A single amino acid change (substitution of glutamate 3 with alanine) in the N-terminal region of rat liver carnitine palmitoyltransferase I abolishes malonyl-CoA inhibition and high affinity binding.
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We have recently shown by deletion mutation analysis that the conserved first 18 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) are essential for malonyl-CoA inhibition and binding (Shi, J., Zhu, H., Arvidson, D. N. , Cregg, J. M., and Woldegiorgis, G. (1998) Biochemistry 37, 11033-11038). To identify specific residue(s) involved in malonyl-CoA binding and inhibition of L-CPTI, we constructed two more deletion mutants, Delta12 and Delta6, and three substitution mutations within the conserved first six amino acid residues. Mutant L-CPTI, lacking either the first six N-terminal amino acid residues or with a change of glutamic acid 3 to alanine, was expressed at steady-state levels similar to wild type and had near wild type catalytic activity. However, malonyl-CoA inhibition of these mutant enzymes was reduced 100-fold, and high affinity malonyl-CoA binding was lost. A mutant L-CPTI with a change of histidine 5 to alanine caused only partial loss of malonyl-CoA inhibition, whereas a mutant L-CPTI with a change of glutamine 6 to alanine had wild type properties. These results demonstrate that glutamic acid 3 and histidine 5 are necessary for malonyl-CoA binding and inhibition of L-CPTI by malonyl-CoA but are not required for catalysis. (+info)
Comparisons of flux control exerted by mitochondrial outer-membrane carnitine palmitoyltransferase over ketogenesis in hepatocytes and mitochondria isolated from suckling or adult rats.
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The primary aim of this paper was to calculate and report flux control coefficients for mitochondrial outer-membrane carnitine palmitoyltransferase (CPT I) over hepatic ketogenesis because its role in controlling this pathway during the neonatal period is of academic importance and immediate clinical relevance. Using hepatocytes isolated from suckling rats as our model system, we measured CPT I activity and carbon flux from palmitate to ketone bodies and to CO2 in the absence and presence of a range of concentrations of etomoxir. (This is converted in situ to etomoxir-CoA which is a specific inhibitor of the enzyme.) From these data we calculated the individual flux control coefficients for CPT I over ketogenesis, CO2 production and total carbon flux (0.51 +/- 0.03; -1.30 +/- 0.26; 0.55 +/- 0.07, respectively) and compared them with equivalent coefficients calculated by similar analyses [Drynan, L., Quant, P.A. & Zammit, V.A. (1996) Biochem. J. 317, 791-795] in hepatocytes isolated from adult rats (0.85 +/- 0.20; 0.23 +/- 0.06; 1.06 +/- 0.29). CPT I exerts significantly less control over ketogenesis in hepatocytes isolated from suckling rats than those from adult rats. In the suckling systems the flux control coefficients for CPT I over ketogenesis specifically and over total carbon flux (< 0.6) are not consistent with the enzyme being rate-limiting. Broadly similar results were obtained and conclusions drawn by reanalysis of previous data {from experiments in mitochondria isolated from suckling or adult rats [Krauss, S., Lascelles, C.V., Zammit, V.A. & Quant, P.A. (1996) Biochem. J. 319, 427-433]} using a different approach of control analysis, although it is not strictly valid to compare flux control coefficients from different systems. Our overall conclusion is that flux control coefficients for CPT I over oxidative fluxes from palmitate (or palmitoyl-CoA) differ markedly according to (a) the metabolic state, (b) the stage of development, (c) the specific pathway studied and (d) the model system. (+info)
Light-dependent changes in redox status of the plastidic acetyl-CoA carboxylase and its regulatory component.
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Plastidic acetyl-CoA carboxylase (ACCase; EC 6.4.1.2), which catalyses the synthesis of malonyl-CoA and is the regulatory enzyme of fatty acid synthesis, is activated by light, presumably under redox regulation. To obtain evidence of redox regulation in vivo, the activity of ACCase was examined in pea chloroplasts isolated from plants kept in darkness (dark-ACCase) or after exposure to light for 1 h (light-ACCase) in the presence or absence of a thiol-reducing agent, dithiothreitol (DTT). The protein level was similar for light-ACCase and dark-ACCase, but the activity of light-ACCase in the absence of DTT was approx. 3-fold that of dark-ACCase. The light-ACCase and dark-ACCase were activated approx. 2-fold and 6-fold by DTT respectively, indicating that light-ACCase was in a much more reduced, active form than the dark-ACCase. This is the first demonstration of the light-dependent reduction of ACCase in vivo. Measurement of the activities of ACCase, carboxyltransferase and biotin carboxylase in the presence and absence of DTT, and the thiol-oxidizing agent, 5, 5'-dithiobis-(2-nitrobenzoic) acid, revealed that the carboxyltransferase reaction, but not the biotin carboxylase reaction, was redox-regulated. The cysteine residue(s) responsible for redox regulation probably reside on the carboxyltransferase component. Measurement of the pH dependence of biotin carboxylase and carboxyltransferase activities in the ACCase suggested that both components affect the activity of ACCase in vivo at a physiological pH range. These results suggest that the activation of ACCase by light is caused partly by the pH-dependent activation of two components and by the reductive activation of carboxyltransferase. (+info)
Cytosolic citrate and malonyl-CoA regulation in rat muscle in vivo.
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In liver, insulin and glucose acutely increase the concentration of malonyl-CoA by dephosphorylating and activating acetyl-CoA carboxylase (ACC). In contrast, in incubated rat skeletal muscle, they appear to act by increasing the cytosolic concentration of citrate, an allosteric activator of ACC, as reflected by increases in the whole cell concentrations of citrate and malate [Saha, A. K., D. Vavvas, T. G. Kurowski, A. Apazidis, L. A. Witters, E. Shafrir, and N. B. Ruderman. Am. J. Physiol. 272 (Endocrinol. Metab. 35): E641-E648, 1997]. We report here that sustained increases in plasma insulin and glucose may also increase the concentration of malonyl-CoA in rat skeletal muscle in vivo by this mechanism. Thus 70 and 125% increases in malonyl-CoA induced in skeletal muscle by infusions of glucose for 1 and 4 days, respectively, and a twofold increase in its concentration during a 90-min euglycemic-hyperinsulinemic clamp were all associated with significant increases in the sum of whole cell concentrations of citrate and/or malate. Similar correlations were observed in muscle of the hyperinsulinemic fa/fa rat, in denervated muscle, and in muscle of rats infused with insulin for 5 h. In muscle of 48-h-starved rats 3 and 24 h after refeeding, increases in malonyl-CoA were not accompanied by consistent increases in the concentrations of malate or citrate. However, they were associated with a decrease in the whole cell concentration of long-chain fatty acyl-CoA (LCFA-CoA), an allosteric inhibitor of ACC. The results suggest that increases in the concentration of malonyl-CoA, caused in rat muscle in vivo by sustained increases in plasma insulin and glucose or denervation, may be due to increases in the cytosolic concentration of citrate. In contrast, during refeeding after starvation, the increase in malonyl-CoA in muscle is probably due to another mechanism. (+info)
Evidence that oleoyl-CoA and ATP-dependent elongations coexist in rapeseed (Brassica napus L.).
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The elongation of different substrates was studied using several subcellular fractions from Brassica napus rapeseed. In the presence of malonyl-CoA, NADH and NADPH, very-long-chain fatty acid (VLCFA) synthesis was observed from either oleoyl-CoA (acyl-CoA elongation) or endogenous primers (ATP-dependent elongation). No activity was detected using oleic acid as precursor. Acyl-CoA and ATP-dependent elongation activities were mainly associated with the 15 000 g/25 min membrane fraction. Reverse-phase TLC analysis showed that the proportions of fatty acids synthesized by these activities were different. Acyl-CoA elongation increased up to 60 microM oleoyl-CoA, and ATP-dependent elongation was maximum at 1 mM ATP. Both activities increased with malonyl-CoA concentration (up to 200 microM). Under all conditions tested, acyl-CoA elongation was higher than ATP-dependent elongation, and, in the presence of both ATP and oleoyl-CoA, the elongation activity was always lower. ATP strongly inhibited acyl-CoA elongation, whereas ATP-dependent elongation was slightly stimulated by low oleoyl-CoA concentrations (up to 15 microM) and decreased in the presence of higher concentrations. CoA (up to 150 microM) had no effect on the ATP-dependent elongation, whereas it inhibited the acyl-CoA elongation. These marked differences strongly support the presence in maturing rapeseed of two different elongating activities differently modulated by ATP and oleoyl-CoA. (+info)
Sequencing and functional expression of the malonyl-CoA-sensitive carnitine palmitoyltransferase from Drosophila melanogaster.
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Using expressed sequence tag data, we obtained a cDNA for a carnitine palmitoyltransferase I (CPT I)-like molecule from Drosophila melanogaster. The cDNA encodes a 782-residue protein that shows 49% and 48% sequence identity with the rat liver and skeletal-muscle isoforms of CPT I respectively. The sequence has two predicted membrane-spanning regions, suggesting that it adopts the same topology as its mammalian counterparts. The sequence contains all the residues that have been shown to be characteristic of carnitine acetyltransferases. Expression in the yeast Pichia pastoris confirmed that the cDNA does encode a CPT enzyme. The activity was found to be associated with a mitochondria-enriched fraction. Kinetic analysis revealed a K(m) for carnitine of 406 microM and a K(m) for palmitoyl-CoA of 105 microM. The CPT activity was very sensitive to inhibition by malonyl-CoA, with an IC(50) of 0.74 microM when the activity was assayed with 35 microM palmitoyl-CoA and 1% (w/v) albumin at pH 7.0. A histidine residue at position 140 in rat liver CPT I has been indicated to be important for inhibition by malonyl-CoA. The equivalent residue (position 136) in Drosophila CPT I is arginine, implying that any basic residue might be compatible with such sensitivity. Evidence is presented that, unlike in mammals, Drosophila has only a single CPT I gene. Sequences suggesting the existence of a splice variant in the 5' untranslated region were found; this was consistent with the existence of two promoters for the CPT I gene. (+info)
The Streptomyces peucetius dpsC gene determines the choice of starter unit in biosynthesis of the daunorubicin polyketide.
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The starter unit used in the biosynthesis of daunorubicin is propionyl coenzyme A (CoA) rather than acetyl-CoA, which is used in the production of most of the bacterial aromatic polyketides studied to date. In the daunorubicin biosynthesis gene cluster of Streptomyces peucetius, directly downstream of the genes encoding the beta-ketoacyl:acyl carrier protein synthase subunits, are two genes, dpsC and dpsD, encoding proteins that are believed to function as the starter unit-specifying enzymes. Recombinant strains containing plasmids carrying dpsC and dpsD, in addition to other daunorubicin polyketide synthase (PKS) genes, incorporate the correct starter unit into polyketides made by these genes, suggesting that, contrary to earlier reports, the enzymes encoded by dpsC and dpsD play a crucial role in starter unit specification. Additionally, the results of a cell-free synthesis of 21-carbon polyketides from propionyl-CoA and malonyl-CoA that used the protein extracts of recombinant strains carrying other daunorubicin PKS genes to which purified DpsC was added suggest that this enzyme has the primary role in starter unit discrimination for daunorubicin biosynthesis. (+info)
Polyketide synthase acyl carrier protein (ACP) as a substrate and a catalyst for malonyl ACP biosynthesis.
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BACKGROUND: Using an acyl-acyl carrier protein (ACP) as a starter unit, type II polyketide synthases (PKSs) generate a wide range of polyketide products by successive decarboxylative condensations with the two-carbon donor malonyl (ACP). In vitro experiments have demonstrated that polyketide biosynthesis in reconstituted PKS systems requires the fatty acid synthase (FAS) enzyme malonyl CoA:ACP acyltransferase (FabD) from streptomycetes. It has also been shown that holo-ACPs from a type II PKS can catalyze self-malonylation in the presence of malonyl CoA and negate this FabD requirement. The relative roles of FabD and ACP self-malonylation in PKS biosynthesis in vivo are still not known. RESULTS: We have examined the ACP specificity of the Streptomyces glaucescens FabD and shown that it reacts specifically with monomeric forms of ACP, with comparable k(cat)/K(M) values for ACPs from both type II PKS and FAS systems. Incubations of tetracenomycin ACP (TcmM) with the Escherichia coli FAS ACP (AcpP) unexpectedly revealed that, in addition to the self-malonylation process, TcmM can catalyze the malonylation of AcpP. The k(cat)/K(M) value for the TcmM-catalyzed malonylation of S. glaucescens FAS ACP is two orders of magnitude smaller than that observed for the FabD-catalyzed process. CONCLUSIONS: The ability of a PKS ACP to catalyze malonylation of a FAS ACP is a surprising finding and demonstrates for the first time that PKS ACPs and FabD can catalyze the same reaction. The differences in the catalytic efficiency of these two proteins rationalizes in vitro observations that FabD-independent polyketide biosynthesis proceeds only at high concentrations of a PKS ACP. (+info)