Medium-chain acyl-CoA Synthetase Research Articles

ACSM1 and ACSM3 regulate fatty acid metabolism to support prostate cancer growth and constrain ferroptosis.

Solid tumors are highly reliant on lipids for energy, growth, and survival. In prostate cancer, the activity of the androgen receptor (AR) is associated with reprogramming of lipid metabolic processes. Here, we identified acyl-CoA synthetase medium chain family members 1 and 3 (ACSM1 and ACSM3) as AR-regulated mediators of prostate cancer metabolism and growth. ACSM1 and ACSM3 were upregulated in prostate tumors compared to non-malignant tissues and other cancer types. Both enzymes enhanced proliferation and protected prostate cancer cells from death in vitro, while silencing ACSM3 led to reduced tumor growth in an orthotopic xenograft model. ACSM1 and ACSM3 were major regulators of the prostate cancer lipidome and enhanced energy production via fatty acid oxidation. Metabolic dysregulation caused by loss of ACSM1/3 led to mitochondrial oxidative stress, lipid peroxidation and cell death by ferroptosis. Conversely, elevated ACSM1/3 activity enabled prostate cancer cells to survive toxic levels of medium chain fatty acids and promoted resistance to ferroptosis-inducing drugs and AR antagonists. Collectively, this study reveals a tumor-promoting function for medium chain acyl-CoA synthetases and positions ACSM1 and ACSM3 as key players in prostate cancer progression and therapy resistance.

The Roles of Coenzyme A Binding Pocket Residues in Short and Medium Chain Acyl-CoA Synthetases.

Short- and medium-chain acyl-CoA synthetases catalyze similar two-step reactions in which acyl substrate and ATP bind to form an enzyme-bound acyl-adenylate, then CoA binds for formation of the acyl-CoA product. We investigated the roles of active site residues in CoA binding in acetyl-CoA synthetase (Acs) and a medium-chain acyl-CoA synthetase (Macs) that uses 2-methylbutyryl-CoA. Three highly conserved residues, Arg193, Arg528, and Arg586 of Methanothermobacter thermautotrophicus Acs (AcsMt), are predicted to form important interactions with the 5'- and 3'-phosphate groups of CoA. Kinetic characterization of AcsMt variants altered at each of these positions indicates these Arg residues play a critical role in CoA binding and catalysis. The predicted CoA binding site of Methanosarcina acetivorans Macs (MacsMa) is structurally more closely related to that of 4-chlorobenzoate:coenzyme A ligase (CBAL) than Acs. Alteration of MacsMa residues Tyr460, Arg490, Tyr525, and Tyr527, which correspond to CoA binding pocket residues in CBAL, strongly affected CoA binding and catalysis without substantially affecting acyl-adenylate formation. Both enzymes discriminate between 3'-dephospho-CoA and CoA, indicating interaction between the enzyme and the 3'-phosphate group is important. Alteration of MacsMa residues Lys461 and Lys519, located at positions equivalent to AcsMt Arg528 and Arg586, respectively, had only a moderate effect on CoA binding and catalysis. Overall, our results indicate the active site architecture in AcsMt and MacsMa differs even though these enzymes catalyze mechanistically similar reactions. The significance of this study is that we have delineated the active site architecture with respect to CoA binding and catalysis in this important enzyme superfamily.

Proteomics analyses of acute kidney injury biomarkers in a rat exertional heat stroke model.

The frequency of exertional heat stroke (EHS) increases with the gradual elevation of global temperatures during summer. Acute kidney injury (AKI) is a common complication of EHS, and its occurrence often indicates the worsening of a patient's condition or a poor prognosis. In this study, a rat model of AKI caused by EHS was established, and the reliability of the model was evaluated by HE staining and biochemical assays. The expression of kidney tissue proteins in the EHS rats was analyzed using label-free liquid chromatography-tandem mass spectrometry. A total of 3,129 differentially expressed proteins (DEPs) were obtained, and 10 key proteins were finally identified, which included three upregulated proteins (Ahsg, Bpgm, and Litaf) and seven downregulated proteins (medium-chain acyl-CoA synthetase 2 (Acsm2), Hadha, Keg1, Sh3glb1, Eif3d, Ambp, and Ddah2). The qPCR technique was used to validate these 10 potential biomarkers in rat kidney and urine. In addition, Acsm2 and Ahsg were double-validated by Western blotting. Overall, this study identified 10 reliable biomarkers that may provide potential targets for the treatment of AKI caused by EHS.

Differential Metabolism of Octanoate by Liver, Heart and Muscle and the Failure of Dietary Octanoate to Rescue Mitochondrial Long‐chain Fatty Acid Oxidation‐Deficient Cardiac Hypertrophy

Long-chain fatty acids are critical energetic substrates for cardiac health. During metabolic and heart disease when cardiac mitochondrial oxidative metabolism is impaired, the use of regularly minor energetic substrates, such as ketones and medium-chain fatty acids, could be beneficial for rescuing cardiac function. Here we tested the ability of dietary octanoate, an eight-carbon medium-chain fatty acid, to rescue cardiac hypertrophy in a mouse model of impaired mitochondrial long-chain fatty acid oxidation, the cardiac Carnitine Palmitoyltransferase 2 deficient mice (Cpt2M-/-). CPT2 is a critical enzyme of the acylcarnitine shuttle that converts acylcarnitine moieties to acyl-CoAs in the mitochondrial matrix for subsequent oxidation. In Cpt2M-/- mice, high octanoate-ketogenic diet failed to alleviate hypertrophy, improve cardiac function, or rescue cardiac accumulation of long-chain acylcarnitines. To understand this lack of phenotypic rescue, we found that heart and skeletal muscle mitochondria were unable to oxidize free octanoate, whereas liver mitochondria readily oxidized free octanoate. Liver mitochondria highly expressed medium-chain acyl-CoA synthetases, enabling octanoate activation for oxidation independent of the acylcarnitine-shuttle, whereas no expression was detected in heart or skeletal muscle. Conversely, the carnitine ester of octanoate, octanoyl-carnitine, was readily oxidized by liver, skeletal muscle, and heart, with heart oxidation rates being 4-fold greater than liver. Furthermore, we found that octanoyl-carnitine oxidation in muscle occurred independent of CPT2 enzyme. These data demonstrate that dietary intervention with the medium chain fatty acid octanoate cannot rescue the impairments caused by CPT2-deficiency in the heart questioning the bioavailability of dietary octanoate for the heart. These data also demonstrate that liver oxidizes free octanoate independent of carnitine, and that cardiac and skeletal muscle depend on carnitine, but not CPT2, for octanoate oxidation.

Octanoate is differentially metabolized in liver and muscle and fails to rescue cardiomyopathy in CPT2 deficiency

Long-chain fatty acid oxidation is frequently impaired in primary and systemic metabolic diseases affecting the heart; thus, therapeutically increasing reliance on normally minor energetic substrates, such as ketones and medium-chain fatty acids, could benefit cardiac health. However, the molecular fundamentals of this therapy are not fully known. Here, we explored the ability of octanoate, an eight-carbon medium-chain fatty acid known as an unregulated mitochondrial energetic substrate, to ameliorate cardiac hypertrophy in long-chain fatty acid oxidation-deficient hearts because of carnitine palmitoyltransferase 2 deletion (Cpt2M−/−). CPT2 converts acylcarnitines to acyl-CoAs in the mitochondrial matrix for oxidative bioenergetic metabolism. In Cpt2M−/− mice, high octanoate-ketogenic diet failed to alleviate myocardial hypertrophy, dysfunction, and acylcarnitine accumulation suggesting that this alternative substrate is not sufficiently compensatory for energy provision. Aligning this outcome, we identified a major metabolic distinction between muscles and liver, wherein heart and skeletal muscle mitochondria were unable to oxidize free octanoate, but liver was able to oxidize free octanoate. Liver mitochondria, but not heart or muscle, highly expressed medium-chain acyl-CoA synthetases, potentially enabling octanoate activation for oxidation and circumventing acylcarnitine shuttling. Conversely, octanoylcarnitine was oxidized by liver, skeletal muscle, and heart, with rates in heart 4-fold greater than liver and, in muscles, was not dependent upon CPT2. Together, these data suggest that dietary octanoate cannot rescue CPT2-deficient cardiac disease. These data also suggest the existence of tissue-specific mechanisms for octanoate oxidative metabolism, with liver being independent of free carnitine availability, whereas cardiac and skeletal muscles depend on carnitine but not on CPT2.

Computational study of the binding mechanism of medium chain acyl-CoA synthetase with substrate in Methanosarcina acetivorans

The acyl-AMP forming family of adenylating enzymes catalyzes the formation of acyl-CoA from an acyl substrate, ATP, and CoA, which is a metabolite of many catabolic and anabolic processes. The medium-chain acyl-CoA synthetase from Methanosarcina acetivorans, designated MacsMa, uses 2-methylbutyrate as its preferred substrate. It is reported that the interaction between the sidechain of Cys298 and Lys256 of this enzyme is important for the catalytic activity. The mutation of these residues resulted in the changes of the structure stability and the reduced or absence catalytic activity. In the present study, the binding mechanism between the substrate 2-methylbutyrate- AMP (2MeBA) and MacsMa were explored by integrating multiple computational methods including molecular docking, molecular dynamics simulations, binding free energy calculation, active site access channel analysis and principal component analysis. The binding free energy between WT, mutated Macs and substrate was calculated by MM-GBSA method, which indicated that the binding affinity between this enzyme and substrate was stronger in the WT than that in the mutated forms (K256L, K256T and C298Y). Per-residue binding free energy decomposition identified some residues, such as Gly327, Phe350, Gly351, Gln352 and Lys461, which are important for the enzyme and substrate binding affinity. The access channels of the mutant system (MacsK256L, MacsK256T and MacsC298Y) were found to be different from those in the wild-type systems. It suggested that K256L and C298Y induced larger flexibility to the overall protein compared with the WT, whereas K256T induced larger flexibility to the partial protein compared with the WT by PCA vector porcupines. This study provides novel insight to understand the substrate binding mechanism of Macs and useful information for the rational enzyme design.

Purification and Gene Cloning of an Enantioselective Thioesterification Enzyme fromBrevibacterium ketoglutamicumKU1073, a Deracemization Bacterium of 2-(4-Chlorophenoxy)propanoic Acid

We succeeded in the purification and gene cloning of a new enzyme, α-methyl carboxylic acid deracemizing enzyme 1 (MCAD1) from Brevibacterium ketoglutamicum KU1073, which catalyzes the (S)-enantioselective thioesterification reaction of 2-(4-chlorophenoxy)propanoic acid (CPPA). The cloned gene of MCAD1 contained an ORF of 1,623 bp, encoding a polypeptide of 540 amino acids. In combination with cofactors ATP, coenzyme A (CoASH), and Mg(2+), MCAD1 demonstrated perfect enantioselectivity toward CPPA. The optimal pH and temperature for reaction were found to be 7.25 and 30 °C. Under these conditions, the K(m) and k(cat) values for (S)-CPPA were 0.92 ± 0.17 mM and 0.28 ± 0.026 s(-1) respectively. The results for substrate specificity revealed that MCAD1 had highest activity toward fatty acid tails with a medium chain-length (C(8)). This result indicates that MCAD1 should be classified into a family of medium-chain acyl-CoA synthetase. This novel activity has never been reported for this family.

Evaluation of a screening method by liquid chromatography-tandem mass spectrometry for estimating effect of drugs on the activation and β-oxidation of fatty acids in mitochondria

Fatty acid metabolism is controlled not only by the acyl-coenzyme A (CoA) synthetases but by some enzymes in the β-oxidation cycle. Medium-chain and long-chain acyl-CoA esters are key metabolites in fatty acid metabolism. We have developed an enzymatic assay method for determining chain shortening of the acyl-CoAs via β-oxidation from palmitic and octanoic acids in liver mitochondria. We have evaluated the assay method for detecting whether drugs influence the activation or the β-oxidation of fatty acids. Liver mitochondria were used for investigating the effect of drugs on fatty acid metabolism. The drugs selected were salicylic acid, diclofenac, valproic acid and paracetamol. Each acyl-CoA formed was analysed by liquid chromatography-tandem mass spectrometry. After less than 5 min of incubation, the levels of acyl-CoAs reflected the acyl-CoA synthetase activity, whereas after 60-min incubation they reflected the activity of some enzymes in the β-oxidation cycle. Salicylic acid, diclofenac and valproic acid inhibited the medium-chain acyl-CoA synthetases, whereas valproic acid only exhibited a weak inhibitory activity toward the β-oxidation of the medium-chain fatty acids. In the case of long-chain fatty acid metabolism, salicylic acid and diclofenac inhibited both the activation and β-oxidation, whereas valproic acid was a weak inhibitor for only the β-oxidation activity. Paracetamol showed hardly any influence on the metabolism of medium-chain and long-chain fatty acids. These findings suggest that salicylic acid, diclofenac, valproic acid and paracetamol exert a different influence on fatty acid metabolism depending on the length of the acyl chain. This assay allows sensitive and selective analysis for predicting the pathways by which drugs exert a greater influence over fatty acid metabolism.

Characterization of an Archaeal Medium-Chain Acyl Coenzyme A Synthetase from Methanosarcina acetivorans

Short- and medium-chain acyl coenzyme A (acyl-CoA) synthetases catalyze the formation of acyl-CoA from an acyl substrate, ATP, and CoA. These enzymes catalyze mechanistically similar two-step reactions that proceed through an enzyme-bound acyl-AMP intermediate. Here we describe the characterization of a member of this enzyme family from the methane-producing archaeon Methanosarcina acetivorans. This enzyme, a medium-chain acyl-CoA synthetase designated Macs(Ma), utilizes 2-methylbutyrate as its preferred substrate for acyl-CoA synthesis but cannot utilize acetate and thus cannot catalyze the first step of acetoclastic methanogenesis in M. acetivorans. When propionate or other less favorable acyl substrates, such as butyrate, 2-methylpropionate, or 2-methylvalerate, were utilized, the acyl-CoA was not produced or was produced at reduced levels. Instead, acyl-AMP and PP(i) were released in the absence of CoA, whereas in the presence of CoA, the intermediate was broken down into AMP and the acyl substrate, which were released along with PP(i). These results suggest that although acyl-CoA synthetases may have the ability to utilize a broad range of substrates for the acyl-adenylate-forming first step of the reaction, the intermediate may not be suitable for the thioester-forming second step. The Macs(Ma) structure has revealed the putative acyl substrate- and CoA-binding pockets. Six residues proposed to form the acyl substrate-binding pocket, Lys(256), Cys(298), Gly(351), Trp(259), Trp(237), and Trp(254), were targeted for alteration. Characterization of the enzyme variants indicates that these six residues are critical in acyl substrate binding and catalysis, and even conservative alterations significantly reduced the catalytic ability of the enzyme.

Correction to The Soluble Acyl−Acyl Carrier Protein Synthetase of Vibrio harveyi B392 Is a Member of the Medium Chain Acyl-CoA Synthetase Family

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionNEXTORIGINAL ARTICLEThis notice is a correctionCorrection to The Soluble Acyl−Acyl Carrier Protein Synthetase of Vibrio harveyi B392 Is a Member of the Medium Chain Acyl-CoA Synthetase FamilyYanfang Jiang, Chi Ho Chan, and John E. Cronan*Cite this: Biochemistry 2010, 49, 1, 247Publication Date (Web):December 16, 2009Publication History Received4 December 2009Published online16 December 2009Published inissue 12 January 2010https://doi.org/10.1021/bi902086mCopyright © 2009 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views361Altmetric-Citations1LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (576 KB) Get e-Alerts Get e-Alerts

The 2.1 Å crystal structure of an acyl‐CoA synthetase from Methanosarcina acetivorans reveals an alternate acyl‐binding pocket for small branched acyl substrates

The acyl-AMP forming family of adenylating enzymes catalyze two-step reactions to activate a carboxylate with the chemical energy derived from ATP hydrolysis. X-ray crystal structures have been determined for multiple members of this family and, together with biochemical studies, provide insights into the active site and catalytic mechanisms used by these enzymes. These studies have shown that the enzymes use a domain rotation of 140 degrees to reconfigure a single active site to catalyze the two partial reactions. We present here the crystal structure of a new medium chain acyl-CoA synthetase from Methanosarcina acetivorans. The binding pocket for the three substrates is analyzed, with many conserved residues present in the AMP binding pocket. The CoA binding pocket is compared to the pockets of both acetyl-CoA synthetase and 4-chlorobenzoate:CoA ligase. Most interestingly, the acyl-binding pocket of the new structure is compared with other acyl- and aryl-CoA synthetases. A comparison of the acyl-binding pocket of the acyl-CoA synthetase from M. acetivorans with other structures identifies a shallow pocket that is used to bind the medium chain carboxylates. These insights emphasize the high sequence and structural diversity among this family in the area of the acyl-binding pocket.

Effect of salicylic acid and diclofenac on the medium‐chain and long‐chain acyl‐CoA formation in the liver and brain of mouse

Medium-chain and long-chain acyl-CoA esters are key metabolites in fatty acid metabolism. Effects of salicylic acid on the in vivo formation of acyl-CoAs in mouse liver and brain were investigated. Further, inhibition of the medium-chain and long-chain acyl-CoA synthetases by salicylic acid and diclofenac was determined in mouse liver and brain mitochondria. Acyl-CoA esters were analyzed by liquid chromatography-tandem mass spectrometry. The amounts of medium-chain acyl-CoAs (C(6), C(8) and C(10)) were less than long-chain acyl-CoAs (C(16:0), C(18:0), C(18:1) and C(20:4)) in both liver and brain. The administration of salicylic acid decreased the levels of both the medium-chain (C(6), C(8) and C(10)) and long-chain acyl-CoAs (C(16:0), C(18:0), C(18:1) and C(20:4)) in liver. In brain, however, only long-chain acyl-CoAs were decreased. The level of salicylyl-CoA detected in brain was about 12% of that in liver. Salicylic acid had a strong inhibitory activity (IC(50) = 0.1 mm) for the liver mitochondrial formation of hexanoyl-CoA from hexanoic acid, whereas diclofenac was weak (IC(50) = 4.4 mm). In contrast, diclofenac (IC(50) = 1.4 mm) inhibited the liver mitochondrial long-chain acyl-CoA synthetases more potently than salicylic acid (IC(50) = 25.5 mm). Similar inhibitory activities for the acyl-CoA synthetases were obtained in the case of the brain and liver mitochondria, except for the weak inhibition of brain medium-chain acyl-CoA synthetases by salicylic acid (IC(50) = 1.8 mm). These findings suggest that salicylic acid and diclofenac exhibit different mechanisms of inhibition of fatty acid metabolism depending on the length of the acyl chain and tissues, and they may contribute to the further understanding of the toxic effects associated with these drugs.

Structural Snapshots for the Conformation-dependent Catalysis by Human Medium-chain Acyl-coenzyme A Synthetase ACSM2A

Acyl-CoA synthetases belong to the superfamily of adenylate-forming enzymes, and catalyze the two-step activation of fatty acids or carboxylate-containing xenobiotics. The carboxylate substrate first reacts with ATP to form an acyl-adenylate intermediate, which then reacts with CoA to produce an acyl-CoA ester. Here, we report the first crystal structure of a medium-chain acyl-CoA synthetase ACSM2A, in a series of substrate/product/cofactor complexes central to the catalytic mechanism. We observed a substantial rearrangement between the N- and C-terminal domains, driven purely by the identity of the bound ligand in the active site. Our structures allowed us to identify the presence or absence of the ATP pyrophosphates as the conformational switch, and elucidated new mechanistic details, including the role of invariant Lys557 and a divalent magnesium ion in coordinating the ATP pyrophosphates, as well as the involvement of a Gly-rich P-loop and the conserved Arg472-Glu365 salt bridge in the domain rearrangement.

Evidence for 26 distinct acyl-coenzyme A synthetase genes in the human genome

Acyl-coenzyme A synthetases (ACSs) catalyze the fundamental, initial reaction in fatty acid metabolism. "Activation" of fatty acids by thioesterification to CoA allows their participation in both anabolic and catabolic pathways. The availability of the sequenced human genome has facilitated the investigation of the number of ACS genes present. Using two conserved amino acid sequence motifs to probe human DNA databases, 26 ACS family genes/proteins were identified. ACS activity in either humans or rodents was demonstrated previously for 20 proteins, but 6 remain candidate ACSs. For two candidates, cDNA was cloned, protein was expressed in COS-1 cells, and ACS activity was detected. Amino acid sequence similarities were used to assign enzymes into subfamilies, and subfamily assignments were consistent with acyl chain length preference. Four of the 26 proteins did not fit into a subfamily, and bootstrap analysis of phylograms was consistent with evolutionary divergence. Three additional conserved amino acid sequence motifs were identified that likely have functional or structural roles. The existence of many ACSs suggests that each plays a unique role, directing the acyl-CoA product to a specific metabolic fate. Knowing the full complement of ACS genes in the human genome will facilitate future studies to characterize their specific biological functions.

Sex-differential expression of ornithine aminotransferase in the mouse kidney

The mouse kidney expresses the gene of ornithine aminotransferase (Oat). Previous works suggest that Oat is differentially expressed in female and male mouse kidney (Alonso E, Rubio V. Biochem J 259: 131-138, 1989; Levillain O, Diaz JJ, Blanchard O, Dechaud H. Endocrinology 146: 950-959, 2005; Manteuffel-Cymborowska M, Chmurzynska W, Peska M, Grzelakowska-Sztabert B. Int J Biochem Cell Biol 27: 287-295, 1995; Natesan S, Reddy SR. Comp Biochem Physiol B Biochem Mol Biol 130: 585-595, 2001; Yu H, Yoo PK, Aguirre CC, Tsoa RW, Kern RM, Grody WW, Cederbaum SD, Iyer RK. J Histochem Cytochem 51: 1151-1160, 2003). This study was designed to provide a detailed description of the sexual dimorphism of Oat expression in the mouse kidney and to test the influence of sex hormones on its regulation. Experiments were performed on male and female Swiss OF1 mice during their postnatal development, at adulthood, and in orchidectomized and ovariectomized mice. Kidneys, dissected renal zones, and mitochondria were used to analyze OAT mRNA and protein levels and measure OAT activity. The results revealed that before puberty, Oat expression was similar between female and male kidneys whereas from puberty until adulthood Oat expression increased in the female kidney, becoming approximately 2.5-fold higher than in the male kidney. This sex-differential expression of Oat was associated with a sex-specific distribution of Oat along the corticopapillary axis and within the nephron. OAT was three- to fourfold more expressed in the female than the male cortex. In males, Oat was highly expressed in the medulla, mainly in the thick ascending limbs. Renal Oat distribution in orchidectomized mice resembled that in the females. Ovariectomy did not influence Oat expression. Sex differences are explained by the physiological increase in plasma testosterone in males. Expression of medium-chain acyl-CoA synthetase protein confirmed this finding. We report sexual dimorphism of Oat expression in the mouse kidney and show that Oat is naturally downregulated in the presence of testosterone.

AMP‐forming acetyl‐CoA synthetases in Archaea show unexpected diversity in substrate utilization

Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1) is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS from Methanothermobacter thermautotrophicus (designated as MT-ACS1) and an ACS from Archaeoglobus fulgidus (designated as AF-ACS2), revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1.

The Soluble Acyl-Acyl Carrier Protein Synthetase of Vibrio harveyi B392 Is a Member of the Medium Chain Acyl-CoA Synthetase Family

The gene encoding the unique soluble acyl-acyl carrier protein synthetase (AasS) of the bioluminescent Vibrio harveyi strain B392 has been isolated by expression cloning in Escherichia coli. This enzyme catalyzes the ATP-dependent acylation of the thiol of acyl carrier protein (ACP) with fatty acids with chain lengths from C4 to C18. The gene (called aasS) encodes a protein of 60 kDa, a hexahistidine-tagged version of which was readily expressed in E. coli and purified in large quantities. Surprisingly, the sequence of the encoded protein was significantly more similar to that of an acyl-CoA synthetase of the distantly related bacterium, Thermus thermophilus, than to that of the membrane-bound acyl-acyl carrier protein synthetase of E. coli, an enzyme that catalyzes the same reaction from a more closely related organism. Indeed, the AasS sequence can readily be modeled on the known crystal structures of the T. thermophilus acyl-CoA synthetase with remarkably high levels of conservation of the catalytic site residues. To test the possible role of AasS in the fatty aldehyde-dependent bioluminescence pathway of V. harveyi, the chromosomal aasS gene of the organism was disrupted by insertion of a kanamycin cassette by homologous recombination. The resulting aasS::kan strains retained low levels of acyl-acyl carrier protein synthetase consistent with prior indications of a second such activity in this bacterium. The mutant strains grew normally and had normal levels of bioluminescence but were deficient in the incorporation of exogenous octanoic acid into the cellular phospholipids of V. harveyi, particularly at low octanoate concentrations. These data indicate that AasS is responsible for a high-affinity and high-capacity uptake system that efficiently converts exogenous fatty acids into acyl-ACP species competent to enter the fatty acid biosynthetic cycle.

Expression analysis of genes involved in fat assimilation in human monocytes

The metabolic syndrome X is characterized by a group of risk factors such as obesity, atherogenic dyslipidemia, hypertension, and insulin resistance. To study the functional alterations resulting from genetic variations, ex vivo studies are one option to be carried out. Since it is not an easy procedure to obtain cells from the related tissues ex vivo, the aim of the present study was to investigate whether monocytes can serve as model cells. The purpose was to check if monocytes are insulin target cells or not and to elucidate the expression of genes involved in fat assimilation. Human monocytes were drawn from venous blood of healthy donors, aged 25 - 30, using density gradient separation and antibody-based magnetic cell sorting of CD14-positive cells. An expression analysis of genes was performed using RT-PCR and Western Blot. Transcripts of the three splice-variants of the Acyl-CoA binding protein (ACBP), the Medium-chain Acyl-CoA Synthetase 1 (MACS1), the Insulin Receptor (INSR) and the Peroxisome Proliferator-activated Receptor gamma (PPARgamma) are consistently expressed in monocytes of all donors. Differences in gene expression between donors are found for two other members of the MACS-family, the fatty acid transport protein 3 (FATP3) and the FATP4. On protein level, we tested for ACBP expression. The ACBP protein is consistently expressed in monocytes of all donors. Human monocytes are insulin target cells and capable of fatty acid metabolism to some extent. Ex vivo-derived monocytes could be used in additional studies for analyzing differences in genotype-dependent expression levels of genes involved in fat assimilation such as ACBP, MACS1 or PPARgamma.

The L513S polymorphism in medium‐chain acyl‐CoA synthetase 2 (MACS2) is associated with risk factors of the metabolic syndrome in a Caucasian study population

Enzymes of the medium-chain acyl-CoA synthetase (MACS) family catalyze the ligation of medium chain fatty acids with CoA to produce medium-chain-acyl-CoA. At least four members of the MACS gene family are clustered on human chromosome 16p12. Association studies in the Japanese Suita cohort of MACS polymorphisms and various phenotypes revealed the contribution of the Leu513Ser polymorphism in MACS2 to multiple risk factors of the metabolic syndrome. Here, we investigated the association between this polymorphism and different risk factors in the Caucasian Metabolic Intervention Cohort Kiel. Seven hundred and sixteen male subjects aged 45-65 years were recruited for a standard oral glucose tolerance test and the postprandial assessment of metabolic parameters after an oral metabolic tolerance test (oMTT; 1017 kcal, 51.6% fat, 29.6% carbohydrates, 11.9% protein). The MACS2 Leu513Ser polymorphism was determined by TaqMan-Assay in 705 subjects. Postprandial triglyceride levels following oMTT [area under the curve (AUC)] were significantly higher in subjects carrying the Ser allele compared to subjects homozygous for the Leu allele (1690 +/- 100 mg x h/dL versus 1514 +/- 39 mg x h/dL, p = 0.04). Significant differences between genotype groups were also found for fasting (108 +/- 1.9 mg/dL versus 104 +/- 0.66 mg/dL, p = 0.04) and postprandial (AUC 535 +/- 11 versus 512 +/- 4.0, p = 0.02) glucose levels as well as for high-density-lipoprotein, body mass index, waist circumference, systolic and diastolic blood pressure. Carriers of the Ser allele also show an increased risk of impaired glucose metabolism (OR: 1.48, 95% confidence interval: 0.98-2.27, p = 0.07), adiposity (1.8, 1.16-2.81, p = 0.01) and hypertension (1.5, 0.99-2.17, p = 0.06). In conclusion, our results suggest an involvement of the MACS2 Leu513Ser polymorphism in the development of the metabolic syndrome in Caucasian population. Additionally, the higher triglyceride and glucose levels after an oMTT support a possible functional impact of the polymorphism in vivo.

Purification, characterization, and mass spectrometric sequencing of a medium chain acyl-CoA synthetase from mouse liver mitochondria and comparisons with the homologues of rat and bovine

Medium chain acyl-CoA synthetases catalyze the first reaction of amino acid conjugation of many xenobiotic carboxylic acids and fatty acid metabolism. This paper reports studies on purification, characterization, and the partial amino acid sequence of mouse liver enzyme. The medium chain acyl-CoA synthetase was isolated from mouse liver mitochondria. The purified enzyme catalyzes this reaction not only for straight medium chain fatty acids but also for aromatic and arylacetic acids. Maximal activity was found with hexanoic acid. High activities were obtained with benzoic acid having methyl, pentyl, and methoxy groups in the para- or meta-positions of the benzene ring. However, the enzyme was less active with valproic acid and ketoprofen. Salicylic acid exhibited no activity. The medium chain acyl-CoA synthetases from mouse and bovine liver mitochondria were subjected to in-gel tryptic digestion, followed by LC–MS/MS sequence analysis. The amino acid sequence of each tryptic peptide of mouse liver mitochondrial medium chain acyl-CoA synthetase differed from that from bovine liver mitochondria only in one or two amino acids. LC–MS/MS analysis provided the information about these differences in amino acid sequences. In addition, we compared the properties of this protein with the homologues from rat and bovine.