Melatonin biosynthesis: the structure of serotonin N-acetyltransferase at 2.5 A resolution suggests a catalytic mechanism.
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Conversion of serotonin to N-acetylserotonin, the precursor of the circadian neurohormone melatonin, is catalyzed by serotonin N-acetyltransferase (AANAT) in a reaction requiring acetyl coenzyme A (AcCoA). AANAT is a globular protein consisting of an eight-stranded beta sheet flanked by five alpha helices; a conserved motif in the center of the beta sheet forms the cofactor binding site. Three polypeptide loops converge above the AcCoA binding site, creating a hydrophobic funnel leading toward the cofactor and serotonin binding sites in the protein interior. Two conserved histidines not found in other NATs are located at the bottom of the funnel in the active site, suggesting a catalytic mechanism for acetylation involving imidazole groups acting as general acid/base catalysts. (+info)
Pyruvate dehydrogenase activation in inactive muscle during and after maximal exercise in men.
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Pyruvate dehydrogenase activity (PDHa) and acetyl-group accumulation were examined in the inactive deltoid muscle in response to maximal leg exercise in men. Seven subjects completed three consecutive 30-s bouts of maximal isokinetic cycling, with 4-min rest intervals between bouts. Biopsies of the deltoid were obtained before exercise, after bouts 1 and 3, and after 15 min of rest recovery. Inactive muscle lactate (LA) and pyruvate (PYR) contents increased more than twofold (P < 0.05) after exercise (bout 3) and remained elevated after 15 min of recovery (P < 0.05). Increased PYR accumulation secondary to LA uptake by the inactive deltoid was associated with greater PDHa, which progressively increased from 0.71 +/- 0.23 mmol. min-1. kg wet wt-1 at rest to a maximum of 1.83 +/- 0.30 mmol. min-1. kg wet wt-1 after bout 3 (P < 0.05) and remained elevated after 15 min of recovery (1.63 +/- 0.24 mmol. min-1. kg wet wt-1; P < 0.05). Acetyl-CoA and acetylcarnitine accumulations were unaltered. Increased PDHa allowed and did not limit the oxidation of LA and PYR in inactive human skeletal muscle after maximal exercise. (+info)
Replenishment and depletion of citric acid cycle intermediates in skeletal muscle. Indication of pyruvate carboxylation.
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The effects of various substrates on the concentrations of free amino acids, citric acid cycle intermediates and acylcarnitines were studies in perfused hindquarter of rat in presence of glucose and insulin in order to assess regulatory mechanisms of the level of citric acid cycle intermediates in skeletal muscle. 1. Acetate and acetoacetate effected a significant increase in the level of citrate cycle intermediates and accumulation of acetylcarnitine. These changes were accompanied by a reduction in the level of alanine. The concentration of AMP was significantly elevated. 2. Muscle mitochondria fixed 14CO2 in the presence of pyruvate. The products were identified as malate or citrate when whole and disintegrated mitochondria were used respectively. The fixation was greatly stimulated by acetylcarnitine. 3. Acetylcarnitine inhibited the production of pyruvate from malate by muscle mitochondria. 4. Perfusion with 2-oxoisocaproate and 2-oxoisovalerate promoted increases in the level of citric cycle intermediates, a drop in both alanine and glutamate, and accumulation of branched-chain acylcarnitines. 2-Oxoisocaproate also caused a reduction of alanine released from the muscle. 5. Perfusion with leucine and valine did not change the concentration of citric acid cycle intermediates, but elevated glutamate and still more the concentration of alanine. 6. It is concluded that citric cycle intermediate level in the perfused resting muscle is modified by a) conditions which change the concentration of acetyl-CoA and thereby modify the rate of pyruvate carboxylation and decarboxylation of malate via malic enzyme b) conditions which change the concentration of pyruvate cause changes in alanine and cycle intermediates in the same direction via transamination reactions c) conditions which change the concentrations of 2-oxoacids which are converted to cycle intermediates via oxidation. (+info)
The role of an iron-sulfur cluster in an enzymatic methylation reaction. Methylation of CO dehydrogenase/acetyl-CoA synthase by the methylated corrinoid iron-sulfur protein.
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This paper focuses on how a methyl group is transferred from a methyl-cobalt(III) species on one protein (the corrinoid iron-sulfur protein (CFeSP)) to a nickel iron-sulfur cluster on another protein (carbon monoxide dehydrogenase/acetyl-CoA synthase). This is an essential step in the Wood-Ljungdahl pathway of anaerobic CO and CO2 fixation. The results described here strongly indicate that transfer of methyl group to carbon monoxide dehydrogenase/acetyl-CoA synthase occurs by an SN2 pathway. They also provide convincing evidence that oxidative inactivation of Co(I) competes with methylation. Under the conditions of our anaerobic assay, Co(I) escapes from the catalytic cycle one in every 100 turnover cycles. Reductive activation of the CFeSP is required to regenerate Co(I) and recruit the protein back into the catalytic cycle. Our results strongly indicate that the [4Fe-4S] cluster of the CFeSP is required for reductive activation. They support the hypothesis that the [4Fe-4S] cluster of the CFeSP does not participate directly in the methyl transfer step but provides a conduit for electron flow from physiological reductants to the cobalt center. (+info)
The structural basis of ordered substrate binding by serotonin N-acetyltransferase: enzyme complex at 1.8 A resolution with a bisubstrate analog.
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Serotonin N-acetyltransferase, a member of the GNAT acetyltransferase superfamily, is the penultimate enzyme in the conversion of serotonin to melatonin, the circadian neurohormone. Comparison of the structures of the substrate-free enzyme and the complex with a bisubstrate analog, coenzyme A-S-acetyltryptamine, demonstrates that acetyl coenzyme A (AcCoA) binding is accompanied by a large conformational change that in turn leads to the formation of the serotonin-binding site. The structure of the complex also provides insight into how the enzyme may facilitate acetyl transfer. A water-filled channel leading from the active site to the surface provides a pathway for proton removal following amine deprotonation. Furthermore, structural and mutagenesis results indicate an important role for Tyr-168 in catalysis. (+info)
Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica.
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Biosynthesis of proteinogenic amino acids in the extremely halophilic archaeon Haloarcula hispanica was explored by using biosynthetically directed fractional 13C labeling with a mixture of 90% unlabeled and 10% uniformly 13C-labeled glycerol. The resulting 13C-labeling patterns in the amino acids were analyzed by two-dimensional 13C,1H correlation spectroscopy. The experimental data provided evidence for a split pathway for isoleucine biosynthesis, with 56% of the total Ile originating from threonine and pyruvate via the threonine pathway and 44% originating from pyruvate and acetyl coenzyme A via the pyruvate pathway. In addition, the diaminopimelate pathway involving diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis and an analysis of the 13C-labeling pattern in tyrosine indicated novel biosynthetic pathways that have so far not been further characterized. For the 17 other proteinogenic amino acids, the data were consistent with data for commonly found biosynthetic pathways. A comparison of our data with the amino acid metabolisms of eucarya and bacteria supports the theory that pathways for synthesis of proteinogenic amino acids were established before ancient cells diverged into archaea, bacteria, and eucarya. (+info)
Melatonin, its precursors, and synthesizing enzyme activities in the human ovary.
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The presence of melatonin (N-acetyl-5-methoxytryptamine) and its precursors, serotonin (5-hydroxytryptamine) and N-acetylserotonin, was demonstrated in extracts of human ovary using reverse-phase high-performance liquid chromatography coupled with fluorometric detection. In addition, activities of two melatonin-synthesizing enzymes, arylalkylamine N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), were found in human ovary homogenates. The apparent Michaelis constants for the substrates of NAT and HIOMT in the human ovary were similar to those reported for the pineal glands of humans and other mammals. These findings strongly suggest that the human ovary, like the pineal gland, may synthesize melatonin from serotonin by the sequential action of NAT and HIOMT. (+info)
Characterization of a novel spermidine/spermine acetyltransferase, BltD, from Bacillus subtilis.
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Overexpression of the BltD gene in Bacillus subtilis causes acetylation of the polyamines spermidine and spermine. BltD is co-regulated with another gene, Blt, which encodes a multidrug export protein whose overexpression facilitates spermidine export [Woolridge, Vazquez-Laslop, Markham, Chevalier, Gerner and Neyfakh (1997) J. Biol. Chem. 272, 8864-8866]. Here we show that BltD acetylates both spermidine and spermine at primary propyl amine moieties, with spermine being the preferred substrate. In the presence of saturating concentrations of acetyl CoA, BltD rapidly acetylates spermine at both the N1 and N12 positions. The Km (app) values for spermine, spermidine and N1-acetylspermine are +info)