofen-CoA having the lowest (1.0 +/- 0.1 microM/min). The relative reactivities of the acyl-CoAs were dependent on the substitution at the carbon atom alpha to the acyl carbon and on the presence of an oxygen atom in a position beta to the acyl carbon and were as follows: phenoxyacetic acid > o-hydroxybenzoic acid--phenoxypropionic acid > arylacetic acid derivatives > 2-methyl-2-phenoxypropionic acid--2-phenylpropionic acid. For each acyl-CoA thioester, the overall hydrolysis rate was determined as the time-dependent formation of parent compound. A linear trend was observed when comparing the reactivities of the acyl-CoAs with glutathione with the corresponding overall hydrolysis rates. Thus, the most reactive compound (MCPA-CoA) was also the compound with the highest rate of hydrolysis and the least reactive compounds (ibuprofen-CoA, clofibryl-CoA) were also the compounds least susceptible to hydrolysis.

Interaction of 3,4-dienoyl-CoA thioesters with medium chain acyl-CoA dehydrogenase: stereochemistry of inactivation of a flavoenzyme.

The medium chain acyl-CoA dehydrogenase is rapidly inhibited by racemic 3,4-dienoyl-CoA derivatives with a stoichiometry of two molecules of racemate per enzyme flavin. Synthesis of R- and S-3,4-decadienoyl-CoA shows that the R-enantiomer is a potent, stoichiometric, inhibitor of the enzyme. alpha-Proton abstraction yields an enolate to oxidized flavin charge-transfer intermediate prior to adduct formation. The crystal structure of the reduced, inactive enzyme shows a single covalent bond linking the C-4 carbon of the 2,4-dienoyl-CoA moiety and the N5 locus of reduced flavin. The kinetics of reversal of adduct formation by release of the conjugated 2,4-diene were evaluated as a function of both acyl chain length and truncation of the CoA moiety. The adduct is most stable with medium chain length allenic inhibitors. However, the adducts with R-3,4-decadienoyl-pantetheine and -N-acetylcysteamine are some 9- and >100-fold more kinetically stable than the full-length CoA thioester. Crystal structures of these reduced enzyme species, determined to 2.4 A, suggest that the placement of H-bonds to the inhibitor carbonyl oxygen and the positioning of the catalytic base are important determinants of adduct stability. The S-3,4-decadienoyl-CoA is not a significant inhibitor of the medium chain dehydrogenase and does not form a detectable flavin adduct. However, the S-isomer is rapidly isomerized to the trans-trans-2,4-conjugated diene. Protein modeling studies suggest that the S-enantiomer cannot approach close enough to the isoalloxazine ring to form a flavin adduct, but can be facilely reprotonated by the catalytic base. These studies show that truncation of CoA thioesters may allow the design of unexpectedly potent lipophilic inhibitors of fatty acid oxidation.