Mechanism of action of beta-oxoacyl-CoA thiolase from rat liver cytosol. Direct evidence for the order of addition of the two acetyl-CoA molecules during the formation of acetoacetyl-CoA.
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ABSTRACT:
SUBMITTER: Jordan PM
PROVIDER: S-EPMC1152102 | biostudies-other | 1983 Jul
REPOSITORIES: biostudies-other
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Project description:Thiolases are vital enzymes which participate in both degradative and biosynthetic pathways. Biosynthetic thiolases catalyze carbon-carbon bond formation by a Claisen condensation reaction. The cytoplasmic acetoacetyl-CoA thiolase from Saccharomyces cerevisiae, ERG10, catalyses carbon-carbon bond formation in the mevalonate pathway. The structure of a S. cerevisiae biosynthetic thiolase has not previously been reported. Here, crystal structures of apo ERG10 and its Cys91Ala variant were solved at resolutions of 2.2 and 1.95 Å, respectively. The structure determined shows that ERG10 shares the characteristic thiolase superfamily fold, with a similar active-site architecture to those of type II thiolases and a similar binding pocket, apart from Ala159 at the entrance to the pantetheine-binding cavity, which appears to be a determinant of the poor binding ability of the substrate. Moreover, comparative binding-pocket analysis of molecule B in the asymmetric unit of the apo structure with that of the CoA-bound complex of human mitochondrial acetoacetyl-CoA thiolase indicates the canonical binding mode of CoA. Furthermore, the steric hindrance revealed in a structural comparison of molecule A with the CoA-bound form raise the possibility of conformational changes that are associated with substrate binding.
| S-EPMC5947687 | biostudies-literature
Project description:Many reactions within a cell are thermodynamically unfavorable. To efficiently run some of those endergonic reactions, nature evolved intermediate-channeling enzyme complexes, in which the products of the first endergonic reactions are immediately consumed by the second exergonic reactions. Based on this concept, we studied how archaea overcome the unfavorable first reaction of isoprenoid biosynthesis-the condensation of two molecules of acetyl-CoA to acetoacetyl-CoA catalyzed by acetoacetyl-CoA thiolases (thiolases). We natively isolated an enzyme complex comprising the thiolase and 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGCS) from a fast-growing methanogenic archaeon, Methanothermococcus thermolithotrophicus HMGCS catalyzes the second reaction in the mevalonate pathway-the exergonic condensation of acetoacetyl-CoA and acetyl-CoA to HMG-CoA. The 380-kDa crystal structure revealed that both enzymes are held together by a third protein (DUF35) with so-far-unknown function. The active-site clefts of thiolase and HMGCS form a fused CoA-binding site, which allows for efficient coupling of the endergonic thiolase reaction with the exergonic HMGCS reaction. The tripartite complex is found in almost all archaeal genomes and in some bacterial ones. In addition, the DUF35 proteins are also important for polyhydroxyalkanoate (PHA) biosynthesis, most probably by functioning as a scaffold protein that connects thiolase with 3-ketoacyl-CoA reductase. This natural and highly conserved enzyme complex offers great potential to improve isoprenoid and PHA biosynthesis in biotechnologically relevant organisms.
| S-EPMC5879682 | biostudies-literature
Project description:Thiolase I and II coexist as part of the glyoxysomal beta-oxidation system in sunflower (Helianthus annuus L.) cotyledons, the only system shown to have both forms. The importance of thiolases can be underscored not only by their ubiquity, but also by their involvement in a wide variety of processes in plants, animals and bacteria. Here we describe the cloning, expression and purification of acetoacetyl CoA thiolase (AACT) in enzymatically active form. Use of the extensive amount of sequence information from the databases facilitated the efficient generation of the gene-specific primers used in the RACE protocols. The recombinant AACT (1233 bp) shares 75% similarity with other plant AACTs. Comparison of specific activity of this recombinant AACT to a previously reported enzyme purified from primary sunflower cotyledon tissue was very similar (263 nkat/mg protein vs 220 nkat/mg protein, respectively). Combining the most pure fractions from the affinity column, the enzyme was purified 88-fold with a 55% yield of the enzymatically active, 47 kDa AACT.
| S-EPMC2793306 | biostudies-literature
Project description:Acetoacetyl-CoA thiolase (AT) is an enzyme that catalyses the CoA-dependent thiolytic cleavage of acetoacetyl-CoA to yield 2 molecules of acetyl-CoA, or the reverse condensation reaction. A full-length cDNA clone pBSGT-3, which has homology to known thiolases, was isolated from Dictyostelium cDNA library. Expression of the protein encoded in pBSGT-3 in Escherichia coli, its thiolase enzyme activity, and the amino acid sequence homology search revealed that pBSGT-3 encodes an AT. The recombinant AT (r-thiolase) was expressed in an active form in an E. coli expression system, and purified to homogeneity by selective ammonium sulfate fractionation and two steps of column chromatography. The purified enzyme exhibited a specific activity of 4.70 mU/mg protein. Its N-terminal sequence was (NH₂)-Arg-Met-Tyr-Thr-Thr-Ala-Lys-Asn-Leu-Glu-, which corresponds to the sequence from positions 15 to 24 of the amino acid sequence deduced from pBSGT-3 clone. The r-thiolase in the inclusion body expressed highly in E. coli was the precursor form, which is slightly larger than the purified r-thiolase. When incubated with the cell-free extract of Dictyostelium cells, the precursor was converted to the same size to the purified r-thiolase, suggesting that the presequence at the N-terminus is removed by a Dictyostelium processing peptidase.
| S-EPMC3014551 | biostudies-literature
Project description:Mitochondrial acetoacetyl-CoA thiolase (T2, encoded by the ACAT1 gene) deficiency is an inherited disorder of ketone body and isoleucine metabolism. It typically manifests with episodic ketoacidosis. The presence of isoleucine-derived metabolites is the key marker for biochemical diagnosis. To date, 105 ACAT1 variants have been reported in 149 T2-deficient patients. The 56 disease-associated missense ACAT1 variants have been mapped onto the crystal structure of T2. Almost all these missense variants concern residues that are completely or partially buried in the T2 structure. Such variants are expected to cause T2 deficiency by having lower in vivo T2 activity because of lower folding efficiency and/or stability. Expression and activity data of 30 disease-associated missense ACAT1 variants have been measured by expressing them in human SV40-transformed fibroblasts. Only two variants (p.Cys126Ser and p.Tyr219His) appear to have equal stability as wild-type. For these variants, which are inactive, the side chains point into the active site. In patients with T2 deficiency, the genotype does not correlate with the clinical phenotype but exerts a considerable effect on the biochemical phenotype. This could be related to variable remaining residual T2 activity in vivo and has important clinical implications concerning disease management and newborn screening.
| S-EPMC6790690 | biostudies-literature
Project description:Fusarium pseudograminearum is a major pathogen for the destructive disease Fusarium crown rot (FCR) of wheat (Triticum aestivum). The cytosolic Acetoacetyl-CoA thiolase II (AACT) is the first catalytic enzyme in the mevalonate pathway that biosynthesizes isoprenoids in plants. However, there has been no investigation of wheat cytosolic AACT genes in defense against pathogens including Fusarium pseudograminearum. Herein, we identified a cytosolic AACT-encoding gene from wheat, named TaAACT1, and demonstrated its positively regulatory role in the wheat defense response to F. pseudograminearum. One haplotype of TaAACT1 in analyzed wheat genotypes was associated with wheat resistance to FCR. The TaAACT1 transcript level was elevated after F. pseudograminearum infection, and was higher in FCR-resistant wheat genotypes than in susceptible wheat genotypes. Functional analysis indicated that knock down of TaAACT1 impaired resistance against F. pseudograminearum and reduced the expression of downstream defense genes in wheat. TaAACT1 protein was verified to localize in the cytosol of wheat cells. TaAACT1 and its modulated defense genes were rapidly responsive to exogenous jasmonate treatment. Collectively, TaAACT1 contributes to resistance to F. pseudograminearum through upregulating the expression of defense genes in wheat. This study sheds new light on the molecular mechanisms underlying wheat defense against FCR.
| S-EPMC10094598 | biostudies-literature
Project description:BackgroundClonorchis sinensis (C. sinensis) inhabits in bile duct of the host. However, the mechanisms involved in why C. sinensis can survive in the bile environment containing lipids have not yet been explored. In this study, C. sinensis acetoacetyl-CoA thiolase (CsACAT), a member of the thiolase family which has a key role in the beta oxidation pathway of fatty acid production, was identified and characterized to understand its potential role in adapting to the bile environment.MethodsThe encoding sequence, conserved domains and spatial structure of CsACAT were identified and analyzed by bioinformatic tools. Recombinant CsACAT (rCsACAT) was obtained using a procaryotic expression system. The expression pattern of CsACAT was confirmed by quantitative real-time PCR, western blotting, and immunofluorescence. Gradients of lecithin were then set to culture C. sinensis adults in vitro and the survival rate of C. sinensis was analyzed, as well as the expression level and enzymatic activity of CsACAT in different lipid environments. Hypercholesteremia rabbit models were established by feeding with a hyperlipidemic diet and then infected intragastrically with C. sinensis. One and a half months later, the worm burdens and the expression level of CsACAT was detected.ResultsCsACAT was confirmed to be a member of the thiolase family and present in the excretory/secretory proteins of C. sinensis. CsACAT was specifically localized at the vitellarium and sub-tegumental muscle layer in adult worms. The mRNA level of CsACAT in eggs was higher than those in adult worms and metacercariae. When adult worms were cultured with higher concentration of lecithin, the expression level and enzyme activity of CsACAT were up-regulated. The survival rate of adult worms was higher than control group. More adult worms were recovered from hypercholesteremia rabbit models. The expression level of CsACAT in these worms was higher than control group.ConclusionsOur results implied that C. sinensis might sense lipid levels and survive better in the bile environment with higher lipid levels. C. sinensis might modulate the expression and enzymatic activity of CsACAT, an enzyme involved in fatty acid metabolism, for energy or physical requirements to adapt to the host.
| S-EPMC4359446 | biostudies-literature
Project description:Leishmania, a protozoan parasite, is responsible for significant morbidity and mortality worldwide, manifesting as cutaneous, mucocutaneous, and visceral leishmaniasis. These diseases pose a substantial burden, especially in impoverished regions with limited access to effective medical treatments. Current therapies are toxic, have low efficacy, and face growing resistance. Understanding the metabolic pathways of Leishmania, particularly those differing from its host, can unveil potential therapeutic targets. In this study, we investigated the acetyl-CoA synthetase (ACS) enzyme from Leishmania infantum (LiAcs1), which, unlike many organisms, also exhibits acetoacetyl-CoA synthetase (KBC) activity. This dual functionality is unique among acyl-CoA/NRPS/luciferase superfamily enzymes and crucial for the parasite's reliance on leucine catabolism, energy production, and sterol biosynthesis. Our biochemical characterization of LiAcs1 revealed its ability to utilize both acetate and acetoacetate substrates. Additionally, LiAcs1 displayed a distinct CoA substrate inhibition pattern, partially alleviated by acetoacetate. Structural analysis provided insights into the substrate binding flexibility of LiAcs1, highlighting a more promiscuous substrate pocket compared to other ACS- or KBC-specific enzymes. Substrate mimetics elucidated its ability to accommodate both small and large AMP-ester derivatives, contributing to its dual ACS/KBC functionality. These findings not only advance our understanding of Leishmania metabolism but also present LiAcs1 as a promising drug target. The dual functionality of LiAcs1 underscores the potential for developing selective inhibitors that could disrupt critical metabolic pathways across Leishmania spp. as it appears this enzyme is highly conserved across this genus. This paves the way for developing novel effective treatments against this devastating disease.
| S-EPMC11584562 | biostudies-literature
Project description:The effects of oleic acid on the activities of cytosolic HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase, AcAc-CoA (acetoacetyl-CoA) thiolase and AcAc-CoA synthetase, as well as microsomal HMG-CoA reductase, all enzymes in the pathway of cholesterol biosynthesis, were studied in the isolated perfused rat liver. Oleic acid bound to bovine serum albumin, or albumin alone, was infused for 4 h at a rate sufficient to sustain an average concentration of 0.61 +/- 0.05 mM fatty acid during the perfusion. Hepatic cytosol and microsomal fractions were isolated at the termination of the perfusion. Oleic acid simultaneously increased the activities of the cytosolic cholesterol-biosynthetic enzymes 1.4-2.7-fold in livers from normal fed rats and from animals fasted for 24 h. These effects were accompanied by increased net secretion by the liver of cholesterol and triacylglycerol in the very-low-density lipoprotein (VLDL). We confirmed the observations reported previously from this laboratory of the stimulation by oleic acid of microsomal HMG-CoA reductase. In cytosols from perfused livers, the increase in AcAc-CoA thiolase activity was characterized by an increase in Vmax. without any change in the apparent Km of the enzyme for AcAc-CoA. In contrast, oleic acid decreased the Km of HMG-CoA synthase for Ac-CoA, without alteration of the Vmax. of the enzyme. The Vmax. of AcAc-CoA synthetase was increased by oleic acid, and there was a trend towards a small increase in the Km of the enzyme for acetoacetate. These data allow us to conclude that the enzymes that supply the HMG-CoA required for hepatic cholesterogenesis are stimulated, as is HMG-CoA reductase, by a physiological substrate, fatty acid, that increases rates of hepatic cholesterol synthesis and cholesterol secretion. Furthermore, we suggest that these effects of fatty acid on hepatic cholesterol metabolism result from stimulation of secretion of triacylglycerol in the VLDL by fatty acids, and the absolute requirement of cholesterol as an important structural surface component of the VLDL necessary for transport of triacylglycerol from the liver.
| S-EPMC1138398 | biostudies-other
Project description:The beta-oxidation of valproic acid (VPA; 2-n-propylpentanoic acid) was investigated in vitro in intact rat liver mitochondria incubated with (3)H-labelled VPA. The metabolism of [4,5-(3)H(2)]VPA and [2-(3)H]VPA was studied by analysing the different acyl-CoA intermediates formed by reverse-phase HPLC with radiochemical detection. Valproyl-CoA, Delta(2(E))-valproyl-CoA,3-hydroxyvalproyl-CoA and 3-oxovalproyl-CoA (labelled and non-labelled) were determined using continuous on-line radiochemical and UV detection. The formation of these intermediates was investigated using the two tritiated precursors in respiratory states 3 and 4. Valproyl-CoA was present at highest concentrations under both conditions. Two distinct labelled peaks were found, which were identified as (3)H(2)O and [4,5-(3)H(2)]3-oxo-VPA. The formation of (3)H(2)O strongly suggested that VPA underwent complete beta-oxidation and that [4,5-(3)H(2)]3-oxo-VPA was formed by hydrolysis of the corresponding thioester. The hypothesis that 3-oxovalproyl-CoA undergoes thiolytic cleavage was investigated further. For this purpose a mito chondrial lysate was incubated with synthetic 3-oxovalproyl-CoA, carnitine and carnitine acetyltransferase for subsequent monitoring of the formation of propionylcarnitine and pentanoylcarnitine using electrospray ionization tandem MS. The detection of these compounds demonstrated unequivocally that the intermediate 3-oxovalproyl-CoA is a substrate of a mitochondrial thiolase, producing propionyl-CoA and pentanoyl-CoA, thus demonstrating the complete beta-oxidation of VPA in the mitochondrion. Our data should lead to a re-evaluation of the generally accepted concept that the biotransformation of VPA by mitochondrial beta-oxidation is incomplete.
| S-EPMC1222442 | biostudies-other