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CTP Synthase Sulfolobus Solfataricus

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CTP Synthase Sulfolobus Solfataricus CTP Synthase from Sulfolobus solfataricus Master Thesis in Biochemistry Iben Havskov Lauritsen June 2010 University of Copenhagen, Department of Biology Supervisor: Kaj Frank Jensen PREFACE This work represents my master thesis in Biochemistry at the University of Copenhagen. Most of the work was carried out at the Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen under supervision by Kaj Frank Jensen. Crystallization was carried out at Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen under supervision by Eva Johansson. The preliminary work I did on solving the structure was done at Department of Chemistry, Technical University of Denmark under supervision by Pernille Harris. She later finished solving the structure, and a paper on the work is about to be submitted. I thank Eva Johansson and Pernille Harris for teaching me how to make protein crystals and how to solve the structure of them. That has been a very exciting part of the project for me. I also thank Lise Schack for endless help and good company in the laboratory. Finally I thank Kaj Frank Jensen for encouraging supervision. _____________________________________________ Iben Havskov Lauritsen June 2010, Copenhagen ABSTRACT CTP synthase from the extreme thermoacidophilic archaeon Sulfolobus solfataricus has been investigated in several ways in this study. CTP synthase is responsible for de novo synthesis of CTP from UTP. The first part of the reaction is the deamination of glutamine to generate ammonia for the second part of the reaction, the CTP synthesis. This work is mostly focused on the kinetics of the first part of the reaction. GTP was found to activate this glutaminase reaction, by both lowering Km and increasing kcat. Furthermore the activating effect of GTP was found to be increased 20-fold in the presence of the substrates ATP and UTP. ATP and UTP also increased the reaction rate of the glutaminase reaction without GTP. The activation by ATP and UTP was investigated in more detail, and it turned out that neither ATP nor UTP alone displayed much activation. But when ATP was present, increasing concentrations of UTP gave increasing activity rates. Both ATP and UTP displayed positive cooperativity. GTP had a different effect on the production of CTP than it had on the activation of the glutaminase reaction; it was an activator of the production of CTP up to 50-100 M GTP, but inhibited the reaction at higher concentrations. This showed that the two reactions of CTP synthase from S. solfataricus are only coupled up to this concentration of GTP. Size analysis by sucrose gradient sedimentation showed that CTP synthase was a dimer that tetramerized in the presence of nucleotides. The protein was crystallized and the structure solved by X-ray diffraction. Crystal structures of CTP synthases from other organisms had already been published, but unlike these, which had crystallized as tetramers, CTP synthase from Sulfolobus solfataricus had crystallized as a dimer. The enzyme was found to be protected from trypsin-proteolysis by the nucleotides ATP, UTP and CTP. It was also found that CTP could be produced with ammonium chloride as ammonia source instead of glutamine. INTRODUCTION Sulfolobus solfataricus Sulfolobus solfataricus is an aerobic, hyperthermophilic, acidophilic crenarchaeon (Archaea) that grows optimally at 80 °C and pH 2-4. It metabolizes sulphur and is able to grow on a variety of carbon sources (2-4). Sulfolobus strains are widely studied, and are model organisms for studying the Crenarchaea branch of the Archaea kingdom, in part because they are relatively easy to culture. Strains of the genus Sulfolobus have been isolated all over the world from acidic solfataric fields, and are basically found everywhere where there is volcanic activity. The strain used in this work, S. solfataricus strain P2, was isolated from a solfataric field near Naples, Pisciarelly (3), and its genome has been sequenced (4). The genes for pyrimidine synthesis are highly conserved and are concentrated in two operon-like structures within 5.5 kb. Only genes encoding carbamoylphosphate synthetase are found elsewhere, clustered with arginine biosynthesis genes (4). Pyrimidine metabolism Nucleotides are essential for all organisms. Besides their role in energy metabolism, they are used for the synthesis of DNA, RNA, phospholipids and several co-enzymes. The purines and the pyrimidines are synthesized via two different pathways. Since the need for nucleotides is universal for all living (growing) organisms, these de novo pathways are essentially identical throughout the biological world. All sequenced bacterial genomes, except some intracellular parasites, encode the enzymes required for de novo biosynthesis of pyrimidine nucleotides (5). Most organisms also have salvage pathways, where nucleotides are synthesized from nucleosides or bases taken up by the cell or from enzymatic breakdown of nucleic acids (6). These salvage pathways differ between organisms. 1 - 2 ATP + HCO pyrA pyrB pyrC 3 Carbamyl phosphate Carbamyl aspartate Dihydroorotate + glutamine pyrD pyrG ndk pyrH CTP UTP UDP UMP pyrF OMP pyrE Orotate ndk udk upp CDP Uridine udp Uracil uraA Uracil from outside the cell cmk cdd codA CMP udk Cytidine rihA Cytosine codB Cytosine from outside the cell Figure 1. Schematic representation of pyrimidine nucleotide de novo and salvage pathways for E. coli. At the top of the figure is shown the unbranched de novo pathway leading ultimately to CTP and below that are shown the salvage reaction pathways. The enzymes encoded by the genes in the de novo pathway are: Carbamoyl phosphate synthase/CP synthase (pyrA); aspartate transcarbamoylase/ATCase ( pyrB); dihydroorotase/DHOase (pyrC); dihydroorotate dehydrogenase/DHODase (pyrD); orotate phosphoribosyltransferase/OPRTase (pyrE); orotidylate decarboxylase/OMP decarboxylase (pyrF);UMP kinase (pyrH); nucleoside diphosphate kinase/NDP kinase (ndk); CTP synthase (pyrG). Enzymes of the salvage pathways are: CMP kinase (cmk); uridine kinase (udk); cytidine deaminase (cdd); uridine phosphorylase (udp); ribonucleoside hydrolase A (rihA); Cytosine deaminase (codA); UPRTase (upp). The cytoplasmic membrane proteins are cytosine permease (codB) and uracil permease (uraA). CTP synthase The pyrimidine de novo pathway is unbranched, and the last step is the amination of UTP to produce CTP (7). This process is catalyzed by CTP synthase/synthetase (CTPS) which is encoded by pyrG in bacteria, ctrA in some gram positive bacteria, URA7 and URA8 in Saccharomyces cerevisiae and CTPS1 and CTPS2 in humans. Most eukaryotes express two isoforms (8). The enzyme has been studied intensively for many years. Especially the Escherichia coli enzyme is well characterized, but a lot of work has also been done on CTPS from S. cerevisiae, Lactococcus lactis and recently also on human CTPS (9). In 2004 the structure of both E. coli and Thermus thermophilus CTPS was solved (8;10), and the structure of the synthase domain of human CTPS has also been published (11). CTP synthase is a Mg2+-demanding, glutamine amidotranferase, weighing approximately 60 kDa. It consists of a single polypeptide chain, and is a homotetramer in its fully active form. The overall reaction of CTP synthase is the deamination of glutamine to glutamate, and the amination of UTP to 2 CTP at the expense of an ATP to ADP dephosphorylation (12). CTPS from L. lactis and S. cerevisiae are also able to catalyze the conversion of dUTP to dCTP. For L. lactis CTPS the reaction is probably not physiologically relevant (13), but it might play a role in the nucleotide metabolism of S. cerevisiae (14). The action of CTPS consists of two separate reactions, taking place in different domains of the enzyme. Figure 2. The reaction catalyzed by CTPS. CTPS catalyzes both hydrolysis of glutamine to glutamate and ammonia and ATP dependent synthesis of CTP from UTP. Picture is reproduced from (10). Glutamine amide transfer domain In the C-terminal end of the enzyme is a class I glutamine amidotransferase (GATase) domain, with the catalytic triad Cys-His-Glu. Here ammonia is generated by glutamine hydrolysis via the formation of a covalent cysteinyl-glutamyl intermediate (8;10;12;15;16) Amidoligase/synthase domain In the N-terminal end of the enzyme is the synthase domain (amidoligase, ALase). This domain structurally resembles dethiobiotin synthase (8;10). Here UTP is activated by ATP-dependent phosphorylation at the 4-position and then reacts with ammonia (12;17-19). The enzyme can use external ammonia instead of nascent ammonia from glutamine hydrolysis (20;21), but this is not thought to be of physiological importance (22). Ammonia tunnel The ammonia from the glutaminase domain is delivered to the synthase domain via a tunnel in the enzyme (8;23-25). Such a tunnel is seen in several other enzymes with GATase domains (26). In most of these enzymes with molecular tunnels, the tunnel is preformed, and 3 exists also in the absence of ligands bound to the active site, but there is at least one example where the tunnel not formed before substrates have bound to their respective active sites (27). In the crystal structures of CTPS from T. thermophilus (10), no ammonia tunnel is seen, but it is suggested that binding of ATP and UTP makes the glutaminase domain approach the synthase domain to make a connecting channel between the two active sites, thereby coupling the two half-reactions. In the structure of E. coli CTPS (8), a tunnel is seen between the two active sites. The tunnel is accessible to solvent close to the GATase site at the proposed GTP binding site, and this is probably where exogenous ammonia enters the active site (8). Regulation of the reaction Perhaps not surprisingly, the reaction is highly regulated. It is a key step of controlling the level of CTP in the cell, and the activity of CTPS is regulated by interaction with all four ribonucleotide triphosphates. It is directly inhibited by its own product, CTP, which act as a negative feedback control.
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