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Liang Tong - One of the best experts on this subject based on the ideXlab platform.
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structural and functional studies of pyruvate Carboxylase regulation by cyclic di amp in lactic acid bacteria
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Philip H Choi, Huong Thi Pham, Joshua J Woodward, Mark S Turner, Liang TongAbstract:Cyclic di-3',5'-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism in Listeria monocytogenes by inhibiting its pyruvate Carboxylase (LmPC), a Biotin-dependent enzyme with Biotin Carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition of Lactococcus lactis PC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. In L. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool in L. lactis is negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP-insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.
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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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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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characterizing the importance of the Biotin Carboxylase domain dimer for staphylococcus aureus pyruvate Carboxylase catalysis
Biochemistry, 2013Co-Authors: Chiyuan Chou, Philip H Choi, Liang TongAbstract:Biotin Carboxylase (BC) is a conserved component among Biotin-dependent Carboxylases and catalyzes the MgATP-dependent carboxylation of Biotin, using bicarbonate as the CO2 donor. Studies with Escherichia coli BC have suggested long-range communication between the two active sites of a dimer, although its mechanism is not well understood. In addition, mutations in the dimer interface can produce stable monomers that are still catalytically active. A homologous dimer for the BC domain is observed in the structure of the tetrameric pyruvate Carboxylase (PC) holoenzyme. We have introduced site-specific mutations into the BC domain dimer interface of Staphylococcus aureus PC (SaPC), equivalent to those used for E. coli BC, and also made chimeras replacing the SaPC BC domain with the E. coli BC subunit (EcBC chimera) or the yeast ACC BC domain (ScBC chimera). We assessed the catalytic activities of these mutants and characterized their oligomerization states by gel filtration and analytical ultracentrifugation...
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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.
Martin St. Maurice - One of the best experts on this subject based on the ideXlab platform.
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REVIEW ARTICLE Structure, mechanism and regulation of pyruvate Carboxylase
2014Co-Authors: Sarawut Jitrapakdee, Martin St. Maurice, John C. Wallace, Ivan Rayment, Wallace W Cleland, Paul V AttwoodAbstract:PC (pyruvate Carboxylase) is a Biotin-containing enzyme that catalyses the HCO3−- and MgATP-dependent carboxylation of pyruvate to form oxaloacetate. This is a very important anaplero-tic reaction, replenishing oxaloacetate withdrawn from the tricar-boxylic acid cycle for various pivotal biochemical pathways. PC is therefore considered as an enzyme that is crucial for intermediary metabolism, controlling fuel partitioning toward gluconeogenesis or lipogenesis and in insulin secretion. The enzyme was dis-covered in 1959 and over the last decade there has been much progress in understanding its structure and function. PC from most organisms is a tetrameric protein that is allosterically regulated by acetyl-CoA and aspartate. High-resolution crystal structures of the holoenzyme with various ligands bound have recently been determined, and have revealed details of the binding sites and the relative positions of the Biotin Carboxylase, carboxyltransferas
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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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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.
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allosteric regulation of the Biotin dependent enzyme pyruvate Carboxylase by acetyl coa
Biochemical Society Transactions, 2012Co-Authors: Abdussalam Adinazada, Sarawut Jitrapakdee, Martin St. Maurice, Tonya N. Zeczycki, Wallace W Cleland, Paul V AttwoodAbstract:The activity of the Biotin-dependent enzyme pyruvate Carboxylase from many organisms is highly regulated by the allosteric activator acetyl-CoA. A number of X-ray crystallographic structures of the native pyruvate Carboxylase tetramer are now available for the enzyme from Rhizobium etli and Staphylococcus aureus. Although all of these structures show that intersubunit catalysis occurs, in the case of the R. etli enzyme, only two of the four subunits have the allosteric activator bound to them and are optimally configured for catalysis of the overall reaction. However, it is apparent that acetyl-CoA binding does not induce the observed asymmetrical tetramer conformation and it is likely that, under normal reaction conditions, all of the subunits have acetyl-CoA bound to them. Thus the activation of the enzyme by acetyl-CoA involves more subtle structural effects, one of which may be to facilitate the correct positioning of Arg353 and Biotin in the Biotin Carboxylase domain active site, thereby promoting Biotin carboxylation and, at the same time, preventing abortive decarboxylation of carboxyBiotin. It is also apparent from the crystal structures that there are allosteric interactions induced by acetyl-CoA binding in the pair of subunits not optimally configured for catalysis of the overall reaction.
Grover L. Waldrop - One of the best experts on this subject based on the ideXlab platform.
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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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Computational Redesign of Bacterial Biotin Carboxylase Inhibitors Using Structure-Based Virtual Screening of Combinatorial Libraries
MDPI AG, 2014Co-Authors: Michal Brylinski, Grover L. WaldropAbstract:As the spread of antibiotic resistant bacteria steadily increases, there is an urgent need for new antibacterial agents. Because fatty acid synthesis is only used for membrane biogenesis in bacteria, the enzymes in this pathway are attractive targets for antibacterial agent development. Acetyl-CoA Carboxylase catalyzes the committed and regulated step in fatty acid synthesis. In bacteria, the enzyme is composed of three distinct protein components: Biotin Carboxylase, Biotin carboxyl carrier protein, and carboxyltransferase. Fragment-based screening revealed that amino-oxazole inhibits Biotin Carboxylase activity and also exhibits antibacterial activity against Gram-negative organisms. In this report, we redesigned previously identified lead inhibitors to expand the spectrum of bacteria sensitive to the amino-oxazole derivatives by including Gram-positive species. Using 9,411 small organic building blocks, we constructed a diverse combinatorial library of 1.2 × 108 amino-oxazole derivatives. A subset of 9 × 106 of these compounds were subjected to structure-based virtual screening against seven Biotin Carboxylase isoforms using similarity-based docking by eSimDock. Potentially broad-spectrum antibiotic candidates were selected based on the consensus ranking by several scoring functions including non-linear statistical models implemented in eSimDock and traditional molecular mechanics force fields. The analysis of binding poses of the top-ranked compounds docked to Biotin Carboxylase isoforms suggests that: (1) binding of the amino-oxazole anchor is stabilized by a network of hydrogen bonds to residues 201, 202 and 204; (2) halogenated aromatic moieties attached to the amino-oxazole scaffold enhance interactions with a hydrophobic pocket formed by residues 157, 169, 171 and 203; and (3) larger substituents reach deeper into the binding pocket to form additional hydrogen bonds with the side chains of residues 209 and 233. These structural insights into drug-Biotin Carboxylase interactions will be tested experimentally in in vitro and in vivo systems to increase the potency of amino-oxazole inhibitors towards both Gram-negative as well as Gram-positive species
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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.
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Constituents of Cinnamon Inhibit Bacterial Acetyl CoA Carboxylase
Planta medica, 2010Co-Authors: Glen Meades, Grover L. Waldrop, Rachel L. Henken, M. Mukhlesur Rahman, S. Douglass Gilman, Guy P.p. Kamatou, Alvaro Viljoen, Simon GibbonsAbstract:Cinnamon bark (Cinnamomum zeylanicum )i s used extensively as an antimicrobial material and currently is being increasingly used in Europe by people with type II diabetes to control their glu- cose levels. In this paper we describe the action of cinnamon oil, its major component, trans-cin- namaldehyde, and an analogue, 4-hydroxy-3-me- thoxy-trans-cinnamaldehyde against bacterial acetyl-CoA Carboxylase in an attempt to elucidate the mechanism of action of this well-known anti- microbial material. These natural products inhib- ited the carboxyltransferase component of Esche- richia coli acetyl-CoA Carboxylase but had no ef- fect on the activity of the Biotin Carboxylase com- ponent. The inhibition patterns indicated that these products bound to the Biotin binding site of carboxyltransferase with trans-cinnamalde- hyde having a Ki value of 3.8 ± 0.6mM. The inhibi- tion of carboxyltransferase by 4-hydroxy-3-me- thoxy-trans-cinnamaldehyde was analyzed with a new assay for this enzyme based on capillary electrophoresis. These results explain, in part, the antibacterial activity of this well-known anti- microbial material. " Cinnamomum zeylanicum l " Lauraceae l " trans‑cinnamaldehyde
Paul V Attwood - One of the best experts on this subject based on the ideXlab platform.
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REVIEW ARTICLE Structure, mechanism and regulation of pyruvate Carboxylase
2014Co-Authors: Sarawut Jitrapakdee, Martin St. Maurice, John C. Wallace, Ivan Rayment, Wallace W Cleland, Paul V AttwoodAbstract:PC (pyruvate Carboxylase) is a Biotin-containing enzyme that catalyses the HCO3−- and MgATP-dependent carboxylation of pyruvate to form oxaloacetate. This is a very important anaplero-tic reaction, replenishing oxaloacetate withdrawn from the tricar-boxylic acid cycle for various pivotal biochemical pathways. PC is therefore considered as an enzyme that is crucial for intermediary metabolism, controlling fuel partitioning toward gluconeogenesis or lipogenesis and in insulin secretion. The enzyme was dis-covered in 1959 and over the last decade there has been much progress in understanding its structure and function. PC from most organisms is a tetrameric protein that is allosterically regulated by acetyl-CoA and aspartate. High-resolution crystal structures of the holoenzyme with various ligands bound have recently been determined, and have revealed details of the binding sites and the relative positions of the Biotin Carboxylase, carboxyltransferas
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allosteric regulation of the Biotin dependent enzyme pyruvate Carboxylase by acetyl coa
Biochemical Society Transactions, 2012Co-Authors: Abdussalam Adinazada, Sarawut Jitrapakdee, Martin St. Maurice, Tonya N. Zeczycki, Wallace W Cleland, Paul V AttwoodAbstract:The activity of the Biotin-dependent enzyme pyruvate Carboxylase from many organisms is highly regulated by the allosteric activator acetyl-CoA. A number of X-ray crystallographic structures of the native pyruvate Carboxylase tetramer are now available for the enzyme from Rhizobium etli and Staphylococcus aureus. Although all of these structures show that intersubunit catalysis occurs, in the case of the R. etli enzyme, only two of the four subunits have the allosteric activator bound to them and are optimally configured for catalysis of the overall reaction. However, it is apparent that acetyl-CoA binding does not induce the observed asymmetrical tetramer conformation and it is likely that, under normal reaction conditions, all of the subunits have acetyl-CoA bound to them. Thus the activation of the enzyme by acetyl-CoA involves more subtle structural effects, one of which may be to facilitate the correct positioning of Arg353 and Biotin in the Biotin Carboxylase domain active site, thereby promoting Biotin carboxylation and, at the same time, preventing abortive decarboxylation of carboxyBiotin. It is also apparent from the crystal structures that there are allosteric interactions induced by acetyl-CoA binding in the pair of subunits not optimally configured for catalysis of the overall reaction.
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novel insights into the Biotin Carboxylase domain reactions of pyruvate Carboxylase from rhizobium etli
Biochemistry, 2011Co-Authors: Tonya N. Zeczycki, Paul V Attwood, Sarawut Jitrapakdee, Martin St. Maurice, Ann L. Menefee, John C. Wallace, Abdussalam Adinazada, Kathy H Surinya, Wallace W ClelandAbstract:The catalytic mechanism of the MgATP-dependent carboxylation of Biotin in the Biotin Carboxylase domain of pyruvate Carboxylase from R. etli (RePC) is common to the Biotin-dependent Carboxylases. The current site-directed mutagenesis study has clarified the catalytic functions of several residues proposed to be pivotal in MgATP-binding and cleavage (Glu218 and Lys245), HCO3– deprotonation (Glu305 and Arg301), and Biotin enolization (Arg353). The E218A mutant was inactive for any reaction involving the BC domain and the E218Q mutant exhibited a 75-fold decrease in kcat for both pyruvate carboxylation and the full reverse reaction. The E305A mutant also showed a 75- and 80-fold decrease in kcat for both pyruvate carboxylation and the full reverse reaction, respectively. While Glu305 appears to be the active site base which deprotonates HCO3–, Lys245, Glu218, and Arg301 are proposed to contribute to catalysis through substrate binding interactions. The reactions of the Biotin Carboxylase and carboxyl transfe...
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activation and inhibition of pyruvate Carboxylase from rhizobium etli
Biochemistry, 2011Co-Authors: Tonya N. Zeczycki, Paul V Attwood, Sarawut Jitrapakdee, Martin St. Maurice, Ann L. Menefee, John C. Wallace, Wallace W ClelandAbstract:While crystallographic structures of the R. etli pyruvate Carboxylase (PC) holoenzyme revealed the location and probable positioning of the essential activator, Mg2+, and nonessential activator, acetyl-CoA, an understanding of how they affect catalysis remains unclear. The current steady-state kinetic investigation indicates that both acetyl-CoA and Mg2+ assist in coupling the MgATP-dependent carboxylation of Biotin in the Biotin Carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. Initial velocity plots of free Mg2+ vs pyruvate were nonlinear at low concentrations of Mg2+ and a nearly complete loss of coupling between the BC and CT domain reactions was observed in the absence of acetyl-CoA. Increasing concentrations of free Mg2+ also resulted in a decrease in the Ka for acetyl-CoA. Acetyl phosphate was determined to be a suitable phosphoryl donor for the catalytic phosphorylation of MgADP, while phosphonoacetate inhibited both the phosphorylation of MgADP by carbam...
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Interaction between the Biotin carboxyl carrier domain and the Biotin Carboxylase domain in pyruvate Carboxylase from Rhizobium etli.
Biochemistry, 2011Co-Authors: A.d. Lietzan, Paul V Attwood, Ann L. Menefee, Tonya N. Zeczycki, Sudhanshu Kumar, John C. Wallace, W. Wallace Cleland, Martin St. MauriceAbstract:Pyruvate Carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered Biotin of PC must gain access to active sites in both the Biotin Carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of Biotin in the Biotin Carboxylase domain. We report the 2.4 A resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the Biotin carboxyl carrier protein domain and the Biotin Carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While Biotin is observed in the Biotin Carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxyBiotin access to the BC domain active site. The binding location for the Biotin carboxyl carrier protein domain demonstrates that tethered Biotin cannot bind in the Biotin Carboxylase domain active site in the same orientation as free Biotin, helping to explain the difference in catalysis observed between tethered Biotin and free Biotin substrates in Biotin Carboxylase enzymes. Electron density located in the Biotin Carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during Biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the Biotin-dependent enzyme family.
Tonya N. Zeczycki - One of the best experts on this subject based on the ideXlab platform.
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Kinetic and Thermodynamic Analysis of Acetyl-CoA Activation of Staphylococcus aureus Pyruvate Carboxylase
2017Co-Authors: Lauren E. Westerhold, Lance C. Bridges, Saame Raza Shaikh, Tonya N. ZeczyckiAbstract:Allosteric regulation of pyruvate Carboxylase (PC) activity is pivotal to maintaining metabolic homeostasis. In contrast, dysregulated PC activity contributes to the pathogenesis of numerous diseases, rendering PC a possible target for allosteric therapeutic development. Recent research efforts have focused on demarcating the role of acetyl-CoA, one of the most potent activators of PC, in coordinating catalytic events within the multifunctional enzyme. Herein, we report a kinetic and thermodynamic analysis of acetyl-CoA activation of the Staphylococcus aureus PC (SaPC)-catalyzed carboxylation of pyruvate to identify novel means by which acetyl-CoA synchronizes catalytic events within the PC tetramer. Kinetic and linked-function analysis, or thermodynamic linkage analysis, indicates that the substrates of the Biotin Carboxylase and carboxyl transferase domain are energetically coupled in the presence of acetyl-CoA. In contrast, both kinetic and energetic coupling between the two domains is lost in the absence of acetyl-CoA, suggesting a functional role for acetyl-CoA in facilitating the long-range transmission of substrate-induced conformational changes within the PC tetramer. Interestingly, thermodynamic activation parameters for the SaPC-catalyzed carboxylation of pyruvate are largely independent of acetyl-CoA. Our results also reveal the possibility that global conformational changes give rise to observed species-specific thermodynamic activation parameters. Taken together, our kinetic and thermodynamic results provide a possible allosteric mechanism by which acetyl-CoA coordinates catalysis within the PC tetramer
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Pyruvate Occupancy in the Carboxyl Transferase Domain of Pyruvate Carboxylase Facilitates Product Release from the Biotin Carboxylase Domain through an Intermolecular Mechanism
2016Co-Authors: Lauren E. Westerhold, Stephanie L. Adams, Hanna L. Bergman, Tonya N. ZeczyckiAbstract:Protein structure, ligand binding, and catalytic turnover contributes to the governance of catalytic events occurring at spatially distinct domains in multifunctional enzymes. Coordination of these catalytic events partially rests on the ability of spatially discrete active sites to communicate with other allosteric and active sites on the same polypeptide chain (intramolecular) or on different polypeptide chains (intermolecular) within the holoenzyme. Often, communication results in long-range effects on substrate binding or product release. For example, pyruvate binding to the carboxyl transferase (CT) domain of pyruvate Carboxylase (PC) increases the rate of product release in the Biotin Carboxylase (BC) domain. In order to address how CT domain ligand occupancy is “sensed” by other domains, we generated functional, mixed hybrid tetramers using the E218A (inactive BC domain) and T882S (low pyruvate binding, low activity) mutant forms of PC. The apparent Ka pyruvate for the pyruvate-stimulated release of Pi catalyzed by the T882S:E218A[1:1] hybrid tetramer was comparable to the wild-type enzyme and nearly 10-fold lower than that for the T882S homotetramer. In addition, the ratio of the rates of oxaloacetate formation to Pi release for the WT:T882S[1:1] and E218A:T882S[1:1] hybrid tetramer-catalyzed reactions was 0.5 and 0.6, respectively, while the T882S homotetramer exhibited a near 1:1 coupling of the two domains, suggesting that the mechanisms coordinating catalytic events is more complicated that we initially assumed. The results presented here are consistent with an intermolecular communication mechanism, where pyruvate binding to the CT domain is “sensed” by domains on a different polypeptide chain within the tetramer
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allosteric regulation of the Biotin dependent enzyme pyruvate Carboxylase by acetyl coa
Biochemical Society Transactions, 2012Co-Authors: Abdussalam Adinazada, Sarawut Jitrapakdee, Martin St. Maurice, Tonya N. Zeczycki, Wallace W Cleland, Paul V AttwoodAbstract:The activity of the Biotin-dependent enzyme pyruvate Carboxylase from many organisms is highly regulated by the allosteric activator acetyl-CoA. A number of X-ray crystallographic structures of the native pyruvate Carboxylase tetramer are now available for the enzyme from Rhizobium etli and Staphylococcus aureus. Although all of these structures show that intersubunit catalysis occurs, in the case of the R. etli enzyme, only two of the four subunits have the allosteric activator bound to them and are optimally configured for catalysis of the overall reaction. However, it is apparent that acetyl-CoA binding does not induce the observed asymmetrical tetramer conformation and it is likely that, under normal reaction conditions, all of the subunits have acetyl-CoA bound to them. Thus the activation of the enzyme by acetyl-CoA involves more subtle structural effects, one of which may be to facilitate the correct positioning of Arg353 and Biotin in the Biotin Carboxylase domain active site, thereby promoting Biotin carboxylation and, at the same time, preventing abortive decarboxylation of carboxyBiotin. It is also apparent from the crystal structures that there are allosteric interactions induced by acetyl-CoA binding in the pair of subunits not optimally configured for catalysis of the overall reaction.
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novel insights into the Biotin Carboxylase domain reactions of pyruvate Carboxylase from rhizobium etli
Biochemistry, 2011Co-Authors: Tonya N. Zeczycki, Paul V Attwood, Sarawut Jitrapakdee, Martin St. Maurice, Ann L. Menefee, John C. Wallace, Abdussalam Adinazada, Kathy H Surinya, Wallace W ClelandAbstract:The catalytic mechanism of the MgATP-dependent carboxylation of Biotin in the Biotin Carboxylase domain of pyruvate Carboxylase from R. etli (RePC) is common to the Biotin-dependent Carboxylases. The current site-directed mutagenesis study has clarified the catalytic functions of several residues proposed to be pivotal in MgATP-binding and cleavage (Glu218 and Lys245), HCO3– deprotonation (Glu305 and Arg301), and Biotin enolization (Arg353). The E218A mutant was inactive for any reaction involving the BC domain and the E218Q mutant exhibited a 75-fold decrease in kcat for both pyruvate carboxylation and the full reverse reaction. The E305A mutant also showed a 75- and 80-fold decrease in kcat for both pyruvate carboxylation and the full reverse reaction, respectively. While Glu305 appears to be the active site base which deprotonates HCO3–, Lys245, Glu218, and Arg301 are proposed to contribute to catalysis through substrate binding interactions. The reactions of the Biotin Carboxylase and carboxyl transfe...
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activation and inhibition of pyruvate Carboxylase from rhizobium etli
Biochemistry, 2011Co-Authors: Tonya N. Zeczycki, Paul V Attwood, Sarawut Jitrapakdee, Martin St. Maurice, Ann L. Menefee, John C. Wallace, Wallace W ClelandAbstract:While crystallographic structures of the R. etli pyruvate Carboxylase (PC) holoenzyme revealed the location and probable positioning of the essential activator, Mg2+, and nonessential activator, acetyl-CoA, an understanding of how they affect catalysis remains unclear. The current steady-state kinetic investigation indicates that both acetyl-CoA and Mg2+ assist in coupling the MgATP-dependent carboxylation of Biotin in the Biotin Carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. Initial velocity plots of free Mg2+ vs pyruvate were nonlinear at low concentrations of Mg2+ and a nearly complete loss of coupling between the BC and CT domain reactions was observed in the absence of acetyl-CoA. Increasing concentrations of free Mg2+ also resulted in a decrease in the Ka for acetyl-CoA. Acetyl phosphate was determined to be a suitable phosphoryl donor for the catalytic phosphorylation of MgADP, while phosphonoacetate inhibited both the phosphorylation of MgADP by carbam...