The Experts below are selected from a list of 438 Experts worldwide ranked by ideXlab platform
Grover L. Waldrop - One of the best experts on this subject based on the ideXlab platform.
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Crystal Structure of Carboxyltransferase from Staphylococcus aureus Bound to the Antibacterial Agent Moiramide B.
Biochemistry, 2016Co-Authors: Molly A. Silvers, Svetlana Pakhomova, David B. Neau, William Silvers, Nicholas Anzalone, Carol M. Taylor, Grover L. WaldropAbstract:The dramatic increase in the prevalence of antibiotic-resistant bacteria has necessitated a search for new antibacterial agents against novel targets. Moiramide B is a natural product, broad-spectrum antibiotic that inhibits the Carboxyltransferase component of acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid synthesis. Herein, we report the 2.6 A resolution crystal structure of moiramide B bound to Carboxyltransferase. An unanticipated but significant finding was that moiramide B bound as the enol/enolate. Crystallographic studies demonstrate that the (4S)-methyl succinimide moiety interacts with the oxyanion holes of the enzyme, supporting the notion that an anionic enolate is the active form of the antibacterial agent. Structure–activity studies demonstrate that the unsaturated fatty acid tail of moiramide B is needed only for entry into the bacterial cell. These results will allow the design of new antibacterial agents against the bacterial form of Carboxyltransferase.
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Crystal Structure of Carboxyltransferase from Staphylococcus aureus Bound to the Antibacterial Agent Moiramide B
2016Co-Authors: Molly A. Silvers, Svetlana Pakhomova, David B. Neau, Nicholas Anzalone, William C. Silvers, Carol M. Taylor, Grover L. WaldropAbstract:The dramatic increase in the prevalence of antibiotic-resistant bacteria has necessitated a search for new antibacterial agents against novel targets. Moiramide B is a natural product, broad-spectrum antibiotic that inhibits the Carboxyltransferase component of acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid synthesis. Herein, we report the 2.6 Å resolution crystal structure of moiramide B bound to Carboxyltransferase. An unanticipated but significant finding was that moiramide B bound as the enol/enolate. Crystallographic studies demonstrate that the (4S)-methyl succinimide moiety interacts with the oxyanion holes of the enzyme, supporting the notion that an anionic enolate is the active form of the antibacterial agent. Structure–activity studies demonstrate that the unsaturated fatty acid tail of moiramide B is needed only for entry into the bacterial cell. These results will allow the design of new antibacterial agents against the bacterial form of Carboxyltransferase
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A tale of two functions: Enzymatic activity and translational repression by Carboxyltransferase. Nucleic Acids Res
2015Co-Authors: Glen Meades, Brian K. Benson, Anne Grove, Grover L. WaldropAbstract:Acetyl-CoA Carboxylase catalyzes the first commit-ted step in fatty acid synthesis. Escherichia coli acetyl-CoA carboxylase is composed of biotin carboxylase, Carboxyltransferase and biotin carboxyl carrier protein functions. The accA and accD genes that code for the a- and b-subunits, respectively, are not in an operon, yet yield an a2b2 Carboxyltransferase. Here, we report that Carboxyltransferase regulates its own translation by binding the mRNA encoding its subunits. This interaction is mediated by a zinc finger on the b-subunit; mutation of the four cysteines to alanine diminished nucleic acid binding and catalytic activity. Carboxyltransferase binds the coding regions of both subunit mRNAs and inhibits transla-tion, an inhibition that is relieved by the substrate acetyl-CoA. mRNA binding reciprocally inhibits catalytic activity. Preferential binding of carboxyl-transferase to RNA in situ was shown using fluorescence resonance energy transfer. We propose an unusual regulatory mechanism by which Carboxyltransferase acts as a ‘dimmer switch ’ to regulate protein production and catalytic activity, while sensing the metabolic state of the cell through acetyl-CoA concentration
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Design, Synthesis, and Antibacterial Properties of Dual-Ligand Inhibitors of Acetyl-CoA Carboxylase
2014Co-Authors: Molly A. Silvers, Carol M. Taylor, Gregory T. Robertson, Grover L. WaldropAbstract:There is an urgent demand for the development of new antibiotics due to the increase in drug-resistant pathogenic bacteria. A novel target is the multifunctional enzyme acetyl-CoA carboxylase (ACC), which catalyzes the first committed step in fatty acid synthesis and consists of two enzymes: biotin carboxylase and Carboxyltransferase. Covalently attaching known inhibitors against these enzymes with saturated hydrocarbon linkers of different lengths generated dual-ligand inhibitors. Kinetic results revealed that the dual-ligands inhibited the ACC complex in the nanomolar range. Microbiology assays showed that the dual-ligand with a 15-carbon linker did not exhibit any antibacterial activity, while the dual-ligand with a 7-carbon linker displayed broad-spectrum antibacterial activity as well as a decreased susceptibility in the development of bacterial resistance. These results suggest that the properties of the linker are vital for antibacterial activity and show how inhibiting two different enzymes with the same compound increases the overall potency while also impeding the development of resistance
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Mathematical modelling of negative feedback regulation by Carboxyltransferase
IET systems biology, 2011Co-Authors: Glen Meades, Grover L. Waldrop, Xiaoyu Cai, N.k. Thalji, M.s. De QueirozAbstract:Acetyl-CoA carboxylase catalyses the first committed step in fatty acid synthesis in all organisms. The chemistry is accomplished in two half-reactions: activation of biotin via carboxylation by biotin carboxylase, followed by the Carboxyltransferase-catalysed transfer of the carboxyl moiety from carboxybiotin to acetyl-CoA to generate malonyl-CoA. The Escherichia coli form of the Carboxyltransferase subunit was recently found to regulate its own activity and expression by binding its own mRNA. By binding acetyl-CoA or the mRNA encoding its own subunits, Carboxyltransferase is able to sense the metabolic state of the cell and attenuate its own translation and enzymatic activity using a negative feedback mechanism. Here, the network of these interactions is modelled mathematically with a set of non-linear differential equations. Numerical simulations of the model show that it qualitatively and quantitatively agrees with the experimental results for both inhibition of Carboxyltransferase by mRNA and attenuation of translation. The modelling of the autoregulatory function of Carboxyltransferase confirms that it is more than isolated interactions, but functions as a single dynamic system.
Martin St. Maurice - One of the best experts on this subject based on the ideXlab platform.
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The role of biotin and oxamate in the Carboxyltransferase reaction of pyruvate carboxylase.
Archives of biochemistry and biophysics, 2014Co-Authors: A.d. Lietzan, Yi Lin, Martin St. MauriceAbstract:Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. During catalysis, carboxybiotin is translocated to the Carboxyltransferase domain where the carboxyl group is transferred to the acceptor substrate, pyruvate. Many studies on the Carboxyltransferase domain of PC have demonstrated an enhanced oxaloacetate decarboxylation activity in the presence of oxamate and it has been shown that oxamate accepts a carboxyl group from carboxybiotin during oxaloacetate decarboxylation. The X-ray crystal structure of the Carboxyltransferase domain from Rhizobium etli PC reveals that oxamate is positioned in the active site in an identical manner to the substrate, pyruvate, and kinetic data are consistent with the oxamate-stimulated decarboxylation of oxaloacetate proceeding through a simple ping-pong bi bi mechanism in the absence of the biotin carboxylase domain. Additionally, analysis of truncated PC enzymes indicates that the BCCP domain devoid of biotin does not contribute directly to the enzymatic reaction and conclusively demonstrates a biotin-independent oxaloacetate decarboxylation activity in PC. These findings advance the description of catalysis in PC and can be extended to the study of related biotin-dependent enzymes.
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functionally diverse biotin dependent enzymes with oxaloacetate decarboxylase activity
Archives of Biochemistry and Biophysics, 2014Co-Authors: A.d. Lietzan, Martin St. MauriceAbstract:Abstract Biotin-dependent enzymes catalyze carboxylation, decarboxylation and transcarboxylation reactions that participate in the primary metabolism of a wide range of organisms. In all cases, the overall reaction proceeds via two half reactions that take place in physically distinct active sites. In the first half-reaction, a carboxyl group is transferred to the 1-N′ of a covalently tethered biotin cofactor. The tethered carboxybiotin intermediate subsequently translocates to a second active site where the carboxyl group is either transferred to an acceptor substrate or, in some bacteria and archaea, is decarboxylated to biotin and CO 2 in order to power the export of sodium ions from the cytoplasm. A homologous Carboxyltransferase domain is found in three enzymes that catalyze diverse overall reactions: carbon fixation by pyruvate carboxylase, decarboxylation and sodium transport by the biotin-dependent oxaloacetate decarboxylase complex, and transcarboxylation by transcarboxylase from Propionibacterium shermanii . Over the past several years, structural data have emerged which have greatly advanced the mechanistic description of these enzymes. This review assembles a uniform description of the Carboxyltransferase domain structure and catalytic mechanism from recent studies of pyruvate carboxylase, oxaloacetate decarboxylase and transcarboxylase, three enzymes that utilize an analogous Carboxyltransferase domain to catalyze the biotin-dependent decarboxylation of oxaloacetate.
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Insights Into the Carboxyltransferase Reaction of Pyruvate Carboxylase From the Structures of Bound Product and Intermediate Analogs
Biochemical and biophysical research communications, 2013Co-Authors: A.d. Lietzan, Martin St. MauriceAbstract:Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The Carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates.
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a substrate induced biotin binding pocket in the Carboxyltransferase domain of pyruvate carboxylase
Journal of Biological Chemistry, 2013Co-Authors: A.d. Lietzan, Martin St. MauriceAbstract:Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the Carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the Carboxyltransferase domain of PC from Rhizobium etli. Structures of the Carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the Carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp590 and Tyr628 and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with Carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes. Background: Biotin-dependent enzymes efficiently coordinate multiple reactions in physically separate active sites. Results: Substrate binding remodels the Carboxyltransferase active site to form a biotin binding pocket. Conclusion: Pyruvate must bind before carboxybiotin can access the Carboxyltransferase active site. Significance: The active sites of biotin-dependent enzymes control biotin access, providing a mechanism to coordinate the overall reaction between multiple active sites.
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A substrate-induced biotin binding pocket in the Carboxyltransferase domain of pyruvate carboxylase.
The Journal of biological chemistry, 2013Co-Authors: A.d. Lietzan, Martin St. MauriceAbstract:Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the Carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the Carboxyltransferase domain of PC from Rhizobium etli. Structures of the Carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the Carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp(590) and Tyr(628) and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with Carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes.
A.d. Lietzan - One of the best experts on this subject based on the ideXlab platform.
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The role of biotin and oxamate in the Carboxyltransferase reaction of pyruvate carboxylase.
Archives of biochemistry and biophysics, 2014Co-Authors: A.d. Lietzan, Yi Lin, Martin St. MauriceAbstract:Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. During catalysis, carboxybiotin is translocated to the Carboxyltransferase domain where the carboxyl group is transferred to the acceptor substrate, pyruvate. Many studies on the Carboxyltransferase domain of PC have demonstrated an enhanced oxaloacetate decarboxylation activity in the presence of oxamate and it has been shown that oxamate accepts a carboxyl group from carboxybiotin during oxaloacetate decarboxylation. The X-ray crystal structure of the Carboxyltransferase domain from Rhizobium etli PC reveals that oxamate is positioned in the active site in an identical manner to the substrate, pyruvate, and kinetic data are consistent with the oxamate-stimulated decarboxylation of oxaloacetate proceeding through a simple ping-pong bi bi mechanism in the absence of the biotin carboxylase domain. Additionally, analysis of truncated PC enzymes indicates that the BCCP domain devoid of biotin does not contribute directly to the enzymatic reaction and conclusively demonstrates a biotin-independent oxaloacetate decarboxylation activity in PC. These findings advance the description of catalysis in PC and can be extended to the study of related biotin-dependent enzymes.
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functionally diverse biotin dependent enzymes with oxaloacetate decarboxylase activity
Archives of Biochemistry and Biophysics, 2014Co-Authors: A.d. Lietzan, Martin St. MauriceAbstract:Abstract Biotin-dependent enzymes catalyze carboxylation, decarboxylation and transcarboxylation reactions that participate in the primary metabolism of a wide range of organisms. In all cases, the overall reaction proceeds via two half reactions that take place in physically distinct active sites. In the first half-reaction, a carboxyl group is transferred to the 1-N′ of a covalently tethered biotin cofactor. The tethered carboxybiotin intermediate subsequently translocates to a second active site where the carboxyl group is either transferred to an acceptor substrate or, in some bacteria and archaea, is decarboxylated to biotin and CO 2 in order to power the export of sodium ions from the cytoplasm. A homologous Carboxyltransferase domain is found in three enzymes that catalyze diverse overall reactions: carbon fixation by pyruvate carboxylase, decarboxylation and sodium transport by the biotin-dependent oxaloacetate decarboxylase complex, and transcarboxylation by transcarboxylase from Propionibacterium shermanii . Over the past several years, structural data have emerged which have greatly advanced the mechanistic description of these enzymes. This review assembles a uniform description of the Carboxyltransferase domain structure and catalytic mechanism from recent studies of pyruvate carboxylase, oxaloacetate decarboxylase and transcarboxylase, three enzymes that utilize an analogous Carboxyltransferase domain to catalyze the biotin-dependent decarboxylation of oxaloacetate.
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Insights Into the Carboxyltransferase Reaction of Pyruvate Carboxylase From the Structures of Bound Product and Intermediate Analogs
Biochemical and biophysical research communications, 2013Co-Authors: A.d. Lietzan, Martin St. MauriceAbstract:Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The Carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates.
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a substrate induced biotin binding pocket in the Carboxyltransferase domain of pyruvate carboxylase
Journal of Biological Chemistry, 2013Co-Authors: A.d. Lietzan, Martin St. MauriceAbstract:Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the Carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the Carboxyltransferase domain of PC from Rhizobium etli. Structures of the Carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the Carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp590 and Tyr628 and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with Carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes. Background: Biotin-dependent enzymes efficiently coordinate multiple reactions in physically separate active sites. Results: Substrate binding remodels the Carboxyltransferase active site to form a biotin binding pocket. Conclusion: Pyruvate must bind before carboxybiotin can access the Carboxyltransferase active site. Significance: The active sites of biotin-dependent enzymes control biotin access, providing a mechanism to coordinate the overall reaction between multiple active sites.
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A substrate-induced biotin binding pocket in the Carboxyltransferase domain of pyruvate carboxylase.
The Journal of biological chemistry, 2013Co-Authors: A.d. Lietzan, Martin St. MauriceAbstract:Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the Carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the Carboxyltransferase domain of PC from Rhizobium etli. Structures of the Carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the Carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp(590) and Tyr(628) and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with Carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes.
Liang Tong - One of the best experts on this subject based on the ideXlab platform.
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a distinct holoenzyme organization for two subunit pyruvate carboxylase
Nature Communications, 2016Co-Authors: Philip H Choi, Yucheng Lin, Minhan Lin, Chiyuan Chou, Lars E P Dietrich, Liang TongAbstract:Pyruvate carboxylase (PC) has important roles in metabolism and is crucial for virulence for some pathogenic bacteria. PC contains biotin carboxylase (BC), Carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP) components. It is a single-chain enzyme in eukaryotes and most bacteria, and functions as a 500 kD homo-tetramer. In contrast, PC is a two-subunit enzyme in a collection of Gram-negative bacteria, with the α subunit containing the BC and the β subunit the CT and BCCP domains, and it is believed that the holoenzyme has α4β4 stoichiometry. We report here the crystal structures of a two-subunit PC from Methylobacillus flagellatus. Surprisingly, our structures reveal an α2β4 stoichiometry, and the overall architecture of the holoenzyme is strikingly different from that of the homo-tetrameric PCs. Biochemical and mutagenesis studies confirm the stoichiometry and other structural observations. Our functional studies in Pseudomonas aeruginosa show that its two-subunit PC is important for colony morphogenesis. Pyruvate carboxylases are homotetrameric enzymes in eukaryotes and most bacteria. Here, the authors report the structure of an unusual two-subunit form of the enzyme from the Gram-negative bacterium Methylobacillus flagellates, revealing an unexpected α2β4stoichiometry.
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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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mechanism for the inhibition of the Carboxyltransferase domain of acetyl coenzyme a carboxylase by pinoxaden
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Yi Seul Kim, Liang TongAbstract:Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and have been targeted for drug development against obesity, diabetes, and other diseases. The Carboxyltransferase (CT) domain of this enzyme is the site of action for three different classes of herbicides, as represented by haloxyfop, tepraloxydim, and pinoxaden. Our earlier studies have demonstrated that haloxyfop and tepraloxydim bind in the CT active site at the interface of its dimer. However, the two compounds probe distinct regions of the dimer interface, sharing primarily only two common anchoring points of interaction with the enzyme. We report here the crystal structure of the CT domain of yeast ACC in complex with pinoxaden at 2.8-A resolution. Despite their chemical diversity, pinoxaden has a similar binding mode as tepraloxydim and requires a small conformational change in the dimer interface for binding. Crystal structures of the CT domain in complex with all three classes of herbicides confirm the importance of the two anchoring points for herbicide binding. The structures also provide a foundation for understanding the molecular basis of the herbicide resistance mutations and cross resistance among the herbicides, as well as for the design and development of new inhibitors against plant and human ACCs.
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A different mechanism for the inhibition of the Carboxyltransferase domain of acetyl-coenzyme A carboxylase by tepraloxydim
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Song Xiang, Matthew M. Callaghan, Keith G. Watson, Liang TongAbstract:Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and are attractive targets for drug discovery. Haloxyfop and tepraloxydim belong to two distinct classes of commercial herbicides and kill sensitive plants by inhibiting the Carboxyltransferase (CT) activity of ACC. Our earlier structural studies showed that haloxyfop is bound near the active site of the CT domain, at the interface of its dimer, and a large conformational change in the dimer interface is required for haloxyfop binding. We report here the crystal structure at 2.3 A resolution of the CT domain of yeast ACC in complex with tepraloxydim. The compound has a different mechanism of inhibiting the CT activity compared to haloxyfop, as well as the mammalian ACC inhibitor CP-640186. Tepraloxydim probes a different region of the dimer interface and requires only small but important conformational changes in the enzyme, in contrast to haloxyfop. The binding mode of tepraloxydim explains the structure-activity relationship of these inhibitors, and provides a molecular basis for their distinct sensitivity to some of the resistance mutations, as compared to haloxyfop. Despite the chemical diversity between haloxyfop and tepraloxydim, the compounds do share two binding interactions to the enzyme, which may be important anchoring points for the development of ACC inhibitors.
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A Symmetrical Tetramer for S. aureus Pyruvate Carboxylase in Complex with Coenzyme A
Structure (London England : 1993), 2009Co-Authors: Song Xiang, Gorka Lasso, David Gil, Mikel Valle, Liang TongAbstract:Pyruvate carboxylase (PC) is a conserved metabolic enzyme with important cellular functions. We report crystallographic and cryo-electron microscopy (EM) studies of Staphylococcus aureus PC (SaPC) in complex with acetyl-CoA, an allosteric activator, and mutagenesis, biochemical, and structural studies of the biotin binding site of its Carboxyltransferase (CT) domain. The disease-causing A610T mutation abolishes catalytic activity by blocking biotin binding to the CT active site, and Thr908 might play a catalytic role in the CT reaction. The crystal structure of SaPC in complex with CoA reveals a symmetrical tetramer, with one CoA molecule bound to each monomer, and cryo-EM studies confirm the symmetrical nature of the tetramer. These observations are in sharp contrast to the highly asymmetrical tetramer of Rhizobium etli PC in complex with ethyl-CoA. Our structural information suggests that acetyl-CoA promotes a conformation for the dimer of the biotin carboxylase domain of PC that might be catalytically more competent.
Peter Dimroth - One of the best experts on this subject based on the ideXlab platform.
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structure function relations in oxaloacetate decarboxylase complex fluorescence and infrared approaches to monitor oxomalonate and na binding effect
PLOS ONE, 2010Co-Authors: Thierry Granjon, Pius Dahinden, Yolanda Auchli, Ofelia Maniti, Rene Buchet, Olivier Marcillat, Peter DimrothAbstract:Background Oxaloacetate decarboxylase (OAD) is a member of the Na+ transport decarboxylase enzyme family found exclusively in anaerobic bacteria. OAD of Vibrio cholerae catalyses a key step in citrate fermentation, converting the chemical energy of the decarboxylation reaction into an electrochemical gradient of Na+ ions across the membrane, which drives endergonic membrane reactions such as ATP synthesis, transport and motility. OAD is a membrane-bound enzyme composed of α, β and γ subunits. The α subunit contains the Carboxyltransferase catalytic site. Methodology/Principal Findings In this report, spectroscopic techniques were used to probe oxomalonate (a competitive inhibitor of OAD with respect to oxaloacetate) and Na+ effects on the enzyme tryptophan environment and on the secondary structure of the OAD complex, as well as the importance of each subunit in the catalytic mechanism. An intrinsic fluorescence approach, Red Edge Excitation Shift (REES), indicated that solvent molecule mobility in the vicinity of OAD tryptophans was more restricted in the presence of oxomalonate. It also demonstrated that, although the structure of OAD is sensitive to the presence of NaCl, oxomalonate was able to bind to the enzyme even in the absence of Na+. REES changes due to oxomalonate binding were also observed with the αγ and α subunits. Infrared spectra showed that OAD, αγ and α subunits have a main component band centered between 1655 and 1650 cm−1 characteristic of a high content of α helix structures. Addition of oxomalonate induced a shift of the amide-I band of OAD toward higher wavenumbers, interpreted as a slight decrease of β sheet structures and a concomitant increase of α helix structures. Oxomalonate binding to αγand α subunits also provoked secondary structure variations, but these effects were negligible compared to OAD complex. Conclusion Oxomalonate binding affects the tryptophan environment of the Carboxyltransferase subunit, whereas Na+ alters the tryptophan environment of the β subunit, consistent with the function of these subunits within the enzyme complex. Formation of a complex between OAD and its substrates elicits structural changes in the α-helical as well as β-strand secondary structure elements.
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Structure-function relations in oxaloacetate decarboxylase complex. Fluorescence and infrared approaches to monitor oxomalonate and Na(+) binding effect.
Public Library of Science (PLoS), 2010Co-Authors: Thierry Granjon, Pius Dahinden, Yolanda Auchli, Ofelia Maniti, Rene Buchet, Olivier Marcillat, Peter DimrothAbstract:BACKGROUND:Oxaloacetate decarboxylase (OAD) is a member of the Na(+) transport decarboxylase enzyme family found exclusively in anaerobic bacteria. OAD of Vibrio cholerae catalyses a key step in citrate fermentation, converting the chemical energy of the decarboxylation reaction into an electrochemical gradient of Na(+) ions across the membrane, which drives endergonic membrane reactions such as ATP synthesis, transport and motility. OAD is a membrane-bound enzyme composed of alpha, beta and gamma subunits. The alpha subunit contains the Carboxyltransferase catalytic site. METHODOLOGY/PRINCIPAL FINDINGS:In this report, spectroscopic techniques were used to probe oxomalonate (a competitive inhibitor of OAD with respect to oxaloacetate) and Na(+) effects on the enzyme tryptophan environment and on the secondary structure of the OAD complex, as well as the importance of each subunit in the catalytic mechanism. An intrinsic fluorescence approach, Red Edge Excitation Shift (REES), indicated that solvent molecule mobility in the vicinity of OAD tryptophans was more restricted in the presence of oxomalonate. It also demonstrated that, although the structure of OAD is sensitive to the presence of NaCl, oxomalonate was able to bind to the enzyme even in the absence of Na(+). REES changes due to oxomalonate binding were also observed with the alphagamma and alpha subunits. Infrared spectra showed that OAD, alphagamma and alpha subunits have a main component band centered between 1655 and 1650 cm(-1) characteristic of a high content of alpha helix structures. Addition of oxomalonate induced a shift of the amide-I band of OAD toward higher wavenumbers, interpreted as a slight decrease of beta sheet structures and a concomitant increase of alpha helix structures. Oxomalonate binding to alphagamma and alpha subunits also provoked secondary structure variations, but these effects were negligible compared to OAD complex. CONCLUSION:Oxomalonate binding affects the tryptophan environment of the Carboxyltransferase subunit, whereas Na(+) alters the tryptophan environment of the beta subunit, consistent with the function of these subunits within the enzyme complex. Formation of a complex between OAD and its substrates elicits structural changes in the alpha-helical as well as beta-strand secondary structure elements
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crystal structure of the Carboxyltransferase domain of the oxaloacetate decarboxylase na pump from vibrio cholerae
Journal of Molecular Biology, 2006Co-Authors: Remo Studer, Pius Dahinden, Yolanda Auchli, Xiaodan Li, Weiwu Wang, Peter DimrothAbstract:Abstract Oxaloacetate decarboxylase is a membrane-bound multiprotein complex that couples oxaloacetate decarboxylation to sodium ion transport across the membrane. The initial reaction catalyzed by this enzyme machinery is the carboxyl transfer from oxaloacetate to the prosthetic biotin group. The crystal structure of the Carboxyltransferase at 1.7 A resolution shows a dimer of α 8 β 8 barrels with an active site metal ion, identified spectroscopically as Zn 2+ , at the bottom of a deep cleft. The enzyme is completely inactivated by specific mutagenesis of Asp17, His207 and His209, which serve as ligands for the Zn 2+ metal ion, or by Lys178 near the active site, suggesting that Zn 2+ as well as Lys178 are essential for the catalysis. In the present structure this lysine residue is hydrogen-bonded to Cys148. A potential role of Lys178 as initial acceptor of the carboxyl group from oxaloacetate is discussed.
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identification of a domain in the α subunit of the oxaloacetate decarboxylase na pump that accomplishes complex formation with the γ subunit
FEBS Journal, 2005Co-Authors: Pius Dahinden, Peter DimrothAbstract:The oxaloacetate decarboxylase Na+ pumps OAD-1 and OAD-2 of Vibrio cholerae are composed of a peripheral α-subunit associated with two integral membrane-bound subunits (β and γ). The α-subunit contains the Carboxyltransferase domain in its N-terminal portion and the biotin-binding domain in its C-terminal portion. The γ-subunit plays a profound role in the assembly of the complex. It interacts with the β-subunit through its N-terminal membrane-spanning region and with the α-subunit through its hydrophilic C-terminal domain. The biochemical and structural requirements for the latter interaction were analysed with OAD-2 expression clones for subunit α-2 and the C-terminal domain of γ-2, termed γ′-2. If the two proteins were synthesized together in Escherichia coli they formed a complex that was stable at neutral pH and dissociated at pH<5.0. An internal stretch of 40 amino acids of α-2 was identified by deletion mutagenesis to be essential for the binding with γ′-2. This portion of the α-subunit is connected via flexible linker peptides to the Carboxyltransferase domain at its N terminus and to the biotin-binding domain at its C terminus. Results of site-directed mutagenesis indicated that a conserved tyrosine (491) and threonine 494 of this peptide contributed significantly to the stability of the complex with γ′-2. This peptide therefore represents a newly identified, separate domain of the α-subunit and has been called the ‘association domain’.
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Oxaloacetate decarboxylase of Archaeoglobus fulgidus: cloning of genes and expression in Escherichia coli.
Archives of microbiology, 2004Co-Authors: Pius Dahinden, Klaas M. Pos, Malgorzata Taralczak, Peter DimrothAbstract:Archaeoglobus fulgidus harbors three consecutive and one distantly located gene with similarity to the oxaloacetate decarboxylase Na+ pump of Klebsiella pneumoniae (KpOadGAB). The water-soluble Carboxyltransferase (AfOadA) and the biotin protein (AfOadC) were readily synthesized in Escherichia coli, but the membrane-bound subunits AfOadB and AfOadG were not. AfOadA was affinity purified from inclusion bodies after refolding and AfOadC was affinity purified from the cytosol. Isolated AfOadA catalyzed the carboxyltransfer from [4-14C]-oxaloacetate to the prosthetic biotin group of AfOadC or the corresponding biotin domain of KpOadA. Conversely, the Carboxyltransferase domain of KpOadA exhibited catalytic activity not only with its pertinent biotin domain but also with AfOadC.