Structure of human carnitine acetyltransferase. Molecular basis for fatty acyl transfer.
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Carnitine acyltransferases are a family of ubiquitous enzymes that play a pivotal role in cellular energy metabolism. We report here the x-ray structure of human carnitine acetyltransferase to a 1.6-A resolution. This structure reveals a monomeric protein of two equally sized alpha/beta domains. Each domain is shown to have a partially similar fold to other known but oligomeric enzymes that are also involved in group-transfer reactions. The unique monomeric arrangement of the two domains constitutes a central narrow active site tunnel, indicating a likely universal feature for all members of the carnitine acyltransferase family. Superimposition of the substrate complex of a related protein, dihydrolipoyl trans-acetylase, reveals that both substrates localize to the active site tunnel of human carnitine acetyltransferase, suggesting the location of the ligand binding sites for carnitine and coenzyme A. Most significantly, this structure provides critical insights into the molecular basis for fatty acyl chain transfer and a possible common mechanism among a wide range of acyltransferases utilizing a catalytic dyad. (+info)
Exercise-trained but not untrained rats maintain free carnitine reserves during acute exercise.
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Exercise training is known to induce physiological adaptations that improve exercise performance and alter patterns of energy substrate utilization to favour fatty acid oxidation. L-Carnitine is an essential cofactor for the oxidation of fatty acids under all physiological conditions, including exercise. This study evaluated the effect of acute exercise on carnitine concentrations in tissue and serum, liver carnitine palmitoyltransferase-I activity and expression, and serum lipids in both trained and untrained rats as compared to non-exercised rats. Serum acyl- and total carnitine was significantly higher in the trained animals, whether exercised or not, suggesting an exercise-induced increase in a renal threshold for carnitine. Untrained rats had significantly higher acylcarnitine in skeletal muscle and an acyl/free carnitine ratio of 0.63 +/- 0.06 compared with 0.31 +/- 0.16 in trained animals receiving an identical acute bout of exercise, demonstrating that untrained animals utilized a significantly higher percentage of free carnitine reserves during exercise. This study suggests that free carnitine reserves may be reduced during exercise in untrained rats, an effect that has the potential to impair both carbohydrate and fat metabolism during exercise. (+info)
Structural model of carnitine palmitoyltransferase I based on the carnitine acetyltransferase crystal.
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CPT I (carnitine palmitoyltransferase I) catalyses the conversion of palmitoyl-CoA into palmitoylcarnitine in the presence of L-carnitine, facilitating the entry of fatty acids into mitochondria. We propose a 3-D (three-dimensional) structural model for L-CPT I (liver CPT I), based on the similarity of this enzyme to the recently crystallized mouse carnitine acetyltransferase. The model includes 607 of the 773 amino acids of L-CPT I, and the positions of carnitine, CoA and the palmitoyl group were assigned by superposition and docking analysis. Functional analysis of this 3-D model included the mutagenesis of several amino acids in order to identify putative catalytic residues. Mutants D477A, D567A and E590D showed reduced L-CPT I activity. In addition, individual mutation of amino acids forming the conserved Ser685-Thr686-Ser687 motif abolished enzyme activity in mutants T686A and S687A and altered K(m) and the catalytic efficiency for carnitine in mutant S685A. We conclude that the catalytic residues are His473 and Asp477, while Ser687 probably stabilizes the transition state. Several conserved lysines, i.e. Lys455, Lys505, Lys560 and Lys561, were also mutated. Only mutants K455A and K560A showed decreases in activity of 50%. The model rationalizes the finding of nine natural mutations in patients with hereditary L-CPT I deficiencies. (+info)
Crystal structure of PapA5, a phthiocerol dimycocerosyl transferase from Mycobacterium tuberculosis.
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Polyketide-associated protein A5 (PapA5) is an acyltransferase that is involved in production of phthiocerol and phthiodiolone dimycocerosate esters, a class of virulence-enhancing lipids produced by Mycobacterium tuberculosis. Structural analysis of PapA5 at 2.75-A resolution reveals a two-domain structure that shares unexpected similarity to structures of chloramphenicol acetyltransferase, dihydrolipoyl transacetylase, carnitine acetyltransferase, and VibH, a non-ribosomal peptide synthesis condensation enzyme. The PapA5 active site includes conserved histidine and aspartic acid residues that are critical to PapA5 acyltransferase activity. PapA5 catalyzes acyl transfer reactions on model substrates that contain long aliphatic carbon chains, and two hydrophobic channels were observed linking the PapA5 surface to the active site with properties consistent with these biochemical activities and substrate preferences. An additional alpha helix not observed in other acyltransferase structures blocks the putative entrance into the PapA5 active site, indicating that conformational changes may be associated with PapA5 activity. PapA5 represents the first structure solved for a protein involved in polyketide synthesis in Mycobacteria. (+info)
Structural and biochemical studies of the substrate selectivity of carnitine acetyltransferase.
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Carnitine acyltransferases catalyze the exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids, and are attractive targets for drug discovery against diabetes and obesity. These enzymes are classified based on their substrate selectivity for short-chain, medium-chain, or long-chain fatty acids. Structural information on carnitine acetyltransferase suggests that residues Met-564 and Phe-565 may be important determinants of substrate selectivity with the side chain of Met-564 located in the putative binding pocket for acyl groups. Both residues are replaced by glycine in carnitine palmitoyltransferases. To assess the functional relevance of this structural observation, we have replaced these two residues with small amino acids by mutagenesis, characterized the substrate preference of the mutants, and determined the crystal structures of two of these mutants. Kinetic studies confirm that the M564G or M564A mutation is sufficient to increase the activity of the enzyme toward medium-chain substrates with hexanoyl-CoA being the preferred substrate for the M564G mutant. The crystal structures of the M564G mutant, both alone and in complex with carnitine, reveal a deep binding pocket that can accommodate the larger acyl group. We have determined the crystal structure of the F565A mutant in a ternary complex with both the carnitine and CoA substrates at a 1.8-A resolution. The F565A mutation has minor effects on the structure or the substrate preference of the enzyme. (+info)
Redesign of carnitine acetyltransferase specificity by protein engineering.
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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. (+info)
Carnitine treatment inhibits increases in cerebral carnitine esters and glutamate detected by mass spectrometry after hypoxia-ischemia in newborn rats.
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BACKGROUND AND PURPOSE: Cerebral ischemic insults disrupt normal respiratory activity in mitochondria. Carnitine plays an essential role in mitochondrial metabolism and in modulating excess acyl-coenzyme A (acyl-CoA) levels. The effects of cerebral ischemia on carnitine metabolism are not well understood, although the newborn may be particularly vulnerable to carnitine deficiency. We used a newborn rat model of hypoxia-ischemia (HI) to test the hypothesis that HI alters acyl-CoA:CoA homeostasis and that this effect can be prevented by treatment with carnitine. METHODS: A total of 120 postnatal day 7 rats were subjected to 70 minutes of HI after treatment with 16 mmol/kg intraperitoneal l-carnitine or diluent. Carnitine, acylcarnitines, and excitatory amino acids were measured by mass spectrometry, and carnitine acetyl transferase activity, superoxide, and levels of the mitochondrial phospholipid cardiolipin (CL) were measured at 2- and 24-hour recovery. RESULTS: HI and hypoxia were associated with a significant increase in the ratio of acyl-CoA:CoA, which was prevented by treatment with carnitine. Carnitine treatment also prevented increases in glutamate, glycine, superoxide, and decrease of CL. CONCLUSIONS: Carnitine metabolic pathways are compromised in HI and hypoxia. The protective effect of carnitine treatment on HI injury may be attributable to maintaining mitochondrial function. (+info)
Serum and tissue carnitine assay based on dialysis.
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Carnitine (L-beta-hydroxy-gamma-trimethylaminobutyric acid) aids mitochondrial energy production by transferring fatty acids across the membranes for beta-oxidation. We describe here a modified enzymatic assay for free serum and tissue carnitine based on dialysis to remove interfering substances in the serum, with subsequent conversion of carnitine to the acyl derivative by carnitine acetyltransferase (EC 2.3.1.7) in the presence of 5,5'-dithiobis-(2-nitrobenzoic acid). The method compared well with a radioenzymatic assay. The reference interval for serum is 28-70 mumol/L. Patients with advanced diabetes and those undergoing valproic acid treatment displayed lower mean values; a statistically significant number of them showed serum carnitine values below the reference interval. The method was also applied to carnitine measurement in cerebrospinal fluid and human tissues. (+info)