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Dolors Serra - One of the best experts on this subject based on the ideXlab platform.
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sensing of nutrients by cpt1c regulates late endosome lysosome anterograde transport and axon growth
eLife, 2019Co-Authors: Marta Palomoguerrero, Rut Fado, Maria Casas, Marta Perezmontero, Miguel Baena, Patrick O Helmer, Jose Luis Dominguez, Aina Roig, Dolors SerraAbstract:Anterograde transport of late endosomes or lysosomes (LE/Lys) is crucial for proper axon growth. However, the role of energetic nutrients has been poorly explored. Malonyl-CoA is a precursor of fatty acids, and its intracellular levels highly fluctuate depending on glucose availability or the energy sensor AMP-activated protein kinase (AMPK). We demonstrate in HeLa cells that carnitine palmitoyltransferase 1C (CPT1C) senses Malonyl-CoA and enhances LE/Lys anterograde transport by interacting with the endoplasmic reticulum protein protrudin and facilitating the transfer of Kinesin-1 from protrudin to LE/Lys. In cultured mouse cortical neurons, glucose deprivation, pharmacological activation of AMPK or inhibition of Malonyl-CoA synthesis decreases LE/Lys abundance at the axon terminal, and shortens axon length in a CPT1C-dependent manner. These results identify CPT1C as a new regulator of anterograde LE/Lys transport in response to Malonyl-CoA changes, and give insight into how axon growth is controlled by nutrients.
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definition by functional and structural analysis of two malonyl coa sites in carnitine palmitoyltransferase 1a
Journal of Biological Chemistry, 2007Co-Authors: Montserrat Morillas, Eduardo Lopezvinas, Assia Bentebibel, Chandrashekaran Gurunathan, Dolores De Arriaga, Dolors SerraAbstract: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.
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alteration of the malonyl coa carnitine palmitoyltransferase i interaction in the β cell impairs glucose induced insulin secretion
Diabetes, 2005Co-Authors: Laura Herrero, Dolors Serra, David Sebastian, Guillermina Asins, Blanca Rubi, Pierre Maechler, Marc Prentki, Fausto G HegardtAbstract:Carnitine palmitoyltransferase I, which is expressed in the pancreas as the liver isoform (LCPTI), catalyzes the rate-limiting step in the transport of fatty acids into the mitochondria for their oxidation. Malonyl-CoA derived from glucose metabolism regulates fatty acid oxidation by inhibiting LCPTI. To examine directly whether the availability of long-chain fatty acyl-CoA (LC-CoA) affects the regulation of insulin secretion in the β-cell and whether Malonyl-CoA may act as a metabolic coupling factor in the β-cell, we infected INS(832/13) cells and rat islets with an adenovirus encoding a mutant form of LCPTI (Ad-LCPTI M593S) that is insensitive to Malonyl-CoA. In Ad-LCPTI M593S–infected INS(832/13) cells, LCPTI activity increased sixfold. This was associated with enhanced fatty acid oxidation, at any glucose concentration, and a 60% suppression of glucose-stimulated insulin secretion (GSIS). In isolated rat islets in which LCPTI M593S was overexpressed, GSIS decreased 40%. The impairment of GSIS in Ad-LCPTI M593S–infected INS(832/13) cells was not recovered when cells were incubated with 0.25 mmol/l palmitate, indicating the deep metabolic influence of a nonregulated fatty acid oxidation system. At high glucose concentration, overexpression of a Malonyl-CoA–insensitive form of LCPTI reduced partitioning of exogenous palmitate into lipid esterification products and decreased protein kinase C activation. Moreover, LCPTI M593S expression impaired KATP channel–independent GSIS in INS(832/13) cells. The LCPTI M593S mutant caused more pronounced alterations in GSIS and lipid partitioning (fat oxidation, esterification, and the level of nonesterified palmitate) than LCPTI wt in INS(832/13) cells that were transduced with these constructs. The results provide direct support for the hypothesis that the Malonyl-CoA/CPTI interaction is a component of a metabolic signaling network that controls insulin secretion.
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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
Journal of Biological Chemistry, 2003Co-Authors: Montserrat Morillas, Guillermina Asins, Fausto G Hegardt, Paulino Gomezpuertas, Alfonso Valencia, Assia Bentebibel, Eva Selles, Nuria Casals, Dolors SerraAbstract:Abstract 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 (Thr314, Asn464, Ala478, Met593, and Cys608, 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 Met593mutants were prepared: M593A and M593E. The catalytic efficiency (V max/K m) of CPT I in mutants A478G and C608A and all Met593 mutants toward carnitine as substrate was clearly increased. In those CPT I proteins in which Met593 had been mutated, the Malonyl-CoA sensitivity was nearly abolished. Mutations in Ala478, Cys608, and Thr314 to their homologous amino acid residues in CPT II, CAT, and ChAT caused various decreases in Malonyl-CoA sensitivity. Ala478 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., Gomez-Puertas, P., Rubi, B., Clotet, J., Arino, J., Valencia, A., Hegardt, F. G., Serra, D., and Asins, G. (2002) J. Biol. Chem. 277, 11473–11480). Met593 may participate in the interaction of Malonyl-CoA in the second affinity site, whose location has not been reported.
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structural model of a malonyl coa binding site of carnitine octanoyltransferase and carnitine palmitoyltransferase i mutational analysis of a malonyl coa affinity domain
Journal of Biological Chemistry, 2002Co-Authors: Montserrat Morillas, Dolors Serra, Fausto G Hegardt, Blanca Rubi, Paulino Gomezpuertas, Josep Clotet, Joaquin Arino, Alfonso Valencia, Guillermina AsinsAbstract:Abstract 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., Gomez-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 His340, implicated together with His131 in COT Malonyl-CoA sensitivity (Morillas, M., Clotet, J., Rubi, B., Serra, D., Asins, G., Arino, J., and Hegardt F. G. (2000) FEBS Lett. 466, 183–186). When we mutated COT His131 the IC50increased, and Malonyl-CoA competed with the substrate decanoyl-CoA. Mutation of COT Ala332, present in the domain 8 amino acids away from His340, decreased the Malonyl-CoA sensitivity of COT. The homologous histidine and alanine residues of L-CPT I, His277, His483, and Ala478 were also mutated, which decreased Malonyl-CoA sensitivity. Natural mutation of Pro479, 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.
Gebre Woldegiorgis - One of the best experts on this subject based on the ideXlab platform.
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substitution of glutamate 3 valine 19 leucine 23 and serine 24 with alanine in the n terminal region of human heart muscle carnitine palmitoyltransferase i abolishes malonyl coa inhibition and binding
Archives of Biochemistry and Biophysics, 2003Co-Authors: Hongfa Zhu, Jianying Shi, Dennis N Arvidson, Michelle Treber, Jia Dai, Gebre WoldegiorgisAbstract:Abstract The muscle isoform of carnitine palmitoyltransferase I (M-CPTI) is 30- to 100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). We have previously shown that deletion of the first 28 N-terminal amino acid residues in M-CPTI abolished malonyl CoA inhibition and high-affinity binding [Biochemistry 39 (2000) 712–717]. To determine the role of specific residues within the first 28 N-terminal amino acids of human heart M-CPTI on malonyl CoA sensitivity and binding, we constructed a series of substitution mutations and a mutant M-CPTI composed of deletion 18 combined with substitution mutations V19A, L23A, and S24A. All mutants had CPT activity similar to that of the wild type. A change of Glu3 to Ala resulted in a 60-fold decrease in malonyl CoA sensitivity and loss of high-affinity malonyl CoA binding. A change of His5 to Ala in M-CPTI resulted in only a 2-fold decrease in malonyl CoA sensitivity and a significant loss in the low- but not high-affinity malonyl CoA binding. Deletion of the first 18 N-terminal residues combined with substitution mutations V19A, L23A, and S24A resulted in a mutant M-CPTI with an over 140-fold decrease in malonyl CoA sensitivity and a significant loss in both high- and low-affinity malonyl CoA binding. This was further confirmed by a combined four-residue substitution of Glu3, Val19, Leu23, and Ser24 with alanine. Our site-directed mutagenesis studies demonstrate that Glu3, Val19, Leu23, and Ser24 in M-CPTI are important for malonyl CoA inhibition and binding, but not for catalysis.
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identification by mutagenesis of conserved arginine and glutamate residues in the c terminal domain of rat liver carnitine palmitoyltransferase i that are important for catalytic activity and malonyl coa sensitivity
Journal of Biological Chemistry, 2003Co-Authors: Michelle Treber, Gebre WoldegiorgisAbstract:Abstract Carnitine palmitoyltransferase I (CPTI) catalyzes the conversion of long chain fatty acyl-CoAs to acylcarnitines in the presence of l-carnitine. To determine the role of the conserved glutamate residue, Glu-603, on catalysis and Malonyl-CoA sensitivity, we separately changed the residue to alanine, histidine, glutamine, and aspartate. Substitution of Glu-603 with alanine or histidine resulted in complete loss of L-CPTI activity. A change of Glu-603 to glutamine caused a significant decrease in catalytic activity and Malonyl-CoA sensitivity. Substitution of Glu-603 with aspartate, a negatively charged amino acid with only one methyl group less than the glutamate residue in the wild type enzyme, resulted in partial loss in CPTI activity and a 15-fold decrease in Malonyl-CoA sensitivity. The mutant L-CPTI with a replacement of the conserved Arg-601 or Arg-606 with alanine also showed over 40-fold decrease in Malonyl-CoA sensitivity, suggesting that these two conserved residues may be important for substrate and inhibitor binding. Since a conservative substitution of Glu-603 to aspartate or glutamine resulted in partial loss of activity and Malonyl-CoA sensitivity, it further suggests that the negative charge and the longer side chain of glutamate are essential for catalysis and Malonyl-CoA sensitivity. We predict that this region of L-CPTI spanning these conserved C-terminal residues may be the region of the protein involved in binding the CoA moiety of palmitoyl-CoA and Malonyl-CoA and/or the putative low affinity acyl-CoA/Malonyl-CoA binding site.
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the first 28 n terminal amino acid residues of human heart muscle carnitine palmitoyltransferase i are essential for malonyl coa sensitivity and high affinity binding
Biochemistry, 2000Co-Authors: Jianying Shi, Hongfa Zhu, Dennis N Arvidson, Gebre WoldegiorgisAbstract:Heart/skeletal muscle carnitine palmitoyltransferase I (M-CPTI) is 30−100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). To determine the role of the N-terminal region of human heart M-CPTI on malonyl CoA sensitivity and binding, a series of deletion mutations were constructed ranging in size from 18 to 83 N-terminal residues. All of the deletions except Δ83 were active. Mitochondria from the yeast strains expressing Δ28 and Δ39 exhibited a 2.5-fold higher activity compared to the wild type, but were insensitive to malonyl CoA inhibition and had complete loss of high-affinity malonyl CoA binding. The high-affinity site (KD1, Bmax1) for binding of malonyl CoA to M-CPTI was completely abolished in the Δ28, Δ39, Δ51, and Δ72 mutants, suggesting that the decrease in malonyl CoA sensitivity observed in these mutants was due to the loss of the high-affinity binding entity of the enzyme. Δ18 showed only a 4-fold loss in malonyl CoA sensitivity but had activity and high-affinity ma...
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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
Journal of Biological Chemistry, 1999Co-Authors: Jianying Shi, Hongfa Zhu, Dennis N Arvidson, Gebre WoldegiorgisAbstract: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, Δ12 and Δ6, 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.
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deletion of the conserved first 18 n terminal amino acid residues in rat liver carnitine palmitoyltransferase i abolishes malonyl coa sensitivity and binding
Biochemistry, 1998Co-Authors: Jianying Shi, Hongfa Zhu, James M Cregg, Dennis N Arvidson, Gebre WoldegiorgisAbstract:To assess the role of the 130 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) on Malonyl-CoA sensitivity and binding, we constructed a series of mutants with deletions of the 18, 35, 52, 73, 83, or 129 most N-terminal amino acid residues. The deletion mutants were expressed in the yeast Pichia pastoris. We determined the effects of these mutations on L-CPTI activity, Malonyl-CoA sensitivity, and binding in isolated mitochondria prepared from the yeast strains expressing the wild-type and deletion mutants. The mutant protein that lacked the first 18 N-terminal amino acid residues, Delta18, had activity and kinetic properties similar to wild-type L-CPTI, but it was almost completely insensitive to Malonyl-CoA inhibition (I50 = 380 microM versus 2.0 microM). In addition, loss of Malonyl-CoA sensitivity in Delta18 was accompanied by a 70-fold decrease in affinity for malonyl CoA (KD = 70 nM versus 1.1 nM) compared to wild-type L-CPTI. Deletion of the first 35, 52, 73, and 83 N-terminal amino acid residues had a similar effect on Malonyl-CoA sensitivity as did the 18-residue deletion mutant, and there was a progressive reduction in the affinity for Malonyl-CoA binding. By contrast, deletion of the first 129 N-terminal amino acid residues resulted in the synthesis of an inactive protein. To our knowledge, this is the first report to demonstrate a critical role for these perfectly conserved first 18 N-terminal amino acid residues of L-CPTI in Malonyl-CoA sensitivity and binding.
Jeremy R Lohman - One of the best experts on this subject based on the ideXlab platform.
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structures of lnmk a bifunctional acyltransferase decarboxylase with substrate analogues reveal the basis for selectivity and stereospecificity
Biochemistry, 2021Co-Authors: Lee M Stunkard, Benjamin J Kick, Jeremy R LohmanAbstract:LnmK stereospecifically accepts (2R)-methylMalonyl-CoA, generating propionyl-S-acyl carrier protein to support polyketide biosynthesis. LnmK and its homologues are the only known enzymes that carry out a decarboxylation (DC) and acyl transfer (AT) reaction in the same active site as revealed by structure-function studies. Substrate-assisted catalysis powers LnmK, as decarboxylation of (2R)-methylMalonyl-CoA generates an enolate capable of deprotonating active site Tyr62, and the Tyr62 phenolate subsequently attacks propionyl-CoA leading to a propionyl-O-LnmK acyl-enzyme intermediate. Due to the inherent reactivity of LnmK and methylMalonyl-CoA, a substrate-bound structure could not be obtained. To gain insight into substrate specificity, stereospecificity, and catalytic mechanism, we determined the structures of LnmK with bound substrate analogues that bear malonyl-thioester isosteres where the carboxylate is represented by a nitro or sulfonate group. The nitro-bearing malonyl-thioester isosteres bind in the nitronate form, with specific hydrogen bonds that allow modeling of the (2R)-methylMalonyl-CoA substrate and rationalization of stereospecificity. The sulfonate isosteres bind in multiple conformations, suggesting the large active site of LnmK allows multiple binding modes. Considering the smaller malonyl group has more conformational freedom than the methylmalonyl group, we hypothesized the active site can entropically screen against catalysis with the smaller Malonyl-CoA substrate. Indeed, our kinetic analysis reveals Malonyl-CoA is accepted at 1% of the rate of methylMalonyl-CoA. This study represents another example of how our nitro- and sulfonate-bearing methylmalonyl-thioester isosteres are of use for elucidating enzyme-substrate binding interactions and revealing insights into catalytic mechanism. Synthesis of a larger panel of analogues presents an opportunity to study enzymes with complicated structure-function relationships such as acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases, and β-ketoacylsynthases.
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sulfonate nitro bearing methylmalonyl thioester isosteres applied to methylmalonyl coa decarboxylase structure function studies
Journal of the American Chemical Society, 2019Co-Authors: Lee M Stunkard, Austin D Dixon, Tyler J Huth, Jeremy R LohmanAbstract:Malonyl-thioesters are reactive centers of Malonyl-CoA and malonyl- S-acyl carrier protein, essential to fatty acid, polyketide and various specialized metabolite biosynthesis. Enzymes that create or use malonyl-thioesters spontaneously hydrolyze or decarboxylate reactants on the crystallographic time frame preventing determination of structure-function relationships. To address this problem, we have synthesized a panel of methylMalonyl-CoA analogs with the carboxylate represented by a sulfonate or nitro and the thioester retained or represented by an ester or amide. Structures of Escherichia coli methylMalonyl-CoA decarboxylase in complex with our analogs affords insight into substrate binding and the catalytic mechanism. Counterintuitively, the negatively charged sulfonate and nitronate functional groups of our analogs bind in an active site hydrophobic pocket. Upon decarboxylation the enolate intermediate is protonated by a histidine preventing CO2-enolate recombination, yielding propionyl-CoA. Activity assays support a histidine catalytic acid and reveal the enzyme displays significant hydrolysis activity. Our structures also provide insight into this hydrolysis activity. Our analogs inhibit decarboxylation/hydrolysis activity with low micromolar Ki values. This study sets precedents for using Malonyl-CoA analogs with carboxyate isosteres to study the complicated structure-function relationships of acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases and β-ketoacylsynthases.
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Sulfonate/Nitro Bearing Methylmalonyl-Thioester Isosteres Applied to MethylMalonyl-CoA Decarboxylase Structure–Function Studies
2019Co-Authors: Lee M Stunkard, Austin D Dixon, Tyler J Huth, Jeremy R LohmanAbstract:Malonyl-thioesters are reactive centers of Malonyl-CoA and malonyl-S-acyl carrier protein, essential to fatty acid, polyketide and various specialized metabolite biosynthesis. Enzymes that create or use malonyl-thioesters spontaneously hydrolyze or decarboxylate reactants on the crystallographic time frame preventing determination of structure–function relationships. To address this problem, we have synthesized a panel of methylMalonyl-CoA analogs with the carboxylate represented by a sulfonate or nitro and the thioester retained or represented by an ester or amide. Structures of Escherichia coli methylMalonyl-CoA decarboxylase in complex with our analogs affords insight into substrate binding and the catalytic mechanism. Counterintuitively, the negatively charged sulfonate and nitronate functional groups of our analogs bind in an active site hydrophobic pocket. Upon decarboxylation the enolate intermediate is protonated by a histidine preventing CO2-enolate recombination, yielding propionyl-CoA. Activity assays support a histidine catalytic acid and reveal the enzyme displays significant hydrolysis activity. Our structures also provide insight into this hydrolysis activity. Our analogs inhibit decarboxylation/hydrolysis activity with low micromolar Ki values. This study sets precedents for using Malonyl-CoA analogs with carboxyate isosteres to study the complicated structure–function relationships of acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases and β-ketoacylsynthases
Jianying Shi - One of the best experts on this subject based on the ideXlab platform.
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substitution of glutamate 3 valine 19 leucine 23 and serine 24 with alanine in the n terminal region of human heart muscle carnitine palmitoyltransferase i abolishes malonyl coa inhibition and binding
Archives of Biochemistry and Biophysics, 2003Co-Authors: Hongfa Zhu, Jianying Shi, Dennis N Arvidson, Michelle Treber, Jia Dai, Gebre WoldegiorgisAbstract:Abstract The muscle isoform of carnitine palmitoyltransferase I (M-CPTI) is 30- to 100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). We have previously shown that deletion of the first 28 N-terminal amino acid residues in M-CPTI abolished malonyl CoA inhibition and high-affinity binding [Biochemistry 39 (2000) 712–717]. To determine the role of specific residues within the first 28 N-terminal amino acids of human heart M-CPTI on malonyl CoA sensitivity and binding, we constructed a series of substitution mutations and a mutant M-CPTI composed of deletion 18 combined with substitution mutations V19A, L23A, and S24A. All mutants had CPT activity similar to that of the wild type. A change of Glu3 to Ala resulted in a 60-fold decrease in malonyl CoA sensitivity and loss of high-affinity malonyl CoA binding. A change of His5 to Ala in M-CPTI resulted in only a 2-fold decrease in malonyl CoA sensitivity and a significant loss in the low- but not high-affinity malonyl CoA binding. Deletion of the first 18 N-terminal residues combined with substitution mutations V19A, L23A, and S24A resulted in a mutant M-CPTI with an over 140-fold decrease in malonyl CoA sensitivity and a significant loss in both high- and low-affinity malonyl CoA binding. This was further confirmed by a combined four-residue substitution of Glu3, Val19, Leu23, and Ser24 with alanine. Our site-directed mutagenesis studies demonstrate that Glu3, Val19, Leu23, and Ser24 in M-CPTI are important for malonyl CoA inhibition and binding, but not for catalysis.
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the first 28 n terminal amino acid residues of human heart muscle carnitine palmitoyltransferase i are essential for malonyl coa sensitivity and high affinity binding
Biochemistry, 2000Co-Authors: Jianying Shi, Hongfa Zhu, Dennis N Arvidson, Gebre WoldegiorgisAbstract:Heart/skeletal muscle carnitine palmitoyltransferase I (M-CPTI) is 30−100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). To determine the role of the N-terminal region of human heart M-CPTI on malonyl CoA sensitivity and binding, a series of deletion mutations were constructed ranging in size from 18 to 83 N-terminal residues. All of the deletions except Δ83 were active. Mitochondria from the yeast strains expressing Δ28 and Δ39 exhibited a 2.5-fold higher activity compared to the wild type, but were insensitive to malonyl CoA inhibition and had complete loss of high-affinity malonyl CoA binding. The high-affinity site (KD1, Bmax1) for binding of malonyl CoA to M-CPTI was completely abolished in the Δ28, Δ39, Δ51, and Δ72 mutants, suggesting that the decrease in malonyl CoA sensitivity observed in these mutants was due to the loss of the high-affinity binding entity of the enzyme. Δ18 showed only a 4-fold loss in malonyl CoA sensitivity but had activity and high-affinity ma...
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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
Journal of Biological Chemistry, 1999Co-Authors: Jianying Shi, Hongfa Zhu, Dennis N Arvidson, Gebre WoldegiorgisAbstract: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, Δ12 and Δ6, 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.
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deletion of the conserved first 18 n terminal amino acid residues in rat liver carnitine palmitoyltransferase i abolishes malonyl coa sensitivity and binding
Biochemistry, 1998Co-Authors: Jianying Shi, Hongfa Zhu, James M Cregg, Dennis N Arvidson, Gebre WoldegiorgisAbstract:To assess the role of the 130 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) on Malonyl-CoA sensitivity and binding, we constructed a series of mutants with deletions of the 18, 35, 52, 73, 83, or 129 most N-terminal amino acid residues. The deletion mutants were expressed in the yeast Pichia pastoris. We determined the effects of these mutations on L-CPTI activity, Malonyl-CoA sensitivity, and binding in isolated mitochondria prepared from the yeast strains expressing the wild-type and deletion mutants. The mutant protein that lacked the first 18 N-terminal amino acid residues, Delta18, had activity and kinetic properties similar to wild-type L-CPTI, but it was almost completely insensitive to Malonyl-CoA inhibition (I50 = 380 microM versus 2.0 microM). In addition, loss of Malonyl-CoA sensitivity in Delta18 was accompanied by a 70-fold decrease in affinity for malonyl CoA (KD = 70 nM versus 1.1 nM) compared to wild-type L-CPTI. Deletion of the first 35, 52, 73, and 83 N-terminal amino acid residues had a similar effect on Malonyl-CoA sensitivity as did the 18-residue deletion mutant, and there was a progressive reduction in the affinity for Malonyl-CoA binding. By contrast, deletion of the first 129 N-terminal amino acid residues resulted in the synthesis of an inactive protein. To our knowledge, this is the first report to demonstrate a critical role for these perfectly conserved first 18 N-terminal amino acid residues of L-CPTI in Malonyl-CoA sensitivity and binding.
Lee M Stunkard - One of the best experts on this subject based on the ideXlab platform.
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structures of lnmk a bifunctional acyltransferase decarboxylase with substrate analogues reveal the basis for selectivity and stereospecificity
Biochemistry, 2021Co-Authors: Lee M Stunkard, Benjamin J Kick, Jeremy R LohmanAbstract:LnmK stereospecifically accepts (2R)-methylMalonyl-CoA, generating propionyl-S-acyl carrier protein to support polyketide biosynthesis. LnmK and its homologues are the only known enzymes that carry out a decarboxylation (DC) and acyl transfer (AT) reaction in the same active site as revealed by structure-function studies. Substrate-assisted catalysis powers LnmK, as decarboxylation of (2R)-methylMalonyl-CoA generates an enolate capable of deprotonating active site Tyr62, and the Tyr62 phenolate subsequently attacks propionyl-CoA leading to a propionyl-O-LnmK acyl-enzyme intermediate. Due to the inherent reactivity of LnmK and methylMalonyl-CoA, a substrate-bound structure could not be obtained. To gain insight into substrate specificity, stereospecificity, and catalytic mechanism, we determined the structures of LnmK with bound substrate analogues that bear malonyl-thioester isosteres where the carboxylate is represented by a nitro or sulfonate group. The nitro-bearing malonyl-thioester isosteres bind in the nitronate form, with specific hydrogen bonds that allow modeling of the (2R)-methylMalonyl-CoA substrate and rationalization of stereospecificity. The sulfonate isosteres bind in multiple conformations, suggesting the large active site of LnmK allows multiple binding modes. Considering the smaller malonyl group has more conformational freedom than the methylmalonyl group, we hypothesized the active site can entropically screen against catalysis with the smaller Malonyl-CoA substrate. Indeed, our kinetic analysis reveals Malonyl-CoA is accepted at 1% of the rate of methylMalonyl-CoA. This study represents another example of how our nitro- and sulfonate-bearing methylmalonyl-thioester isosteres are of use for elucidating enzyme-substrate binding interactions and revealing insights into catalytic mechanism. Synthesis of a larger panel of analogues presents an opportunity to study enzymes with complicated structure-function relationships such as acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases, and β-ketoacylsynthases.
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sulfonate nitro bearing methylmalonyl thioester isosteres applied to methylmalonyl coa decarboxylase structure function studies
Journal of the American Chemical Society, 2019Co-Authors: Lee M Stunkard, Austin D Dixon, Tyler J Huth, Jeremy R LohmanAbstract:Malonyl-thioesters are reactive centers of Malonyl-CoA and malonyl- S-acyl carrier protein, essential to fatty acid, polyketide and various specialized metabolite biosynthesis. Enzymes that create or use malonyl-thioesters spontaneously hydrolyze or decarboxylate reactants on the crystallographic time frame preventing determination of structure-function relationships. To address this problem, we have synthesized a panel of methylMalonyl-CoA analogs with the carboxylate represented by a sulfonate or nitro and the thioester retained or represented by an ester or amide. Structures of Escherichia coli methylMalonyl-CoA decarboxylase in complex with our analogs affords insight into substrate binding and the catalytic mechanism. Counterintuitively, the negatively charged sulfonate and nitronate functional groups of our analogs bind in an active site hydrophobic pocket. Upon decarboxylation the enolate intermediate is protonated by a histidine preventing CO2-enolate recombination, yielding propionyl-CoA. Activity assays support a histidine catalytic acid and reveal the enzyme displays significant hydrolysis activity. Our structures also provide insight into this hydrolysis activity. Our analogs inhibit decarboxylation/hydrolysis activity with low micromolar Ki values. This study sets precedents for using Malonyl-CoA analogs with carboxyate isosteres to study the complicated structure-function relationships of acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases and β-ketoacylsynthases.
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Sulfonate/Nitro Bearing Methylmalonyl-Thioester Isosteres Applied to MethylMalonyl-CoA Decarboxylase Structure–Function Studies
2019Co-Authors: Lee M Stunkard, Austin D Dixon, Tyler J Huth, Jeremy R LohmanAbstract:Malonyl-thioesters are reactive centers of Malonyl-CoA and malonyl-S-acyl carrier protein, essential to fatty acid, polyketide and various specialized metabolite biosynthesis. Enzymes that create or use malonyl-thioesters spontaneously hydrolyze or decarboxylate reactants on the crystallographic time frame preventing determination of structure–function relationships. To address this problem, we have synthesized a panel of methylMalonyl-CoA analogs with the carboxylate represented by a sulfonate or nitro and the thioester retained or represented by an ester or amide. Structures of Escherichia coli methylMalonyl-CoA decarboxylase in complex with our analogs affords insight into substrate binding and the catalytic mechanism. Counterintuitively, the negatively charged sulfonate and nitronate functional groups of our analogs bind in an active site hydrophobic pocket. Upon decarboxylation the enolate intermediate is protonated by a histidine preventing CO2-enolate recombination, yielding propionyl-CoA. Activity assays support a histidine catalytic acid and reveal the enzyme displays significant hydrolysis activity. Our structures also provide insight into this hydrolysis activity. Our analogs inhibit decarboxylation/hydrolysis activity with low micromolar Ki values. This study sets precedents for using Malonyl-CoA analogs with carboxyate isosteres to study the complicated structure–function relationships of acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases and β-ketoacylsynthases