Carnitine Octanoyltransferase Research Articles

Carnitine octanoyltransferase is important for the assimilation of exogenous acetyl-L-carnitine into acetyl-CoA in mammalian cells

In eukaryotes, carnitine is best known for its ability to shuttle esterified fatty acids across mitochondrial membranes for β-oxidation. It also returns to the cytoplasm, in the form of acetyl-L-carnitine (LAC), some of the resulting acetyl groups for posttranslational protein modification and lipid biosynthesis. While dietary LAC supplementation has been clinically investigated, its effects on cellular metabolism are not well understood. To explain how exogenous LAC influences mammalian cell metabolism, we synthesized isotope-labeled forms of LAC and its analogs. In cultures of glucose-limited U87MG glioma cells, exogenous LAC contributed more robustly to intracellular acetyl-CoA pools than did β-hydroxybutyrate, the predominant circulating ketone body in mammals. The fact that most LAC-derived acetyl-CoA is cytosolic is evident from strong labeling of fatty acids in U87MGcells by exogenous 13C2-acetyl-L-carnitine. We found that the addition of d3-acetyl-L-carnitine increases the supply of acetyl-CoA for cytosolic posttranslational modifications due to its strong kinetic isotope effect on acetyl-CoA carboxylase, the first committed step in fatty acid biosynthesis. Surprisingly, whereas cytosolic carnitine acetyltransferase is believed to catalyze acetyl group transfer from LAC to coenzyme A, CRAT-/- U87MGcells were unimpaired in their ability to assimilate exogenous LAC into acetyl-CoA. We identified carnitine octanoyltransferase as the key enzyme in this process, implicating a role for peroxisomes in efficient LAC utilization. Our work has opened the door to further biochemical investigations of a new pathway for supplying acetyl-CoA to certain glucose-starved cells.

Peroxisomal gene and protein expression increase in response to a high-lipid challenge in human skeletal muscle

Peroxisomes are essential for lipid metabolism and disruption of liver peroxisomal function results in neonatal death. Little is known about how peroxisomal content and activity respond to changes in the lipid environment in human skeletal muscle (HSkM). AimsWe hypothesized and tested that increased peroxisomal gene/protein expression and functionality occur in HSkM as an adaptive response to lipid oversupply. Materials and methodsHSkM biopsies, derived from a total of sixty-two subjects, were collected for 1) examining correlations between peroxisomal proteins and intramyocellular lipid content (IMLC) as well as between peroxisomal functionality and IMLC, 2) assessing peroxisomal gene expression in response to acute- or 7-day high fat meal (HFM), and in human tissue derived primary myotubes for 3) treating with high fatty acids to induce peroxisomal adaptions. IMLC were measured by both biochemical analyses and fluorescent staining. Peroxisomal membrane protein PMP70 and biogenesis gene (PEX) expression were assessed using western blotting and realtime qRT-PCR respectively. 1-14C radiolabeled lignocerate and palmitate oxidation assays were performed for peroxisomal and mitochondrial functionality respectively. Results1) Under fasting conditions, HSkM tissue demonstrated a significant correlation (P ≪ 0.05) between IMCL and the peroxisomal biogenesis factor 19 (PEX19) protein as well as between lipid content and palmitate and lignocerate complete oxidation. 2) Similarly, post-HFM, additional PEX genes (Pex19, PEX11A, and PEX5) were significantly (P ≪ 0.05) upregulated. 3) Increments in PMP70, carnitine octanoyl transferase (CrOT), PGC-1α, and ERRα mRNA were observed post-fatty acid incubation in HSkM cells. PMP70 protein was significantly (P ≪ 0.05) elevated 48-h post lipid treatment. ConclusionsThese results are the first to associate IMLC with peroxisomal gene/protein expression and function in HSkM suggesting an adaptive role for peroxisomes in lipid metabolism in this tissue.

Significance of l-carnitine for human health.

Carnitine acyltransferases catalyze the reversible transfer of acyl groups from acyl-coenzyme A esters to l-carnitine, forming acyl-carnitine esters that may be transported across cell membranes. l-Carnitine is a wáter-soluble compound that humans may obtain both by food ingestion and endogenous synthesis from trimethyl-lysine. Most l-carnitine is intracellular, being present predominantly in liver, skeletal muscle, heart and kidney. The organic cation transporter-2 facilitates l-carnitine uptake inside cells. Congenital dysfunction of this transporter causes primary l-carnitine deficiency. Carnitine acetyltransferase is involved in the export of excess acetyl groups from the mitochondria and in acetylation reactions that regulate gene transcription and enzyme activity. Carnitine octanoyltransferase is a peroxysomal enzyme required for the complete oxidation of very long-chain fatty acids and phytanic acid, a branched-chain fatty acid. Carnitine palmitoyltransferase-1 is a transmembrane protein located on the outer mitochondrial membrane where it catalyzes the conversion of acyl-coenzyme A esters to acyl-carnitine esters. Carnitine acyl-carnitine translocase transports acyl-carnitine esters across the inner mitochondrial membrane in exchange for free l-carnitine that exits the mitochondrial matrix. Carnitine palmitoyltransferase-2 is anchored on the matrix side of the inner mitochondrial membrane, where it converts acyl-carnitine esters back to acyl-coenzyme A esters, which may be used in metabolic pathways, such as mitochondrial β-oxidation. l-Carnitine enhances nonoxidative glucose disposal under euglycemic hyperinsulinemic conditions in both healthy individuals and patients with type 2 diabetes, suggesting that l-carnitine strengthens insulin effect on glycogen storage. The plasma level of acyl-carnitine esters, primarily acetyl-carnitine, increases during diabetic ketoacidosis, fasting, and physical activity, particularly high-intensity exercise. Plasma concentration of free l-carnitine decreases simultaneously under these conditions. © 2017 IUBMB Life, 69(8):578-594, 2017.

TXNIP regulates myocardial fatty acid oxidation via miR-33a signaling.

Myocardial fatty acid β-oxidation is critical for the maintenance of energy homeostasis and contractile function in the heart, but its regulation is still not fully understood. While thioredoxin-interacting protein (TXNIP) has recently been implicated in cardiac metabolism and mitochondrial function, its effects on β-oxidation have remained unexplored. Using a new cardiomyocyte-specific TXNIP knockout mouse and working heart perfusion studies, as well as loss- and gain-of-function experiments in rat H9C2 and human AC16 cardiomyocytes, we discovered that TXNIP deficiency promotes myocardial β-oxidation via signaling through a specific microRNA, miR-33a. TXNIP deficiency leads to increased binding of nuclear factor Y (NFYA) to the sterol regulatory element binding protein 2 (SREBP2) promoter, resulting in transcriptional inhibition of SREBP2 and its intronic miR-33a. This allows for increased translation of the miR-33a target genes and β-oxidation-promoting enzymes, carnitine octanoyl transferase (CROT), carnitine palmitoyl transferase 1 (CPT1), hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase-β (HADHB), and AMPKα and is associated with an increase in phospho-AMPKα and phosphorylation/inactivation of acetyl-CoA-carboxylase. Thus, we have identified a novel TXNIP-NFYA-SREBP2/miR-33a-AMPKα/CROT/CPT1/HADHB pathway that is conserved in mouse, rat, and human cardiomyocytes and regulates myocardial β-oxidation.

Changes in carnitine octanoyltransferase activity induce alteration in fatty acid metabolism

The peroxisomal beta oxidation of very long chain fatty acids (VLCFA) leads to the formation of medium chain acyl-CoAs such as octanoyl-CoA. Today, it seems clear that the exit of shortened fatty acids produced by the peroxisomal beta oxidation requires their conversion into acyl-carnitine and the presence of the carnitine octanoyltransferase (CROT). Here, we describe the consequences of an overexpression and a knock down of the CROT gene in terms of mitochondrial and peroxisomal fatty acids metabolism in a model of hepatic cells. Our experiments showed that an increase in CROT activity induced a decrease in MCFA and VLCFA levels in the cell. These changes are accompanied by an increase in the level of mRNA encoding enzymes of the peroxisomal beta oxidation. In the same time, we did not observe any change in mitochondrial function. Conversely, a decrease in CROT activity had the opposite effect. These results suggest that CROT activity, by controlling the peroxisomal amount of medium chain acyls, may control the peroxisomal oxidative pathway.

Short- and medium-chain carnitine acyltransferases and acyl-CoA thioesterases in mouse provide complementary systems for transport of beta-oxidation products out of peroxisomes.

Peroxisomes metabolize a variety of lipids, acting as a chain-shortening system that produces acyl-CoAs of varying chain lengths, including acetyl-CoA and propionyl-CoA. It is, however, still largely unknown how beta-oxidation products exit peroxisomes and where they are further metabolized. Peroxisomes contain carnitine acetyltransferase (CRAT) and carnitine octanoyltransferase (CROT) that produce carnitine esters for transport out of peroxisomes, together with recently characterized acyl-CoA thioesterases (ACOTs) that produce free fatty acids. Here we have performed tissue expression profiling of the short- and medium-chain carnitine acyltransferases Crat, Crot and the short- and medium-chain thioesterases (Acot12) and (Acot5), and show that they are largely expressed in different tissues, suggesting that they do not compete for the same substrates but rather provide complementary systems for transport of metabolites across the peroxisomal membrane. These data also explain earlier observed tissue differences in peroxisomal production of acetyl-CoA/acetyl-carnitine/acetate and underscores the differences in peroxisome function in various organs.

Definition by Functional and Structural Analysis of Two Malonyl-CoA Sites in Carnitine Palmitoyltransferase 1A

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine in the presence of l-carnitine, thus facilitating the entry of fatty acids to mitochondria, in a process that is physiologically inhibited by malonyl-CoA. To examine the mechanism of CPT1 liver isoform (CPT1A) inhibition by malonyl-CoA, we constructed an in silico model of both its NH2- and COOH-terminal domains. Two malonyl-CoA binding sites were found. One of these, the "CoA site" or "A site," is involved in the interactions between NH2- and COOH-terminal domains and shares the acyl-CoA hemitunnel. The other, the "opposite-to-CoA site" or "O site," is on the opposite side of the enzyme, in the catalytic channel. The two sites share the carnitine-binding locus. To prevent the interaction between NH2- and COOH-terminal regions, we produced CPT1A E26K and K561E mutants. A double mutant E26K/K561E (swap), which was expected to conserve the interaction, was also produced. Inhibition assays showed a 12-fold decrease in the sensitivity (IC50) toward malonyl-CoA for CPT1A E26K and K561E single mutants, whereas swap mutant reverts to wild-type IC50 value. We conclude that structural interaction between both domains is critical for enzyme sensitivity to malonyl-CoA inhibition at the "A site." The location of the "O site" for malonyl-CoA binding was supported by inhibition assays of expressed R243T mutant. The model is also sustained by kinetic experiments that indicated linear mixed type malonyl-CoA inhibition for carnitine. Malonyl-CoA alters the affinity of carnitine, and there appears to be an exponential inverse relation between carnitine Km and malonyl-CoA IC50.

Mutagenesis of Specific Amino Acids Converts Carnitine Acetyltransferase into Carnitine Palmitoyltransferase

Carnitine acyltransferases catalyze the exchange of acyl groups between carnitine and CoA. The members of the family can be classified on the basis of their acyl-CoA selectivity. Carnitine acetyltransferases (CrATs) are very active toward short-chain acyl-CoAs but not toward medium- or long-chain acyl-CoAs. Previously, we identified an amino acid residue (Met(564) in rat CrAT) that was critical to fatty acyl-chain-length specificity. M564G-mutated CrAT behaved as if its natural substrates were medium-chain acyl-CoAs, similar to that of carnitine octanoyltransferase (COT). To extend the specificity of rat CrAT to other substrates, we have performed new mutations. Using in silico molecular modeling procedures, we have now identified a second putative amino acid involved in acyl-CoA specificity (Asp(356) in rat CrAT). The double CrAT mutant D356A/M564G showed 6-fold higher activity toward palmitoyl-CoA than that of the single CrAT mutant M564G and a new activity toward stearoyl-CoA. We show that by performing two amino acid replacements a CrAT can be converted into a pseudo carnitine palmitoyltransferase (CPT) in terms of substrate specificity. To change CrAT specificity from carnitine to choline, we also prepared a mutant CrAT that incorporates four amino acid substitutions (A106M/T465V/T467N/R518N). The quadruple mutant shifted the catalytic discrimination between l-carnitine and choline in favor of the latter substrate and showed a 9-fold increase in catalytic efficiency toward choline compared with that of the wild-type. Molecular in silico docking supports kinetic data for the positioning of substrates in the catalytic site of CrAT mutants.

The Crystal Structure of Carnitine Palmitoyltransferase 2 and Implications for Diabetes Treatment

Carnitine palmitoyltransferases 1 and 2 (CPTs) facilitate the import of long-chain fatty acids into mitochondria. Modulation of the catalytic activity of the CPT system is currently under investigation for the development of novel drugs against diabetes mellitus. We report here the 1.6 A resolution structure of the full-length mitochondrial membrane protein CPT-2. The structure of CPT-2 in complex with the generic CPT inhibitor ST1326 ([R]-N-[tetradecylcarbamoyl]-aminocarnitine), a substrate analog mimicking palmitoylcarnitine and currently in clinical trials for diabetes mellitus treatment, was solved at 2.5 A resolution. These structures of CPT-2 provide insight into the function of residues involved in substrate binding and determination of substrate specificity, thereby facilitating the rational design of antidiabetic drugs. We identify a sequence insertion found in CPT-2 that mediates membrane localization. Mapping of mutations described for CPT-2 deficiency, a hereditary disorder of lipid metabolism, implies effects on substrate recognition and structural integrity of CPT-2.

Activity and mRNA levels of enzymes involved in hepatic fatty acid oxidation in mice fed citrus flavonoids

Objective We investigated the physiologic activity of citrus flavonoids, naringenin, and hesperetin in affecting the activity and mRNA levels of enzymes involved in hepatic fatty acid oxidation and serum and liver lipid levels in male ICR mice. Methods Six-week-old male ICR mice were fed experimental diets containing 1% naringenin or hesperetin and a control diet free of citrus flavonoids for 21 d. Results Naringenin caused a significant 56.1% increase in the hepatic cyanide-insensitive palmitoyl–coenzyme A oxidation rate. This compound also caused significant increases ranging from 9.8% to 55.6% in activity levels of various enzymes involved in hepatic fatty acid oxidation. These increases were accompanied by upregulation of gene expression of enzymes involved in peroxisomal fatty acid oxidation including increases of 46.8% to 88.7% of carnitine octanoyltransferase, acyl–coenzyme A oxidase, bifunctional enzyme, and 3-ketoacyl–coenzyme A thiolase. Messenger RNA levels of mitochondrial proteins involved in fatty acid oxidation except for trifunctional enzyme subunit-β were not affected by naringenin. Naringenin also significantly increased more than five-fold the mRNA level of microsomal cytochrome P-450 IV A1 involved in ω-oxidation of fatty acids. However, hesperetin did not affect any parameter of hepatic fatty acid oxidation. Naringenin, but not hesperetin, significantly lowered serum levels of triacylglycerol, cholesterol, phospholipids, and free fatty acid. Conclusion We demonstrated that naringenin has the physiologic effect of increasing hepatic fatty acid oxidation through upregulation of gene expression of enzymes involved in peroxisomal β-oxidation in mice. The change may account for the propensity of this compound to lower serum lipid levels.

The G553M Mutant of Peroxisomal Carnitine Octanoyltransferase Catalyses Acetyl Transfer and Acetyl-CoA Hydrolysis

The structure of carnitine acetyltransferase revealed a putative binding site for longer acyl chains but access was blocked by methionine 564 (G. Jogl and L. Tong (2003) Cell 112, 113–122). The equivalent residue in all long chain carnitine acyltransferases is a conserved glycine. Mutation of glycine 553 to methionine in bovine COT resulted in loss of activity with all acyl-CoA substrates except acetyl-CoA, supporting the hypothesis that the methionine blocks access for longer acyl chains. The kinetic characteristics of acetyl transfer to carnitine were identical in the native and mutant enzyme. However, rapid acetyl-CoA hydrolysis in the mutant but not the wild-type indicates perturbation of the catalytic site.

A phytol-enriched diet induces changes in fatty acid metabolism in mice both via PPARα-dependent and -independent pathways

Branched-chain fatty acids (such as phytanic and pristanic acid) are ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in vitro. To investigate the effects of these physiological compounds in vivo, wild-type and PPARalpha-deficient (PPARalpha-/-) mice were fed a phytol-enriched diet. This resulted in increased plasma and liver levels of the phytol metabolites phytanic and pristanic acid. In wild-type mice, plasma fatty acid levels decreased after phytol feeding, whereas in PPARalpha-/- mice, the already elevated fatty acid levels increased. In addition, PPARalpha-/- mice were found to be carnitine deficient in both plasma and liver. Dietary phytol increased liver free carnitine in wild-type animals but not in PPARalpha-/- mice. Investigation of carnitine biosynthesis revealed that PPARalpha is likely involved in the regulation of carnitine homeostasis. Furthermore, phytol feeding resulted in a PPARalpha-dependent induction of various peroxisomal and mitochondrial beta-oxidation enzymes. In addition, a PPARalpha-independent induction of catalase, phytanoyl-CoA hydroxylase, carnitine octanoyltransferase, peroxisomal 3-ketoacyl-CoA thiolase, and straight-chain acyl-CoA oxidase was observed. In conclusion, branched-chain fatty acids are physiologically relevant ligands of PPARalpha in mice. These findings are especially relevant for disorders in which branched-chain fatty acids accumulate, such as Refsum disease and peroxisome biogenesis disorders.

Crystal Structure of Mouse Carnitine Octanoyltransferase and Molecular Determinants of Substrate Selectivity*[boxs

Carnitine acyltransferases have crucial functions in fatty acid metabolism. Members of this enzyme family show distinctive substrate preferences for short-, medium- or long-chain fatty acids. The molecular mechanism for this substrate selectivity is not clear as so far only the structure of carnitine acetyltransferase has been determined. To further our understanding of these important enzymes, we report here the crystal structures at up to 2.0-A resolution of mouse carnitine octanoyltransferase alone and in complex with the substrate octanoylcarnitine. The structures reveal significant differences in the acyl group binding pocket between carnitine octanoyltransferase and carnitine acetyltransferase. Amino acid substitutions and structural changes produce a larger hydrophobic pocket that binds the octanoyl group in an extended conformation. Mutation of a single residue (Gly-553) in this pocket can change the substrate preference between short- and medium-chain acyl groups. The side chains of Cys-323 and Met-335 at the bottom of this pocket assume dual conformations in the substrate complex, and mutagenesis studies suggest that the Met-335 residue is important for catalysis.

Structure and Function of Carnitine Acyltransferases

Carnitine acyltransferases catalyze the exchange of acyl groups between carnitine and coenzyme A (CoA). These enzymes include carnitine acetyltransferase (CrAT), carnitine octanoyltransferase (CrOT), and carnitine palmitoyltransferases (CPTs). CPT-I and CPT-II are crucial for the beta-oxidation of long-chain fatty acids in the mitochondria by enabling their transport across the mitochondrial membrane. The activity of CPT-I is inhibited by malonyl-CoA, a crucial regulatory mechanism for fatty acid oxidation. Mutation or dysregulation of the CPT enzymes has been linked to many serious, even fatal human diseases, and these enzymes are promising targets for the development of therapeutic agents against type 2 diabetes and obesity. We have determined the crystal structures of murine CrAT, alone and in complex with its substrate carnitine or CoA. The structure contains two domains. Surprisingly, these two domains share the same backbone fold, which is also similar to that of chloramphenicol acetyltransferase and dihydrolipoyl transacetylase. The active site is located at the interface between the two domains, in a tunnel that extends through the center of the enzyme. Carnitine and CoA are bound in this tunnel, on opposite sides of the catalytic His343 residue. The structural information provides a molecular basis for understanding the catalysis by carnitine acyltransferases and for designing their inhibitors. In addition, our structural information suggests that the substrate carnitine may assist the catalysis by stabilizing the oxyanion in the reaction intermediate.

Redesign of Carnitine Acetyltransferase Specificity by Protein Engineering

In eukaryotes, L-carnitine is involved in energy metabolism by facilitating beta-oxidation of fatty acids. Carnitine acetyltransferases (CrAT) catalyze the reversible conversion of acetyl-CoA and carnitine to acetylcarnitine and free CoA. To redesign the specificity of rat CrAT toward its substrates, we mutated Met564. The M564G mutated CrAT showed higher activity toward longer chain acyl-CoAs: activity toward myristoyl-CoA was 1250-fold higher than that of the wild-type CrAT, and lower activity toward its natural substrate, acetyl-CoA. Kinetic constants of the mutant CrAT showed modification in favor of longer acyl-CoAs as substrates. In the reverse case, mutation of the orthologous glycine (Gly553) to methionine in carnitine octanoyltransferase (COT) decreased activity toward its natural substrates, medium- and long-chain acyl-CoAs, and increased activity toward short-chain acyl-CoAs. Another CrAT mutant, M564A, was prepared and tested in the same way, with similar results. We conclude that Met564 blocks the entry of medium- and long-chain acyl-CoAs to the catalytic site of CrAT. Three-dimensional models of wild-type and mutated CrAT and COT support this hypothesis. We show for the first time that a single amino acid is able to determine the substrate specificity of CrAT and COT.

Gene expression profiles in liver regeneration with oval cell induction

The liver has the unique ability to regenerate even in adulthood. While mature hepatocytes can proliferate by themselves, stem cells also play a critical role in liver regeneration and oval cells are considered to be the progeny of activated hepatic stem cells. We herein investigated the gene expression profiles in the conditions inducing oval cells, using microarray analysis. Two approaches were used to induce oval cells. In the first, animals were treated with a combination of an N-2-acetylaminofluorene (AAF)-containing diet and partial hepatectomy (PHx). In the second, animals were supplied with chow containing a 1:1 mixture of choline-deficient and normal diets, as well as 0.075% ethionine in drinking water. Using in-house cDNA microarrays consisting of 2304 cDNA clones from the mouse liver, 69 and 89 genes, respectively, were found to be up-regulated in these two models. Six genes, i.e., those for insulin-like growth factor binding protein-1, CYP4a14, carnitine octanoyltransferase, osteopontin, and two expressed sequence tags (ESTs) were up-regulated in these models but not in ordinary model with PHx alone. They might be specifically activated in the induction of oval cells, and help to clarify the nature of stem cell stimulation that occurs during liver regeneration.

Gene expression profile of cardiomyocytes in hypertrophic heart induced by continuous norepinephrine infusion in the rats.

Catecholamines play an important role in the development of cardiac hypertrophy. To observe cardiomyocyte-specific gene expression changes induced by catecholamines in vivo, left ventricular cardiomyocytes were isolated from male Sprague-Dawley rats after continuous infusion of norepinephrine (NE; 0.2 mg/kg per hour intravenously) for 0.5, 1, 2, 3 and 7 days. The gene expression profiles of these cells during different NE infusion stages were assessed by using a cDNA microarray, and the microarray data were further analyzed by a clustering method. Cardiac hypertrophy was induced upon continuous NE infusion, with the peak at 3 days. Meanwhile, manifest changes in gene expression profile within cardiomyocytes over the time course were revealed, most of the genes never having been reported to be involved in cardiac hypertrophy. The number of genes displaying differential expression also peaked at the middle stage of infusion (2-3 days), and the majority of the signaling molecules were found differentially expressed mainly at this stage, including phosphatidylinositol 3-kinase, calcium/calmodulin-dependent protein kinase II and non-receptor tyrosine kinases, etc. The tumor suppressor p53 was found up-regulated at very early (0.5 days) and late stages (7 days) of NE infusion. Self-organization clustering analysis revealed subsets of coordinate regulated genes. One set consisted of several enzymes involved in energy metabolism, including carnitine octanoyltransferase, ATP synthase subunit c, pancreatic lipase and glycogen phosphorylase, possessing a similar expression pattern with a rapidly elevated expression level at the early stage of NE infusion. This is the first study to provide transcriptional information for cardiomyocytes, a single cell type, in the heart during the development of cardiac hypertrophy in vivo, and may provide accurate clues to elaborate hypotheses about the evolution of this pathology.

Identification of Conserved Amino Acid Residues in Rat Liver Carnitine Palmitoyltransferase I Critical for Malonyl-CoA Inhibition: MUTATION OF METHIONINE 593 ABOLISHES MALONYL-CoA INHIBITION

Carnitine palmitoyltransferase (CPT) I, which catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine facilitating its transport through the mitochondrial membranes, is inhibited by malonyl-CoA. By using the SequenceSpace algorithm program to identify amino acids that participate in malonyl-CoA inhibition in all carnitine acyltransferases, we found 5 conserved amino acids (Thr(314), Asn(464), Ala(478), Met(593), and Cys(608), rat liver CPT I coordinates) common to inhibitable malonyl-CoA acyltransferases (carnitine octanoyltransferase and CPT I), and absent in noninhibitable malonyl-CoA acyltransferases (CPT II, carnitine acetyltransferase (CAT) and choline acetyltransferase (ChAT)). To determine the role of these amino acid residues in malonyl-CoA inhibition, we prepared the quintuple mutant CPT I T314S/N464D/A478G/M593S/C608A as well as five single mutants CPT I T314S, N464D, A478G, M593S, and C608A. In each case the CPT I amino acid selected was mutated to that present in the same homologous position in CPT II, CAT, and ChAT. Because mutant M593S nearly abolished the sensitivity to malonyl-CoA, two other Met(593) mutants were prepared: M593A and M593E. The catalytic efficiency (V(max)/K(m)) of CPT I in mutants A478G and C608A and all Met(593) mutants toward carnitine as substrate was clearly increased. In those CPT I proteins in which Met(593) had been mutated, the malonyl-CoA sensitivity was nearly abolished. Mutations in Ala(478), Cys(608), and Thr(314) to their homologous amino acid residues in CPT II, CAT, and ChAT caused various decreases in malonyl-CoA sensitivity. Ala(478) is located in the structural model of CPT I near the catalytic site and participates in the binding of malonyl-CoA in the low affinity site (Morillas, M., Gómez-Puertas, P., Rubi, B., Clotet, J., Ariño, J., Valencia, A., Hegardt, F. G., Serra, D., and Asins, G. (2002) J. Biol. Chem. 277, 11473-11480). Met(593) may participate in the interaction of malonyl-CoA in the second affinity site, whose location has not been reported.

Structural Model of a Malonyl-CoA-binding Site of Carnitine Octanoyltransferase and Carnitine Palmitoyltransferase I: MUTATIONAL ANALYSIS OF A MALONYL-CoA AFFINITY DOMAIN

Carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) I, which facilitate the transport of medium- and long-chain fatty acids through the peroxisomal and mitochondrial membranes, are physiologically inhibited by malonyl-CoA. Using an "in silico" macromolecular docking approach, we built a model in which malonyl-CoA could be attached near the catalytic core. This disrupts the positioning of the acyl-CoA substrate in the channel in the model reported for both proteins (Morillas, M., Gómez-Puertas, P., Roca, R., Serra, D., Asins, G., Valencia, A., and Hegardt, F. G. (2001) J. Biol. Chem. 276, 45001-45008). The putative malonyl-CoA domain contained His(340), implicated together with His(131) in COT malonyl-CoA sensitivity (Morillas, M., Clotet, J., Rubi, B., Serra, D., Asins, G., Ariño, J., and Hegardt F. G. (2000) FEBS Lett. 466, 183-186). When we mutated COT His(131) the IC(50) increased, and malonyl-CoA competed with the substrate decanoyl-CoA. Mutation of COT Ala(332), present in the domain 8 amino acids away from His(340), decreased the malonyl-CoA sensitivity of COT. The homologous histidine and alanine residues of L-CPT I, His(277), His(483), and Ala(478) were also mutated, which decreased malonyl-CoA sensitivity. Natural mutation of Pro(479), which is also located in the malonyl-CoA predicted site, to Leu in a patient with human L-CPT I hereditary deficiency, modified malonyl-CoA sensitivity. We conclude that this malonyl-CoA domain is present in both COT and L-CPT I proteins and might be the site at which malonyl-CoA interacts with the substrate acyl-CoA. Other malonyl-CoA non-inhibitable members of the family, CPT II and carnitine acetyltransferase, do not contain this domain.

Structural Model of the Catalytic Core of Carnitine Palmitoyltransferase I and Carnitine Octanoyltransferase (COT): MUTATION OF CPT I HISTIDINE 473 AND ALANINE 381 AND COT ALANINE 238 IMPAIRS THE CATALYTIC ACTIVITY

Carnitine palmitoyltransferase I (CPT I) and carnitine octanoyltransferase (COT) catalyze the conversion of long- and medium-chain acyl-CoA to acylcarnitines in the presence of carnitine. We propose a common three-dimensional structural model for the catalytic domain of both, based on fold identification for 200 amino acids surrounding the active site through a threading approach. The model is based on the three-dimensional structure of the rat enoyl-CoA hydratase, established by x-ray diffraction analysis. The study shows that the structural model of 200 amino acids of the catalytic site is practically identical in CPT I and COT with identical distribution of 4 beta-sheets and 6 alpha-helices. Functional analysis of the model was done by site-directed mutagenesis. When the critical histidine residue 473 in CPT I (327 in COT), localized in the acyl-CoA pocket in the model, was mutated to alanine, the catalytic activity was abolished. Mutation of the conserved alanine residue to aspartic acid, A381D (in CPT I) and A238D (in COT), which are 92/89 amino acids far from the catalytic histidine, respectively (but very close to the acyl-CoA pocket in the structural model), decreased the activity by 86 and 80%, respectively. The K(m) for acyl-CoA increased 6-8-fold, whereas the K(m) for carnitine hardly changed. The inhibition of the mutant CPT I by malonyl-CoA was not altered. The structural model explains the loss of activity reported for the CPT I mutations R451A, W452A, D454G, W391A, del R395, P479L, and L484P, all of which occur in or near the modeled catalytic domain.