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Nicolas Glansdorff - One of the best experts on this subject based on the ideXlab platform.
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New experimental approaches for investigating interactions between Pyrococcus furiosus carbamate kinase and Carbamoyltransferases, enzymes involved in the channeling of thermolabile carbamoyl phosphate
Hindawi Limited, 2005Co-Authors: Jan Massant, Nicolas GlansdorffAbstract:A somewhat neglected but essential aspect of the molecular physiology of hyperthermophiles is the protection of thermolabile metabolites and coenzymes. An example is carbamoyl phosphate (CP), a precursor of pyrimidines and arginine, which is an extremely labile and potentially toxic intermediate. The first evidence for a biologically significant interaction between carbamate kinase (CK) and ornithine Carbamoyltransferase (OTC) from Pyrococcus furiosus was provided by affinity electrophoresis and co-immunoprecipitation in combination with cross-linking (Massant et al. 2002). Using the yeast two-hybrid system, Hummel-Dreyer chromatography and isothermal titration calorimetry, we obtained additional concrete evidence for an interaction between CK and OTC, the first evidence for an interaction between CK and Aspartate Carbamoyltransferase (ATC) and an estimate of the binding constant between CK and ATC. The physical interaction between CK and OTC or ATC may prevent thermodenaturation of CP in the aqueous cytoplasmic environment. Here we emphasize the importance of developing experimental approaches to investigate the mechanism of thermal protection of metabolic intermediates by metabolic channeling and the molecular basis of transient protein–protein interactions in the physiology of hyperthermophiles
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crystal structure of t state Aspartate Carbamoyltransferase of the hyperthermophilic archaeon sulfolobus acidocaldarius
Journal of Molecular Biology, 2004Co-Authors: Dirk De Vos, Nicolas Glansdorff, Christianne Legrain, Filip Van Petegem, Han Remaut, Jozef Van BeeumenAbstract:Abstract Aspartate Carbamoyltransferase (ATCase) is a model enzyme for understanding allosteric effects. The dodecameric complex exists in two main states (T and R) that differ substantially in their quaternary structure and their affinity for various ligands. Many hypotheses have resulted from the structure of the Escherichia coli ATCase, but so far other crystal structures to test these have been lacking. Here, we present the tertiary and quaternary structure of the T state ATCase of the hyperthermophilic archaeon Sulfolobus acidocaldarius ( Sa ATC T ), determined by X-ray crystallography to 2.6 A resolution. The quaternary structure differs from the E. coli ATCase, by having altered interfaces between the catalytic (C) and regulatory (R) subunits, and the presence of a novel C1–R2 type interface. Conformational differences in the 240s loop region of the C chain and the C-terminal region of the R chain affect intersubunit and interdomain interfaces implicated previously in the allosteric behavior of E. coli ATCase. The allosteric-zinc binding domain interface is strengthened at the expense of a weakened R1–C4 type interface. The increased hydrophobicity of the C1–R1 type interface may stabilize the quaternary structure. Catalytic trimers of the S. acidocaldarius ATCase are unstable due to a drastic weakening of the C1–C2 interface. The hyperthermophilic ATCase presents an interesting example of how an allosteric enzyme can adapt to higher temperatures. The structural rearrangement of this thermophilic ATCase may well promote its thermal stability at the expense of changes in the allosteric behavior.
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Using Quaternary Structures to Assess the Evolutionary History of Proteins: The Case of the Aspartate Carbamoyltransferase
Molecular biology and evolution, 2003Co-Authors: Bernard Labedan, D. G. Naumoff, Nicolas GlansdorffAbstract:Many evolutionary scenarios describing the history of proteins are based solely on phylogenetic studies. We have designed a new approach that allows ascertainment of such questionable scenarios by taking into account quaternary structures: we used Aspartate Carbamoyltransferase (ATCase) as a case study. Prokaryotic ATCases correspond to different classes of quaternary structures according to the mode of association of the catalytic PyrB subunit with other polypeptides, either the PyrI regulatory subunit (class B) or a dihydroorotase (class A), which may be active (PyrC, subclass A1) or inactive (PyrC', subclass A2). Class C is uniquely made up of trimers of PyrB. The PyrB phylogenetic tree is not congruent with the tree of life, but it became coherent when we recognized the existence of two families of ATCases, ATC I and ATC II. Remarkably, a very strong correlation was found between the pattern of PyrB phylogenetic clustering and the different classes of quaternary structures of ATCases. All class B ATCases form a clade in family ATC II, which also contains all eukaryotic sequences. In contrast, family ATC I is made up of classes A and C. These results suggest unexpected common ancestry for prokaryotic B and eukaryotic ATCases on the one hand, and for A and C on the other. Thus, the emergence of specific quaternary structures appears to have been a more recent event than the separation into the ATC I and ATC II families. We propose that different evolutionary constraints, depending on the identity of the partners interacting in the different kinds of holoenzymes, operated in a concerted way on the ancestral pyrB genes and the respective associated genes pyrI or pyrC, so as to maintain appropriate inter-polypeptides interactions at the level of quaternary structure. The process of coevolution of genes encoding proteins interacting in various holoenzymes has been assessed by calculating the correlation coefficient between their respective phylogenetic trees. Our approach integrating data obtained from the separate fields of structural biology and molecular evolution could be useful in other cases where pure statistical data need to receive independent confirmation.
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Aspartate Carbamoyltransferase from the thermoacidophilic archaeon sulfolobus acidocaldarius
FEBS Journal, 1999Co-Authors: Virginie Durbecq, Daniel Charlier, Thialin Thiatoong, Vincent Villeret, Martine Roovers, Ruddy Wattiez, Christianne Legrain, Nicolas GlansdorffAbstract:The genes coding for Aspartate Carbamoyltransferase (ATCase) in the extremely thermophilic archaeon Sulfolobus acidocaldarius have been cloned by complementation of a pyrBI deletion mutant of Escherichia coli. Sequencing revealed the existence of an enterobacterial-like pyrBI operon encoding a catalytic chain of 299 amino acids (34 kDa) and a regulatory chain of 170 amino acids (17.9 kDa). The deduced amino acid sequences of the pyrB and pyrI genes showed 27.6–50% identity with archaeal and enterobacterial ATCases. The recombinant S. acidocaldarius ATCase was purified to homogeneity, allowing the first detailed studies of an ATCase isolated from a thermophilic organism. The recombinant enzyme displayed the same properties as the ATCase synthesized in the native host. It is highly thermostable and exhibits Michaelian saturation kinetics for carbamoylphosphate (CP) and positive homotropic cooperative interactions for the binding of l-Aspartate. Moreover, it is activated by nucleoside triphosphates whereas the catalytic subunits alone are inhibited. The holoenzyme purified from recombinant E. coli cells or present in crude extract of the native host have an Mr of 340 000 as estimated by gel filtration, suggesting that it has a quaternary structure similar to that of E. coli ATCase. Only monomers could be found in extracts of recombinant E. coli or Saccharomyces cerevisiae cells expressing the pyrB gene alone. In the presence of CP these monomers assembled into trimers. The stability of S. acidocaldarius ATCase and the allosteric properties of the enzyme are discussed in function of a modeling study.
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Aspartate Carbamoyltransferase from the thermoacidophilic archaeon sulfolobus acidocaldarius cloning sequence analysis enzyme purification and characterization
FEBS Journal, 1999Co-Authors: Virginie Durbecq, Daniel Charlier, Thialin Thiatoong, Vincent Villeret, Martine Roovers, Ruddy Wattiez, Christianne Legrain, Nicolas GlansdorffAbstract:The genes coding for Aspartate Carbamoyltransferase (ATCase) in the extremely thermophilic archaeon Sulfolobus acidocaldarius have been cloned by complementation of a pyrBI deletion mutant of Escherichia coli. Sequencing revealed the existence of an enterobacterial-like pyrBI operon encoding a catalytic chain of 299 amino acids (34 kDa) and a regulatory chain of 170 amino acids (17.9 kDa). The deduced amino acid sequences of the pyrB and pyrI genes showed 27.6–50% identity with archaeal and enterobacterial ATCases. The recombinant S. acidocaldarius ATCase was purified to homogeneity, allowing the first detailed studies of an ATCase isolated from a thermophilic organism. The recombinant enzyme displayed the same properties as the ATCase synthesized in the native host. It is highly thermostable and exhibits Michaelian saturation kinetics for carbamoylphosphate (CP) and positive homotropic cooperative interactions for the binding of l-Aspartate. Moreover, it is activated by nucleoside triphosphates whereas the catalytic subunits alone are inhibited. The holoenzyme purified from recombinant E. coli cells or present in crude extract of the native host have an Mr of 340 000 as estimated by gel filtration, suggesting that it has a quaternary structure similar to that of E. coli ATCase. Only monomers could be found in extracts of recombinant E. coli or Saccharomyces cerevisiae cells expressing the pyrB gene alone. In the presence of CP these monomers assembled into trimers. The stability of S. acidocaldarius ATCase and the allosteric properties of the enzyme are discussed in function of a modeling study.
Peggy J. Farnham - One of the best experts on this subject based on the ideXlab platform.
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start site selection at the tata less carbamoyl phosphate synthase glutamine hydrolyzing Aspartate Carbamoyltransferase dihydroorotase promoter
Journal of Biological Chemistry, 1994Co-Authors: Richard Kollmar, Kristine A. Sukow, Stephen K. Sponagle, Peggy J. FarnhamAbstract:Abstract Transcription of the carbamoyl-phosphate synthase (glutamine-hydrolyzing)/Aspartate Carbamoyltransferase/dihydroorotase (CAD) gene from the Syrian hamster, Mesocricetus auratus, starts at a single major site. We characterized the cis-acting elements that position RNA polymerase II at the correct start site in the CAD promoter. Sequence alignment showed that the CAD promoter lacks a TATA box, but contains a consensus initiator. Mutational analysis of the CAD promoter demonstrated that the sequences between -81 and +26 were sufficient for accurate and efficient transcription in vitro and in vivo; binding sites for the transcription factor Sp1 around -70 and -49 were necessary for transcriptional activity. The binding site at -49 directed initiation about 50 base pairs downstream. A ubiquitous activator protein, Honk, bound to the CAD promoter between -30 and -12, but did not participate in start site selection. The sequences around +1, which contain the consensus initiator, contributed to promoter activity; however, the presence of a consensus initiator in this region was neither necessary nor sufficient for transcription. We concluded from these results that the Sp1 binding site at -49 substituted for the missing TATA box and played a major role in start site selection at the CAD promoter.
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Heat sensitivity and Sp1 activation of complex formation at the Syrian hamster carbamoyl-phosphate synthase (glutamine-hydrolyzing)/Aspartate Carbamoyltransferase/dihydroorotase promoter in vitro.
The Journal of biological chemistry, 1992Co-Authors: Richard Kollmar, Mary J. Lindstrom, Peggy J. FarnhamAbstract:Abstract To study the regulation of transcription of the carbamoyl-phosphate synthase (glutamine-hydrolyzing)/Aspartate Carbamoyltransferase/dihydroorotase (CAD) gene from the Syrian hamster, Mesocricetus auratus, we developed a homologous in vitro transcription system on the basis of nuclear extract from Syrian hamster kidney cells. We optimized the reaction temperature and the concentrations of DNA template, KCl, and MgCl2 simultaneously with the response surface method and found an unusually low temperature optimum of 20 degrees C. We therefore investigated whether CAD transcription in vitro depended on a heat-labile component of nuclear extract. Preincubating extract alone at 30 degrees C reduced transcription from the CAD promoter but not from the major late promoter of adenovirus 2. The formation of stable initiation complexes at the CAD promoter was diminished in heat-treated extract; run-off transcripts, however, accumulated at the same rate as in untreated extract. The heat sensitivity of complex formation correlated with the heat sensitivity of DNA binding by transcription factor Sp1, which binds to two sites in the CAD promoter; moreover, both preformed initiation complexes and DNA-bound Sp1 were heat-resistant. Adding purified Sp1 to heat-treated extract restored complex formation. We propose that Sp1 activates CAD transcription by stabilizing initiation complexes at the CAD promoter.
Richard Kollmar - One of the best experts on this subject based on the ideXlab platform.
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start site selection at the tata less carbamoyl phosphate synthase glutamine hydrolyzing Aspartate Carbamoyltransferase dihydroorotase promoter
Journal of Biological Chemistry, 1994Co-Authors: Richard Kollmar, Kristine A. Sukow, Stephen K. Sponagle, Peggy J. FarnhamAbstract:Abstract Transcription of the carbamoyl-phosphate synthase (glutamine-hydrolyzing)/Aspartate Carbamoyltransferase/dihydroorotase (CAD) gene from the Syrian hamster, Mesocricetus auratus, starts at a single major site. We characterized the cis-acting elements that position RNA polymerase II at the correct start site in the CAD promoter. Sequence alignment showed that the CAD promoter lacks a TATA box, but contains a consensus initiator. Mutational analysis of the CAD promoter demonstrated that the sequences between -81 and +26 were sufficient for accurate and efficient transcription in vitro and in vivo; binding sites for the transcription factor Sp1 around -70 and -49 were necessary for transcriptional activity. The binding site at -49 directed initiation about 50 base pairs downstream. A ubiquitous activator protein, Honk, bound to the CAD promoter between -30 and -12, but did not participate in start site selection. The sequences around +1, which contain the consensus initiator, contributed to promoter activity; however, the presence of a consensus initiator in this region was neither necessary nor sufficient for transcription. We concluded from these results that the Sp1 binding site at -49 substituted for the missing TATA box and played a major role in start site selection at the CAD promoter.
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Heat sensitivity and Sp1 activation of complex formation at the Syrian hamster carbamoyl-phosphate synthase (glutamine-hydrolyzing)/Aspartate Carbamoyltransferase/dihydroorotase promoter in vitro.
The Journal of biological chemistry, 1992Co-Authors: Richard Kollmar, Mary J. Lindstrom, Peggy J. FarnhamAbstract:Abstract To study the regulation of transcription of the carbamoyl-phosphate synthase (glutamine-hydrolyzing)/Aspartate Carbamoyltransferase/dihydroorotase (CAD) gene from the Syrian hamster, Mesocricetus auratus, we developed a homologous in vitro transcription system on the basis of nuclear extract from Syrian hamster kidney cells. We optimized the reaction temperature and the concentrations of DNA template, KCl, and MgCl2 simultaneously with the response surface method and found an unusually low temperature optimum of 20 degrees C. We therefore investigated whether CAD transcription in vitro depended on a heat-labile component of nuclear extract. Preincubating extract alone at 30 degrees C reduced transcription from the CAD promoter but not from the major late promoter of adenovirus 2. The formation of stable initiation complexes at the CAD promoter was diminished in heat-treated extract; run-off transcripts, however, accumulated at the same rate as in untreated extract. The heat sensitivity of complex formation correlated with the heat sensitivity of DNA binding by transcription factor Sp1, which binds to two sites in the CAD promoter; moreover, both preformed initiation complexes and DNA-bound Sp1 were heat-resistant. Adding purified Sp1 to heat-treated extract restored complex formation. We propose that Sp1 activates CAD transcription by stabilizing initiation complexes at the CAD promoter.
Jozef Van Beeumen - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of sulfolobus acidocaldarius Aspartate Carbamoyltransferase in complex with its allosteric activator ctp
Biochemical and Biophysical Research Communications, 2008Co-Authors: Daniele De Vos, Tony Aerts, Filip Van Petegem, Jozef Van BeeumenAbstract:Aspartate Carbamoyltransferase (ATCase) is a paradigm for allosteric regulation of enzyme activity. B-class ATCases display very similar homotropic allosteric behaviour, but differ extensively in their heterotropic patterns. The ATCase from the thermoacidophilic archaeon Sulfolobus acidocaldarius, for example, is strongly activated by its metabolic pathway's end product CTP, in contrast with Escherichia coli ATCase which is inhibited by CTP. To investigate the structural basis of this property, we have solved the crystal structure of the S. acidocaldarius enzyme in complex with CTP. Structure comparison reveals that effector binding does not induce similar large-scale conformational changes as observed for the E. coli ATCase. However, shifts in sedimentation coefficients upon binding of the bi-substrate analogue PALA show the existence of structurally distinct allosteric states. This suggests that the so-called "Nucleotide-Perturbation model" for explaining heterotropic allosteric behaviour, which is based on global conformational strain, is not a general mechanism of B-class ATCases.
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structural investigation of cold activity and regulation of Aspartate Carbamoyltransferase from the extreme psychrophilic bacterium moritella profunda
Journal of Molecular Biology, 2007Co-Authors: Dirk De Vos, Paco Hulpiau, Bjorn Vergauwen, Jozef Van BeeumenAbstract:Aspartate Carbamoyltransferase (EC 2.1.3.2) is extensively studied as a model for cooperativity and allosteric regulation. The structure of the Escherichia coli enzyme has been thoroughly analyzed by X-ray crystallography, and recently the crystal structures of two hyperthermophilic ATCases of the same structural class have been characterized. We here report the detailed functional and structural investigation of the ATCase from the psychrophilic deep sea bacterium Moritella profunda. Our analysis indicates that the enzyme conforms to the E. coli model in that two allosteric states exist that are influenced by similar homotropic interactions. The heterotropic properties differ in that CTP and UTP inhibit the holoenzyme, but ATP seems to exhibit a dual regulatory pattern, activating the enzyme at low concentrations and inhibiting it in the mM range. The crystal structure of the unliganded M. profunda ATCase shows resemblance to a more extreme T state reported previously for an E. coli ATCase mutant. A detailed molecular analysis reveals potential features of adaptation to cold activity and cold regulation. Moreover, M. profunda ATCase presents similarities with certain mutants of E. coli ATCase altered in their kinetic properties or temperature relationships. Finally, structural and functional comparison of ATCases across the full physiological temperature range agrees with an important, but fundamentally different role for electrostatics in protein adaptation at both extremes, i.e. an increased stability through the formation of ion pairs and ion pair networks at high physiological temperatures, and an increased flexibility through enhanced protein solvation at low temperatures.
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Expression, purification, crystallization and preliminary X-ray crystallographic studies of a cold-adapted Aspartate Carbamoyltransferase from Moritella profunda
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2005Co-Authors: Dirk De Vos, Paco Hulpiau, Bjorn Vergauwen, Savvas N. Savvides, Jozef Van BeeumenAbstract:Aspartate Carbamoyltransferase (ATCase) catalyzes the carbamoylation of the α-amino group of l-Aspartate by carbamoyl phosphate (CP) to yield N-carbamoyl-l-Aspartate and orthophosphate in the first step of de novo pyrimidine biosynthesis. Apart from its key role in nucleotide metabolism, the enzyme is generally regarded as a model system in the study of proteins exhibiting allosteric behaviour. Here, the successful preparation, crystallization and diffraction data collection of the ATCase from the psychrophilic bacterium Moritella profunda are reported. To date, there is no structural representative of a cold-adapted ATCase. The structure of M. profunda ATCase is thus expected to provide important insights into the molecular basis of allosteric activity at low temperatures. Furthermore, through comparisons with the recently reported structure of an extremely thermostable ATCase from Sulfolobus acidocaldarius, it is hoped to contribute to general principles governing protein adaptation to extreme environments. A complete native data to 2.85 A resolution showed that the crystal belongs to space group P3221, with unit-cell parameters a = 129.25, b = 129.25, c = 207.23 A, α = β = 90, γ = 120°, and that it contains three catalytic and three regulatory subunits per asymmetric unit. The three-dimensional structure of the Escherichia coli ATCase was sufficient to solve the structure of the M. profunda ATCase via the molecular-replacement method and to obtain electron density of good quality.
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crystal structure of t state Aspartate Carbamoyltransferase of the hyperthermophilic archaeon sulfolobus acidocaldarius
Journal of Molecular Biology, 2004Co-Authors: Dirk De Vos, Nicolas Glansdorff, Christianne Legrain, Filip Van Petegem, Han Remaut, Jozef Van BeeumenAbstract:Abstract Aspartate Carbamoyltransferase (ATCase) is a model enzyme for understanding allosteric effects. The dodecameric complex exists in two main states (T and R) that differ substantially in their quaternary structure and their affinity for various ligands. Many hypotheses have resulted from the structure of the Escherichia coli ATCase, but so far other crystal structures to test these have been lacking. Here, we present the tertiary and quaternary structure of the T state ATCase of the hyperthermophilic archaeon Sulfolobus acidocaldarius ( Sa ATC T ), determined by X-ray crystallography to 2.6 A resolution. The quaternary structure differs from the E. coli ATCase, by having altered interfaces between the catalytic (C) and regulatory (R) subunits, and the presence of a novel C1–R2 type interface. Conformational differences in the 240s loop region of the C chain and the C-terminal region of the R chain affect intersubunit and interdomain interfaces implicated previously in the allosteric behavior of E. coli ATCase. The allosteric-zinc binding domain interface is strengthened at the expense of a weakened R1–C4 type interface. The increased hydrophobicity of the C1–R1 type interface may stabilize the quaternary structure. Catalytic trimers of the S. acidocaldarius ATCase are unstable due to a drastic weakening of the C1–C2 interface. The hyperthermophilic ATCase presents an interesting example of how an allosteric enzyme can adapt to higher temperatures. The structural rearrangement of this thermophilic ATCase may well promote its thermal stability at the expense of changes in the allosteric behavior.
David R. Evans - One of the best experts on this subject based on the ideXlab platform.
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Functional linkage between the glutaminase and synthetase domains of carbamoyl-phosphate synthetase. Role of serine 44 in carbamoyl-phosphate synthetase-Aspartate Carbamoyltransferase-dihydroorotase (cad).
The Journal of biological chemistry, 1999Co-Authors: Anura Hewagama, Hedeel I. Guy, John F. Vickrey, David R. EvansAbstract:Mammalian carbamoyl-phosphate synthetase is part of carbamoyl-phosphate synthetase-Aspartate Carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that also catalyzes the second and third steps of pyrimidine biosynthesis. Carbamoyl phosphate synthesis requires the concerted action of the glutaminase (GLN) and carbamoyl-phosphate synthetase domains of CAD. There is a functional linkage between these domains such that glutamine hydrolysis on the GLN domain does not occur at a significant rate unless ATP and HCO3−, the other substrates needed for carbamoyl phosphate synthesis, bind to the synthetase domain. The GLN domain consists of catalytic and attenuation subdomains. In the separately cloned GLN domain, the catalytic subdomain is down-regulated by interactions with the attenuation domain, a process thought to be part of the functional linkage. Replacement of Ser44 in the GLN attenuation domain with alanine increases thekcat/Km for glutamine hydrolysis 680-fold. The formation of a functional hybrid between the mammalian Ser44 GLN domain and the Escherichia coli carbamoyl-phosphate synthetase large subunit had little effect on glutamine hydrolysis. In contrast, ATP and HCO3− did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted. In accord with this interpretation, the rate of glutamine hydrolysis and carbamoyl phosphate synthesis were no longer coordinated. Approximately 3 times more glutamine was hydrolyzed by the Ser44 → Ala mutant than that needed for carbamoyl phosphate synthesis. Ser44, the only attenuation subdomain residue that extends into the GLN active site, appears to be an integral component of the regulatory circuit that phases glutamine hydrolysis and carbamoyl phosphate synthesis.
