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Liang Tong - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the 500 kda yeast acetyl coa Carboxylase holoenzyme dimer
Nature, 2015Co-Authors: Jia Wei, Liang TongAbstract:Acetyl-CoA Carboxylase (ACC) has crucial roles in fatty acid metabolism and is an attractive target for drug discovery against diabetes, cancer and other diseases. Saccharomyces cerevisiae ACC (ScACC) is crucial for the production of very-long-chain fatty acids and the maintenance of the nuclear envelope. ACC contains biotin Carboxylase (BC) and carboxyltransferase (CT) activities, and its biotin is linked covalently to the biotin carboxyl carrier protein (BCCP). Most eukaryotic ACCs are 250-kilodalton (kDa), multi-domain enzymes and function as homodimers and higher oligomers. They contain a unique, 80-kDa central region that shares no homology with other proteins. Although the structures of the BC, CT and BCCP domains and other biotin-dependent Carboxylase holoenzymes are known, there is currently no structural information on the ACC holoenzyme. Here we report the crystal structure of the full-length, 500-kDa holoenzyme dimer of ScACC. The structure is remarkably different from that of the other biotin-dependent Carboxylases. The central region contains five domains and is important for positioning the BC and CT domains for catalysis. The structure unexpectedly reveals a dimer of the BC domain and extensive conformational differences compared to the structure of the BC domain alone, which is a monomer. These structural changes reveal why the BC domain alone is catalytically inactive and define the molecular mechanism for the inhibition of eukaryotic ACC by the natural product soraphen A and by phosphorylation of a Ser residue just before the BC domain core in mammalian ACC. The BC and CT active sites are separated by 80 A, and the entire BCCP domain must translocate during catalysis.
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structure and function of a single chain multi domain long chain acyl coa Carboxylase
Nature, 2014Co-Authors: Timothy H Tran, Yushan Hsiao, Chiyuan Chou, Lars E P Dietrich, Thomas Walz, Liang TongAbstract:A novel biotin-dependent Carboxylase with a preference for long-chain acyl-CoA substrates and a unique holoenzyme architecture is described. Mutations in biotin-dependent Carboxylases have been linked to serious metabolic diseases in humans, and acetyl-CoA Carboxylase is a target for drug discovery against diabetes, cancer and other diseases. In this manuscript, the authors identified and characterized an unusual biotin-dependent Carboxylase found in bacteria. The enzyme has a preference for long-chain acyl-CoA substrates and a unique holoenzyme architecture. X-ray crystal and cryo-electron microscopy structures reveal a large hexameric enzyme in which the domains of each monomer are extensively swapped within the holoenzyme, suggesting that each cycle of catalysis requires the participation of domains from four different monomers. Functional studies in Pseudomonas aeruginosa suggest that the enzyme is involved in the utilization of specific carbon and nitrogen sources. Biotin-dependent Carboxylases are widely distributed in nature and have important functions in the metabolism of fatty acids, amino acids, carbohydrates, cholesterol and other compounds1,2,3,4,5,6. Defective mutations in several of these enzymes have been linked to serious metabolic diseases in humans, and acetyl-CoA Carboxylase is a target for drug discovery in the treatment of diabetes, cancer and other diseases7,8,9. Here we report the identification and biochemical, structural and functional characterizations of a novel single-chain (120 kDa), multi-domain biotin-dependent Carboxylase in bacteria. It has preference for long-chain acyl-CoA substrates, although it is also active towards short-chain and medium-chain acyl-CoAs, and we have named it long-chain acyl-CoA Carboxylase. The holoenzyme is a homo-hexamer with molecular mass of 720 kDa. The 3.0 A crystal structure of the long-chain acyl-CoA Carboxylase holoenzyme from Mycobacterium avium subspecies paratuberculosis revealed an architecture that is strikingly different from those of related biotin-dependent Carboxylases10,11. In addition, the domains of each monomer have no direct contact with each other. They are instead extensively swapped in the holoenzyme, such that one cycle of catalysis involves the participation of four monomers. Functional studies in Pseudomonas aeruginosa suggest that the enzyme is involved in the utilization of selected carbon and nitrogen sources.
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Structure and function of biotin-dependent Carboxylases
Cellular and Molecular Life Sciences, 2013Co-Authors: Liang TongAbstract:Biotin-dependent Carboxylases include acetyl-CoA Carboxylase (ACC), propionyl-CoA Carboxylase (PCC), 3-methylcrotonyl-CoA Carboxylase (MCC), geranyl-CoA Carboxylase, pyruvate Carboxylase (PC), and urea Carboxylase (UC). They contain biotin Carboxylase (BC), carboxyltransferase (CT), and biotin-carboxyl carrier protein components. These enzymes are widely distributed in nature and have important functions in fatty acid metabolism, amino acid metabolism, carbohydrate metabolism, polyketide biosynthesis, urea utilization, and other cellular processes. ACCs are also attractive targets for drug discovery against type 2 diabetes, obesity, cancer, microbial infections, and other diseases, and the plastid ACC of grasses is the target of action of three classes of commercial herbicides. Deficiencies in the activities of PCC, MCC, or PC are linked to serious diseases in humans. Our understanding of these enzymes has been greatly enhanced over the past few years by the crystal structures of the holoenzymes of PCC, MCC, PC, and UC. The structures reveal unanticipated features in the architectures of the holoenzymes, including the presence of previously unrecognized domains, and provide a molecular basis for understanding their catalytic mechanism as well as the large collection of disease-causing mutations in PCC, MCC, and PC. This review will summarize the recent advances in our knowledge on the structure and function of these important metabolic enzymes.
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an unanticipated architecture of the 750 kda α6β6 holoenzyme of 3 methylcrotonyl coa Carboxylase
Nature, 2012Co-Authors: Christine S Huang, Hong Z Zhou, Liang TongAbstract:3-Methylcrotonyl-CoA Carboxylase (MCC), a member of the biotin-dependent Carboxylase superfamily, is essential for the metabolism of leucine, and deficient mutations in this enzyme are linked to methylcrotonylglycinuria (MCG) and other serious diseases in humans. MCC has strong sequence conservation with propionyl-CoA Carboxylase (PCC), and their holoenzymes are both 750-kilodalton (kDa) α(6)β(6) dodecamers. Therefore the architecture of the MCC holoenzyme is expected to be highly similar to that of PCC. Here we report the crystal structures of the Pseudomonas aeruginosa MCC (PaMCC) holoenzyme, alone and in complex with coenzyme A. Surprisingly, the structures show that the architecture and overall shape of PaMCC are markedly different when compared to PCC. The α-subunits show trimeric association in the PaMCC holoenzyme, whereas they have no contacts with each other in PCC. Moreover, the positions of the two domains in the β-subunit of PaMCC are swapped relative to those in PCC. This structural information establishes a foundation for understanding the disease-causing mutations of MCC and provides new insights into the catalytic mechanism and evolution of biotin-dependent Carboxylases. The large structural differences between MCC and PCC also have general implications for the relationship between sequence conservation and structural similarity.
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acetyl coenzyme a Carboxylase crucial metabolic enzyme and attractive target for drug discovery
Cellular and Molecular Life Sciences, 2005Co-Authors: Liang TongAbstract:Acetyl-coenzyme A Carboxylases (ACCs) have crucial roles in fatty acid metabolism in most living organisms. Mice deficient in ACC2 have continuous fatty acid oxidation and reduced body fat and body weight, validating this enzyme as a target for drug development against obesity, diabetes and other symptoms of the metabolic syndrome. ACC is a biotin-dependent enzyme and catalyzes the carboxylation of acetyl-CoA to produce malonyl-CoA through its two catalytic activities, biotin Carboxylase (BC) and carboxyltransferase (CT). ACC is a multi-subunit enzyme in most prokaryotes, whereas it is a large, multi-domain enzyme in most eukaryotes. The activity of the enzyme can be controlled at the transcriptional level as well as by small molecule modulators and covalent modification. This review will summarize the structural information that is now available for both the BC and CT enzymes, as well as the molecular mechanism of action of potent ACC inhibitors. The current intense research on these enzymes could lead to the development of novel therapies against metabolic syndrome and other diseases.
Grover L. Waldrop - One of the best experts on this subject based on the ideXlab platform.
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Complex Formation and Regulation of Escherichia coli Acetyl-CoA Carboxylase
Biochemistry, 2013Co-Authors: Tyler C. Broussard, Amanda E. Price, Susan M. Laborde, Grover L. WaldropAbstract:Acetyl-CoA Carboxylase is a biotin-dependent enzyme that catalyzes the regulated step in fatty acid synthesis. The bacterial form has three separate components: biotin Carboxylase, biotin carboxyl carrier protein (BCCP), and carboxyltransferase. Catalysis by acetyl-CoA Carboxylase proceeds via two half-reactions. In the first half-reaction, biotin Carboxylase catalyzes the ATP-dependent carboxylation of biotin, which is covalently attached to BCCP, to form carboxybiotin. In the second half-reaction, carboxyltransferase transfers the carboxyl group from carboxybiotin to acetyl-CoA to form malonyl-CoA. All biotin-dependent Carboxylases are proposed to have a two-site ping-pong mechanism in which the Carboxylase and transferase activities are separate and do not interact. This posits two hypotheses: either biotin Carboxylase and BCCP undergo the first half-reaction, BCCP dissociates, and then BCCP binds to carboxyltransferase, or all three constituents form an enzyme complex. To determine which hypothesis is correct, a steady-state enzyme kinetic analysis of Escherichia coli acetyl-CoA Carboxylase was conducted. The results indicated the two active sites of acetyl-CoA Carboxylase interact. Both in vitro and in vivo pull-down assays demonstrated that the three components of E. coli acetyl-CoA Carboxylase form a multimeric complex and that complex formation is unaffected by acetyl-CoA, AMPPNP, and mRNA encoding carboxyltransferase. The implications of these findings for the regulation of acetyl-CoA Carboxylase and fatty acid biosynthesis are discussed.
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The Three-Dimensional Structure of the Biotin Carboxylase-Biotin Carboxyl Carrier Protein Complex of E. coli Acetyl-CoA Carboxylase
Structure (London England : 1993), 2013Co-Authors: Tyler C. Broussard, Amanda E. Price, Matthew J. Kobe, Svetlana Pakhomova, David B. Neau, Tyler S. Champion, Grover L. WaldropAbstract:Acetyl-coenzyme A (acetyl-CoA) Carboxylase is a biotin-dependent, multifunctional enzyme that catalyzes the regulated step in fatty acid synthesis. The Escherichia coli enzyme is composed of a homodimeric biotin Carboxylase (BC), biotinylated biotin carboxyl carrier protein (BCCP), and an α2β2 heterotetrameric carboxyltransferase. This enzyme complex catalyzes two half-reactions to form malonyl-coenzyme A. BC and BCCP participate in the first half-reaction, whereas carboxyltransferase and BCCP are involved in the second. Three-dimensional structures have been reported for the individual subunits; however, the structural basis for how BCCP reacts with the Carboxylase or transferase is unknown. Therefore, we report here the crystal structure of E. coli BCCP complexed with BC to a resolution of 2.49 A. The protein-protein complex shows a unique quaternary structure and two distinct interfaces for each BCCP monomer. These BCCP binding sites are unique compared to phylogenetically related biotin-dependent Carboxylases and therefore provide novel targets for developing antibiotics against bacterial acetyl-CoA Carboxylase.
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Inhibition of biotin Carboxylase by a reaction intermediate analog: implications for the kinetic mechanism.
Biochemical and Biophysical Research Communications, 1999Co-Authors: Carol Z. Blanchard, David R. Amspacher, Robert M. Strongin, Grover L. WaldropAbstract:Abstract The first committed step in long-chain fatty acid synthesis is catalyzed by the multienzyme complex acetyl CoA Carboxylase. One component of the acetyl CoA Carboxylase complex is biotin Carboxylase which catalyzes the ATP-dependent carboxylation of biotin. The Escherichia coli form of biotin Carboxylase can be isolated from the other components of the acetyl CoA Carboxylase complex such that enzymatic activity is retained. The synthesis of a reaction intermediate analog inhibitor of biotin Carboxylase has been described recently ( Organic Lett. 1, 99–102, 1999). The inhibitor is formed by coupling phosphonoacetic acid to the 1′-N of biotin. In this paper the characterization of the inhibition of biotin Carboxylase by this reaction-intermediate analog is described. The analog showed competitive inhibition versus ATP with a slope inhibition constant of 8 mM. Noncompetitive inhibition was found for the analog versus biotin. Phosphonoacetate exhibited competitive inhibition with respect to ATP and noncompetitive inhibition versus bicarbonate. Biotin was found to be a noncompetitive substrate inhibitor of biotin Carboxylase. These data suggested that biotin Carboxylase had an ordered addition of substrates with ATP binding first followed by bicarbonate and then biotin.
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Synthesis of a reaction intermediate analogue of biotin-dependent Carboxylases via a selective derivatization of biotin.
Organic Letters, 1999Co-Authors: David R. Amspacher, Carol Z. Blanchard, Marcelo C. Saraiva, Grover L. Waldrop, Frank R. Fronczek, Robert M. StronginAbstract:An efficient and practical synthesis of 1, a unique reaction intermediate analogue of biotin-dependent Carboxylases, is described. The synthesis features a selective acylation of the 1‘-N of biotin. Target 1 inhibits the activity of the biotin Carboxylase component of acetyl CoA Carboxylase. It is the first known biotin-derived inhibitor of biotin Carboxylase and should promote new kinetic and structural studies of the biotin-dependent Carboxylases.
Robert Haselkorn - One of the best experts on this subject based on the ideXlab platform.
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Wheat acetyl-CoA Carboxylase
Plant molecular biology, 1993Co-Authors: Piotr Gornicki, Robert HaselkornAbstract:The acetyl-CoA Carboxylase present in both wheat germ and total wheat leaf protein contains ca. 220 kDa subunits. It is the major biotin-dependent Carboxylase present in wheat chloroplasts. Active acetyl-CoA Carboxylase purified from wheat germ is a homodimer with an apparent molecular mass of ca. 500 kDa. The enzyme from wheat germ or from wheat chloroplasts is sensitive to the herbicide haloxyfop at micromolar levels. The incorporation of 14C-acetate into fatty acids in freshly cut wheat seedling leaves provides a convenient in vivo assay for both acetyl-CoA Carboxylase and haloxyfop.
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Genes for two subunits of acetyl coenzyme A Carboxylase of Anabaena sp. strain PCC 7120: biotin Carboxylase and biotin carboxyl carrier protein.
Journal of bacteriology, 1993Co-Authors: Piotr Gornicki, L A Scappino, Robert HaselkornAbstract:Genes for two subunits of acetyl-coenzyme A Carboxylase, biotin Carboxylase and biotin carboxyl carrier protein, have been cloned from Anabaena sp. strain PCC 7120. The two proteins are 181 and 447 amino acids long and show 40 and 57% identity to the corresponding Escherichia coli proteins, respectively. The sequence of the biotinylation site in Anabaena sp. strain PCC 7120 is MetLysLeu, not the MetLysMet found in other sequences of biotin-dependent Carboxylases. The amino acid sequence of biotin Carboxylase is also very similar (32 to 47% identity) to the sequence of the biotin Carboxylase domain of other biotin-dependent Carboxylases. Genes for these two subunits of acetyl-coenzyme A Carboxylase are not linked in Anabaena sp. strain PCC 7120, contrary to the situation in E. coli, in which they are in one operon.
Georg Fuchs - One of the best experts on this subject based on the ideXlab platform.
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carboxylation mechanism and stereochemistry of crotonyl coa Carboxylase reductase a carboxylating enoyl thioester reductase
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Tobias J Erb, Georg Fuchs, Volker Brecht, Michael Muller, Birgit E AlberAbstract:Chemo- and stereoselective reductions are important reactions in chemistry and biology, and reductases from biological sources are increasingly applied in organic synthesis. In contrast, Carboxylases are used only sporadically. We recently described crotonyl-CoA Carboxylase/reductase, which catalyzes the reduction of (E)-crotonyl-CoA to butyryl-CoA but also the reductive carboxylation of (E)-crotonyl-CoA to ethylmalonyl-CoA. In this study, the complete stereochemical course of both reactions was investigated in detail. The pro-(4R) hydrogen of NADPH is transferred in both reactions to the re face of the C3 position of crotonyl-CoA. In the course of the carboxylation reaction, carbon dioxide is incorporated in anti fashion at the C2 atom of crotonyl-CoA. For the reduction reaction that yields butyryl-CoA, a solvent proton is added in anti fashion instead of the CO(2). Amino acid sequence analysis showed that crotonyl-CoA Carboxylase/reductase is a member of the medium-chain dehydrogenase/reductase superfamily and shares the same phylogenetic origin. The stereospecificity of the hydride transfer from NAD(P)H within this superfamily is highly conserved, although the substrates and reduction reactions catalyzed by its individual representatives differ quite considerably. Our findings led to a reassessment of the stereospecificity of enoyl(-thioester) reductases and related enzymes with respect to their amino acid sequence, revealing a general pattern of stereospecificity that allows the prediction of the stereochemistry of the hydride transfer for enoyl reductases of unknown specificity. Further considerations on the reaction mechanism indicated that crotonyl-CoA Carboxylase/reductase may have evolved from enoyl-CoA reductases. This may be useful for protein engineering of enoyl reductases and their application in biocatalysis.
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Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic Crenarchaeota and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation
Journal of bacteriology, 1999Co-Authors: Cástor Menéndez, Zsuzsa Bauer, Harald Huber, Nasser Gad'on, Karl-otto Stetter, Georg FuchsAbstract:The pathway of autotrophic CO2 fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA Carboxylase and propionyl-CoA Carboxylase as well as phosphoenolpyruvate Carboxylase. The specific enzyme activities of the Carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO2-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either 14CO2 or [14C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed for C. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus and Acidianus infernus, which showed acetyl-CoA and propionyl-CoA Carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA Carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA Carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula, S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA Carboxylase and propionyl-CoA Carboxylase are proposed to be the main CO2 fixation enzymes, and phosphoenolpyruvate Carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus.
Joanne Stubbe - One of the best experts on this subject based on the ideXlab platform.
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evidence for the direct transfer of the carboxylate of n5 carboxyaminoimidazole ribonucleotide n5 cair to generate 4 carboxy 5 aminoimidazole ribonucleotide catalyzed by escherichia coli pure an n5 cair mutase
Biochemistry, 1999Co-Authors: E Meyer, T J Kappock, C Osuji, Joanne StubbeAbstract:Formation of 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) in the purine pathway in most prokaryotes requires ATP, HCO3-, aminoimidazole ribonucleotide (AIR), and the gene products PurK and PurE. PurK catalyzes the conversion of AIR to N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) in a reaction that requires both ATP and HCO3-. PurE catalyzes the unusual rearrangement of N5-CAIR to CAIR. To investigate the mechanism of this rearrangement, [4,7-13C]-N5-CAIR and [7-14C]-N5-CAIR were synthesized and separately incubated with PurE in the presence of ATP, aspartate, and 4-(N-succinocarboxamide)-5-aminoimidazole ribonucleotide (SAICAR) synthetase (PurC). The SAICAR produced was isolated and analyzed by NMR spectroscopy or scintillation counting, respectively. The PurC trapping of CAIR as SAICAR was required because of the reversibility of the PurE reaction. Results from both experiments reveal that the carboxylate group of the carbamate of N5-CAIR is transferred directly to generate CAIR without equilibration with CO2/HCO3- in solution. The mechanistic implications of these results relative to the PurE-only (CO2- and AIR-requiring) AIR Carboxylases are discussed.