acyl-CoA Thioesters Research Articles

The mechanisms of human hotdog-fold thioesterase 2 (hTHEM2) substrate recognition and catalysis illuminated by a structure and function based analysis.

The focus of this paper is the hotdog-fold thioesterase THEM2 from human (hTHEM2; Swiss-Prot entry Q9NPJ3 ). In an earlier communication (Cheng, Z., Song, F., Shan, X., Wei, Z., Wang, Y., Dunaway-Mariano, D., and Gong, W. (2006) Crystal structure of human thioesterase superfamily member 2, Biochem. Biophys. Res. Commun. 349, 172-177) we reported the apo crystal structure of hTHEM2. Herein, we report the results of an extensive hTHEM2 substrate screen, the structure determination of hTHEM2 complexed with the inert substrate analogue undecan-2-one-CoA (in which OC-CH(2)-S substitutes for OC-S) and the kinetic analysis of active site mutants. The work described in this paper represents the first reported structure-function based analysis of a human hotdog-fold thioesterase. The catalytic mechanism proposed involves the Asp65/Ser83 assisted attack of a water molecule at the Gly57/Asn50 polarized thioester CO and the Asn50 assisted departure of the thiolate leaving group. Thioesterase activity was observed with acyl-CoAs but not with the human acyl-ACP or with an acyl-Cys peptide. The medium-to-long-chain fatty acyl-CoAs displayed the smallest K(m) values. The substrate specificity profile was analyzed within the context of the liganded enzyme to define the structural determinants of substrate recognition. Based on the results of this structure-function analysis we hypothesize that the physiological role of hTHEM2 involves catalysis of the hydrolysis of cytosolic medium-to-long-chain acyl-CoA thioesters.

The selective COX-2 inhibitor celecoxib modulates sphingolipid synthesis

Sphingolipids such as ceramides (Cers) play important roles in cell proliferation, apoptosis, and cell cycle regulation. An increased Cer level is linked to the cytotoxic effects of several chemotherapeutics. Various selective cyclooxygenase-2 (COX-2) inhibitors induce anti-proliferative effects in tumor cells. We addressed the possible interaction of the selective COX-2 inhibitors, coxibs, with the sphingolipid pathway as an explanation of their anti-proliferative effects. Sphingolipids were measured using liquid chromatography tandem mass spectrometry. Treatment of various cancer cell lines with celecoxib significantly increased sphinganine, C(16:0)-, C(24:0)-, C(24:1)-dihydroceramide (dhCer) and led to a depletion of C(24:0)-, C(24:1)-Cer in a time- and concentration-dependent manner, whereas other coxibs had no effect. Using (13)C,(15)N-labeled l-serine, we demonstrated that the augmented dhCers after celecoxib treatment originate from de novo synthesis. Celecoxib inhibited the dihydroceramide desaturase (DEGS) in vivo with an IC(50) of 78.9 +/- 1.5 muM and increased total Cer level about 2-fold, indicating an activation of sphingolipid biosynthesis. Interestingly, inhibition of the sphingolipid biosynthesis by specific inhibitors of l-serine palmitoyltransferase diminished the anti-proliferative potency of celecoxib. In conclusion, induction of de novo synthesis of sphingolipids and inhibition of DEGS contribute to the anti-proliferative effects of celecoxib.

Metabolism of protocatechuic acid influences fatty acid oxidation in rat heart: New anti-angina mechanism implication

Protocatechuic acid (PA), a structurally typical phenolic acid in danshen, shows anti-angina efficacy. But until now, besides scavenging of oxygen free radicals, the understanding of its anti-angina mechanism has been limited. In our study, based on a novel metabolic route of PA identified in rat heart and its influence on fatty acid oxidation (FAO), we proposed a new mechanism for its anti-angina. In detail, three metabolites, catechol methylated metabolite, acyl-coenzyme (CoA) thioester and glycine conjugation, were identified in rat heart. A novel metabolic pathway was confirmed based on several metabolic systems incubated with heart mitochondria, cytosol, microsomes and homogenate. Results indicated that PA was firstly methylated in microsomes and cytosol, which was regarded as the prerequisite step for further metabolism and could be inhibited by tolcapone, and then the resulting methylated metabolite (vanillic acid) diffused into mitochondria where it was converted into acyl-CoA thioester, in similar with FAO. In addition, part of the acyl-CoA thioester was transformed into glycine conjugation, a step also localized within mitochondria. Furthermore, based on isolated rat heart perfusion, it was found that PA markedly decreased FAO, which was shown by higher residual fatty acid level in perfusate ( p < 0.05) and lower acy-CoA/CoA ratio in heart ( p < 0.05). The FAO inhibiting effect of PA could be largely reversed by its methylation inhibitor tolcapone, indicating the effect was closely related with the identified metabolic pathway of PA in heart. The decrease of FAO may switch heart energy substrate preference from fatty acid to glucose, which is beneficial for ischemia heart.

Formation and biliary excretion of glutathione conjugates of bile acids in the rat as shown by liquid chromatography/electrospray ionization–linear ion trap mass spectrometry

Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are reactive acyl-linked metabolites that have been shown to undergo transacylation-type reactions with the thiol group of glutathione (GSH), leading to the formation of thioester-linked GSH conjugates. In the current study, we examined the transformation of cholyl-adenylate (CA–AMP) and cholyl-coenzyme A thioester (CA–CoA) into a cholyl- S-acyl GSH (CA–GSH) conjugate by rat hepatic glutathione S-transferase (GST). The reaction product was analyzed by liquid chromatography (LC)/electrospray ionization (ESI)–linear ion trap mass spectrometry (MS). The GST-catalyzed formation of CA–GSH occurred with both CA–AMP and CA–CoA. Ursodeoxycholic acid, lithocholic acid, and 2,2,4,4- 2H 4-labeled lithocholic acid were administered orally to biliary fistula rats, and their corresponding GSH conjugates were identified in bile by LC/ESI–MS 2. These in vitro and in vivo studies confirm a new mode of BA conjugation in which BAs are transformed into their GSH conjugates via their acyl-linked intermediary metabolites by the catalytic action of GST in the liver, and the GSH conjugates are then excreted into the bile.

AMPK activation by long chain fatty acyl analogs

The antidiabetic efficacy of first-line insulin sensitizers (e.g., metformin, glitazones) is accounted for by activation of AMP-activated protein kinase (AMPK). Long chain fatty acids (LCFA) activate AMPK, but their putative antidiabetic efficacy is masked by their β-oxidized or esterified lipid products. Substituted α,ω-dicarboxylic acids of 14–18 carbon atoms in length (MEDICA analogs) are not metabolized beyond their acyl-CoA thioesters, and may therefore simulate AMPK activation by LCFA while avoiding LCFA turnover into β-oxidized or esterified lipid products. MEDICA analogs are shown here to activate AMPK and some of its downstream targets in vivo, in cultured cells and in a cell-free system consisting of the (α 1β 1γ 1)AMPK recombinant and LKB1-MO25-STRAD (AMPK-kinase) recombinant proteins. AMPK activation by MEDICA is accompanied by normalizing the hyperglycemia-hyperinsulinemia of diabetic db/db mice in vivo with suppression of hepatic glucose production in cultured liver cells. Activation of AMPK by MEDICA or LCFA is accounted for by (a) decreased intracellular ATP/AMP ratio and energy charge by the free acid, (b) activation of LKB1 phosphorylation of AMPK(Thr172) by the acyl-CoA thioester. The two activation modes are complementary since LKB1/AMPK activation by the CoA-thioester is fully evident under conditions of excess AMP. MEDICA analogs may expand the arsenal of AMPK activators used for treating diabetes type 2.

Thematic Review Series: Sphingolipids. Biodiversity of sphingoid bases (“sphingosines”) and related amino alcohols

“Sphingosin” was first described by J. L. W. Thudichum in 1884 and structurally characterized as 2S,3R,4E-2-aminooctadec-4-ene-1,3-diol in 1947 by Herb Carter, who also proposed the designation of “lipides derived from sphingosine as sphingolipides.” This category of amino alcohols is now known to encompass hundreds of compounds that are referred to as sphingoid bases and sphingoid base-like compounds, which vary in chain length, number, position, and stereochemistry of double bonds, hydroxyl groups, and other functionalities. Some have especially intriguing features, such as the tail-to-tail combination of two sphingoid bases in the α,ω-sphingoids produced by sponges. Most of these compounds participate in cell structure and regulation, and some (such as the fumonisins) disrupt normal sphingolipid metabolism and cause plant and animal disease. Many of the naturally occurring and synthetic sphingoid bases are cytotoxic for cancer cells and pathogenic microorganisms or have other potentially useful bioactivities; hence, they offer promise as pharmaceutical leads. This thematic review gives an overview of the biodiversity of the backbones of sphingolipids and the broader field of naturally occurring and synthetic sphingoid base-like compounds.

Metabolic activation of carboxylic acids

Background: Carboxylic acids constitute a large and heterogeneous class of both endogenous and xenobiotic compounds. A number of carboxylic acid drugs have been associated with adverse reactions, linked to the metabolic activation of the carboxylic acid moiety of the compounds, i.e., formation of acyl-glucuronides and acyl-CoA thioesters. Objective: The objective is to give an overview of the current knowledge on metabolic activation of carboxylic acids and how such metabolites may play a role in adverse reactions and toxicity. Methods: Literature concerning the formation and disposition of acyl glucuronides and acyl-CoA thioesters was searched. Also included were papers on the chemical reactivity of acyl glutathione-thioesters, and literature concerning possible links between metabolic activation of carboxylic acids and reported cellular and clinical effects. Results/conclusion: This review demonstrates that metabolites of carboxylic acid drugs must be considered chemically reactive, and that the current knowledge about metabolic activation of this compound class can be a good starting-point for further studies on the consequences of chemically reactive metabolites.

Discovery of Amide (Peptide) Bond Synthetic Activity in Acyl-CoA Synthetase

Acyl-CoA synthetase, which is one of the acid-thiol ligases (EC 6.2.1), plays key roles in metabolic and regulatory processes. This enzyme forms a carbon-sulfur bond in the presence of ATP and Mg(2+), yielding acyl-CoA thioesters from the corresponding free acids and CoA. This enzyme belongs to the superfamily of adenylate-forming enzymes, whose three-dimensional structures are analogous to one another. We here discovered a new reaction while studying the short-chain acyl-CoA synthetase that we recently reported (Hashimoto, Y., Hosaka, H., Oinuma, K., Goda, M., Higashibata, H., and Kobayashi, M. (2005) J. Biol. Chem. 280, 8660-8667). When l-cysteine was used as a substrate instead of CoA, N-acyl-l-cysteine was surprisingly detected as a reaction product. This finding demonstrated that the enzyme formed a carbon-nitrogen bond (EC 6.3.1 acid-ammonia (or amide) ligase (amide synthase); EC 6.3.2 acid-amino acid ligase (peptide synthase)) comprising the amino group of the cysteine and the carboxyl group of the acid. N-Acyl-d-cysteine, N-acyl-dl-homocysteine, and N-acyl-l-cysteine methyl ester were also synthesized from the corresponding cysteine analog substrates by the enzyme. Furthermore, this unexpected enzyme activity was also observed for acetyl-CoA synthetase and firefly luciferase, indicating the generality of the new reaction in the superfamily of adenylate-forming enzymes.

Divergence of Function in the Hot Dog Fold Enzyme Superfamily: The Bacterial Thioesterase YciA

Thioesters play a central role in the cells where they participate in metabolism, membrane synthesis, signal transduction, and gene regulation. Thioesters are converted to the thiol and carboxylic acid components by thioesterase-catalyzed hydrolysis. Here we examine the biochemical and biological function of the hot dog fold thioesterase YciA (EcYciA) from Escherichia coli and its close sequence homologue HI0827 from Haemophilus influenzae (HiYciA). The quaternary structure of HiYciA was determined, using equilibrium sedimentation techniques, to be a homohexamer. Mass spectral and (31)P NMR analysis of purified HiYciA revealed a bound CoA ligand. Kinetic analyses showed that CoA is a strong feedback inhibitor. YciA thioesterase activity toward acyl-CoA substrates was determined using steady-state kinetic methods. The k cat and k cat/ K m values obtained reveal a striking combination of high catalytic efficiency and low substrate specificity. The substrate activity of propionyl-s- N-acetylcysteine was found to be negligible and that of n-butyryl-pantetheinephosphate low, and therefore, it is evident YciA does not target acylated ACPs or other acylated proteins as substrates. The results from bioinformatic analysis of the biological distribution and genome contexts of yciAs are reported. We conclude that YciA is responsible for the efficient, "seemingly" indiscriminant, CoA-regulated hydrolysis of cellular acyl-CoA thioesters in a wide range of bacteria and hypothesize that this activity may support membrane biogenesis.

The acyl-CoA thioesterase I is regulated by PPARα and HNF4α via a distal response element in the promoter

The cytosolic acyl-coenzyme A thioesterase I (Acot1) is an enzyme that hydrolyzes long-chain acyl-CoAs of C(12)-C(20)-CoA in chain length to the free fatty acid and CoA. Acot1 was shown previously to be strongly upregulated at the mRNA and protein level in rodents by fibrates. In this study, we show that Acot1 mRNA levels were increased by 90-fold in liver by treatment with Wy-14,643 and that Acot1 mRNA was also increased by 15-fold in the liver of hepatocyte nuclear factor 4alpha (HNF4alpha) knockout animals. Our study identified a direct repeat 1 (DR1) located in the Acot1 gene promoter in mouse, which binds the peroxisome proliferator-activated receptor alpha (PPARalpha) and HNF4alpha. Chromatin immunoprecipitation (ChIP) assay showed that the identified DR1 bound PPARalpha/retinoid X receptor alpha (RXRalpha) and HNF4alpha, whereas the binding in ChIP was abrogated in the PPARalpha and HNF4alpha knockout mouse models. Reporter gene assays showed activation of the Acot1 promoter in cells by the PPARalpha agonist Wy-14,643 after cotransfection with PPARalpha/RXRalpha. However, transfection with a plasmid containing HNF4alpha also resulted in an increase in promoter activity. Together, these data show that Acot1 is under regulation by an interplay between HNF4alpha and PPARalpha.

Investigating the in vitro metabolism of fipexide: characterization of reactive metabolites using liquid chromatography/mass spectrometry

The in vitro metabolism of the nootropic drug fipexide was studied using different liquid chromatography/mass spectrometry (LC/MS) techniques. This drug has been withdrawn from the market due to toxic effects. No previous reports have investigated the possible involvement of reactive metabolites in the toxicity of fipexide. The hydrolysis of this drug leads to the formation of two potentially toxic species, 3,4-methylenedioxybenzylpiperazine (MDBP) and 4-chlorophenoxyacetic acid (4-CPA). Here, we investigate the in vitro metabolism of fipexide in human, rat, mouse and dog, as well as of MDBP and 4-CPA in human and rat, while focusing on the formation of reactive metabolites. A combination of LC/MS analyses on a hybrid quadrupole-linear ion trap instrument and accurate mass data from QqTOF measurements was employed for the characterization of these metabolites. Microsomal metabolites of fipexide were MDBP, 4-CPA, fipexide N-oxide or hydroxyl, demethylenated fipexide and other minor ones, all of which were investigated by tandem mass spectrometry. Reactive metabolites were detected using several trapping procedures with small molecules such as glutathione, its ethyl ester derivative and N-acetylcysteine. The demethylenated metabolite, a catechol, formed its corresponding ortho-quinone, which readily reacts with these nucleophiles. MDBP was studied in a similar manner, due to its ability to form an analogous catechol. Because of its acidic nature, 4-CPA was assessed for possible acylglucuronide and acyl-CoA thioester metabolites, which could also be involved in bioactivation pathways. Several important metabolites were identified as potential mediators of toxicity via protein binding.

Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters

A new approach to determine in vivo pools of coenzyme A (CoA) and short chain acyl-CoA thioesters is reported. The metabolites released by extraction with trichloroacetic acid are recovered and quantitatively dephosphorylated by treatment with shrimp alkaline phosphatase. Following phosphatase removal, the dephosphorylated CoA metabolites are quantitatively rephosphorylated by treatment with [γ- 33P]ATP plus a dephospho-CoA kinase. The resulting radioactive CoA metabolites are then separated by reverse-phase high-performance liquid chromatography and quantitated by scintillation counting. Due to the enzymatic radiophosphorylation, the assay is specific for CoA and its short chain thioesters and is sensitive to sub-picomole levels of these compounds.

Amino acid conjugation: contribution to the metabolism and toxicity of xenobiotic carboxylic acids

Despite being the first conjugation reaction demonstrated in humans, amino acid conjugation as a route of metabolism of xenobiotic carboxylic acids is not well characterised. This is principally due to the small number and limited structural diversity of xenobiotic substrates for amino acid conjugation. Unlike CYP and uridine 5′-diphosphate glucuronosyltransferase, which are localised in the endoplasmic reticulum, the enzymes of amino acid conjugation reside in mitochondria. Unique among drug metabolism pathways, amino acid conjugation involves initial formation of a xenobiotic acyl-CoA thioester that is then conjugated principally with glycine in humans. However, formation of the xenobiotic acyl-CoA thioester does not always infer subsequent amino acid conjugation. Evidence is presented that in the absence of glycine conjugation substrates that form acyl-CoA thioesters perturb mitochondrial function. This review discusses literature on the enzymes involved and the concept that xenobiotic substrate selectivity provides a barrier to protect the metabolic integrity of the mitochondria.

Evidence for two‐step regulation of pheromone biosynthesis by the pheromone biosynthesis‐activating neuropeptide in the moth Heliothis virescens

The control of pheromone biosynthesis by the neuropeptide PBAN was investigated in the moth Heliothis virescens. When decapitated females were injected with [2-(14)C] acetate, females co-injected with PBAN produced significantly greater quantities of radiolabeled fatty acids in their pheromone gland than females co-injected with saline. This indicates that PBAN controls an enzyme involved in the synthesis of fatty acids, probably acetyl CoA carboxylase. Decapitated females injected with PBAN showed a rapid increase in native pheromone, and a slower increase in the pheromone precursor, (Z)-11-hexadecenoate. Total native palmitate and stearate (both pheromone intermediates) showed a significant decrease after PBAN injection, before their titers were later restored to initial levels. In contrast, the acyl-CoA thioesters of these two saturated fatty acids increased during the period when their total titers decreased. When a mixture of labeled palmitic and heptadecanoic (an acid that cannot be converted to pheromone) acids was applied to the gland, PBAN-injected females produced greater quantities of labeled pheromone and precursor than did saline-injected ones. The two acids showed similar time-course patterns, with no difference in total titers of each of the respective acids between saline- and PBAN-injected females. When labeled heptadecanoic acid was applied to the gland alone, there was no difference in titers of either total heptadecanoate or of heptadecanoyl-CoA between PBAN- and saline-injected females, suggesting that PBAN does not directly control the storage or liberation of fatty acids in the gland, at least for this fatty acid. Overall, these data indicate that PBAN also controls a later step involved in pheromone biosynthesis, perhaps the reduction of acyl-CoA moieties. The control by PBAN of two enzymes, near the beginning and end of the pheromone biosynthetic process, would seem to allow for more efficient utilization of fatty acids and pheromone than control of only one enzyme.

Structural Characterization of a β-Diketone Hydrolase from the Cyanobacterium Anabaena sp. PCC 7120 in Native and Product-Bound Forms, a Coenzyme A-Independent Member of the Crotonase Suprafamily,

The gene alr4455 from the well-studied cyanobacterium Anabaena sp. PCC 7120 encodes a crotonase orthologue that displays beta-diketone hydrolase activity. Anabaena beta-diketone hydrolase (ABDH), in common with 6-oxocamphor hydrolase (OCH) from Rhodococcus sp. NCIMB 9784, catalyzes the desymmetrization of bicyclo[2.2.2]octane-2,6-dione to yield [(S)-3-oxocyclohexyl]acetic acid, a reaction unusual among the crotonase superfamily as the substrate is not an acyl-CoA thioester. The structure of ABDH has been determined to a resolution of 1.5 A in both native and ligand-bound forms. ABDH forms a hexamer similar to OCH and features one active site per enzyme monomer. The arrangement of side chains in the active site indicates that while the catalytic chemistry may be conserved in OCH orthologues, the structural determinants of substrate specificity are different. In the active site of ligand-bound forms that had been cocrystallized with the bicyclic diketone substrate bicyclo[2.2.2]octane-2,6-dione was found the product of the asymmetric enzymatic retro-Claisen reaction [(S)-3-oxocyclohexyl]acetic acid. The structures of ABDH in both native and ligand-bound forms reveal further details about structural variation and modes of coenzyme A-independent activity within the crotonases and provide further evidence of a wider suprafamily of enzymes that have recruited the crotonase fold for the catalysis of reactions other than those regularly attributed to canonical superfamily members.

Overexpression of Rat Long Chain Acyl-CoA Synthetase 1 Alters Fatty Acid Metabolism in Rat Primary Hepatocytes

Long chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for both anabolic and catabolic pathways. We have hypothesized that each of the five ACSL isoforms partitions FA toward specific downstream pathways. Acsl1 mRNA is increased in cells under both lipogenic and oxidative conditions. To elucidate the role of ACSL1 in hepatic lipid metabolism, we overexpressed an Acsl1 adenovirus construct (Ad-Acsl1) in rat primary hepatocytes. Ad-ACSL1, located on the endoplasmic reticulum but not on mitochondria or plasma membrane, increased ACS specific activity 3.7-fold. With 100 or 750 mum [1-(14)C]oleate, Ad-Acsl1 increased oleate incorporation into diacylglycerol and phospholipids, particularly phosphatidylethanolamine and phosphatidylinositol, and decreased incorporation into cholesterol esters and secreted triacylglycerol. Ad-Acsl1 did not alter oleate incorporation into triacylglycerol, beta-oxidation products, or total amount of FA metabolized. In pulse-chase experiments to examine the effects of Ad-Acsl1 on lipid turnover, more labeled triacylglycerol and phospholipid, but less labeled diacylglycerol, remained in Ad-Acsl1 cells, suggesting that ACSL1 increased reacylation of hydrolyzed oleate derived from triacylglycerol and diacylglycerol. In addition, less hydrolyzed oleate was used for cholesterol ester synthesis and beta-oxidation. The increase in [1,2,3-(3)H]glycerol incorporation into diacylglycerol and phospholipid was similar to the increase with [(14)C]oleate labeling suggesting that ACSL1 increased de novo synthesis. Labeling Ad-Acsl1 cells with [(14)C]acetate increased triacylglycerol synthesis but did not channel endogenous FA away from cholesterol ester synthesis. Thus, consistent with the hypothesis that individual ACSLs partition FA, Ad-Acsl1 increased FA reacylation and channeled FA toward diacylglycerol and phospholipid synthesis and away from cholesterol ester synthesis.

The 1.8 Å resolution structure of hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL) fromPseudomonas fluorescens, an enzyme that catalyses the transformation of feruloyl-coenzyme A to vanillin

The crystal structure of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) from Pseudomonas fluorescens AN103 has been solved to 1.8 A resolution. HCHL is a member of the crotonase superfamily and catalyses the hydration of the acyl-CoA thioester of ferulic acid [3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid] and the subsequent retro-aldol cleavage of the hydrated intermediate to yield vanillin (4-hydroxy-3-methoxy-benzaldehyde). The structure contains 12 molecules in the asymmetric unit, in which HCHL assumes a hexameric structure of two stacked trimers. The substrate, feruloyl-CoA, was modelled into the active site based on the structure of enoyl-CoA hydratase bound to the feruloyl-CoA-like substrate 4-(N,N-dimethylamino)-cinnamoyl-CoA (PDB code 1ey3). Feruloyl-CoA was bound in this model between helix 3 of the A subunit and helix 9 of the B subunit. A highly ordered structural water in the HCHL structure coincided with the thioester carbonyl of feruloyl-CoA in the model, suggesting that the oxyanion hole for stabilization of a thioester-derived enolate, characteristic of coenzyme-A dependent members of the crotonase superfamily, is conserved. The model also suggested that a strong hydrogen bond between the phenolic hydroxyl groups of feruloyl-CoA and BTyr239 may be an important determinant of the enzyme's ability to discriminate between the natural substrate and cinnamoyl-CoA, which is not a substrate.

FUNCTIONAL IMPLICATIONS OF MULTIPLE FadD PROTEINS IN THE BIOLOGY OF MYCOBACTERIUM TUBERCULOSIS

Mycobacterium tuberculosis H37Rv genome sequencing revealed an unanticipated variety of genes homologous to proteins involved in β-oxidation machinery. Identification of 34 fadD homologous was quite remarkable and it was tempting to speculate that many of these might not even be expressed in mycobacterial lifecycle. Based on the biochemical function of mycobacterial fadD proteins, we have revealed that there are atleast two distinct families of fadD enzymes. Fatty acyl-AMP ligase (FAAL) activates fatty acids as fatty acyl-adenylates while Fatty acyl-CoA ligase (FACL) activates fatty acids to fatty acyl-CoA thioester analogues. Furthermore, we have demonstrated that FAAL directly transfer fatty acids to cognate polyketide synthase modules to produce complex lipids which are hallmarks of mycobacteria. Recent genome-wide expression studies have emphasized the functional significance of FadD proteins under different conditions. Since these proteins play an important role in mycobacterial biology, we have designed specific inhibitors that may be further developed as antimycobacterial agents. This work was supported by grants from DBT, India to National Institute of Immunology, CSIR to PA and Wellcome Trust International Senior Research Fellowship to RSG.

Crystallographic Trapping of the Glutamyl-CoA Thioester Intermediate of Family I CoA Transferases

Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 A resolution, respectively. YdiF is organized into tetramers, with each monomer having an open alpha/beta structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent gamma-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.

ACYL-COENZYME A FORMATION OF SIMVASTATIN IN MOUSE LIVER PREPARATIONS

Formation of an acyl-CoA thioester has been proposed, but not directly demonstrated, to be a key step in mediating both lactonization and atypical beta-oxidation of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors. Here, we describe studies to characterize formation of acyl-CoA thioesters in vitro in mouse liver preparations using the hydroxy acid form of simvastatin (SVA) as a model substrate. With an optimized chromatography method, three new products were detected in addition to the dehydration product (P1) and the lactone form of simvastatin, which have been characterized previously (Prueksaritanont et al., 2001). Based on high-pressure liquid chromatography analysis, UV spectroscopy, mass spectrometry, and NMR spectral characterization, two metabolites were identified as acyl-CoA thioester conjugates of SVA and P1, respectively, whereas the third metabolite (M1) was confirmed to be the L-beta-hydroxy isomer of simvastatin. M1 was probably formed by stereospecific hydration, a previously reported reaction, and subsequent lactonization of P1-S-acyl CoA. Among all the mouse liver subcellular fractions, microsomes exhibited the highest capacity to catalyze the CoASH-dependent metabolism of SVA, whereas such activity was totally absent in cytosol. Together, these results provide direct experimental evidence that SVA (and conceivably other statins as well) is able to form an acyl-CoA thioester, possibly by microsomal long-chain acyl-CoA synthetase(s), leading to formation of two parallel metabolic pathways, one resulting in the two diastereomers of statin lactones (simvastatin and M1) and the other to the beta-oxidation pathway of statin hydroxy acids.