Research Doctorate - Doctor of Philosopy (PhD) / Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated acutely by a combination of phosphorylation of three key serine (Ser) residues (Ser19, Ser31 and Ser40), and feedback inhibition by the catecholamines. Phosphorylation of Ser40 directly increases TH activity by relieving feedback inhibition of the enzyme. The phosphorylation of Ser19 or Ser31 can potentiate the phosphorylation of Ser40 in a process known as hierarchical phosphorylation. The 2 major human TH isoforms, hTH1 and hTH2, are differentially regulated by hierarchical phosphorylation in vitro. In this study, the human neuroblastoma SH-SY5Y cell line has been transfected with hTH1 and hTH2, and it has been demonstrated that phosphorylation of Ser31 potentiates the phosphorylation of Ser40 in hTH1. Phosphorylation of the equivalent Ser31 residue in hTH2 was not detectable, and thus this enzyme is not subject to Ser31-mediated hierarchical phosphorylation of Ser40 in situ. This is the first study to demonstrate that hTH1 and hTH2 are differentially regulated in situ. In addition, we have examined the nature of feedback inhibition of TH by the catecholamines. In addition to the high affinity, non-dissociable dopamine binding that is relieved by Ser40 phosphorylation, we have identified a second low affinity, readily dissociable binding site which regulates TH activity both in vitro and in situ regardless of the phosphorylation state of the enzyme. This low affinity binding site responds to changes in cytosolic catecholamine levels in situ in order to regulate TH activity. This work has contributed to our understanding of the complex nature of the regulation of TH activity.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19883号 / 理博第4210号 / 新制||理||1605(附属図書館) / 32960 / 京都大学大学院理学研究科化学専攻 / (主査)教授 三木 邦夫, 教授 杉山 弘, 教授 藤井 紀子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Thesis advisor: Evan R. Kantrowitz / The enzyme aspartate transcarbamoylase (ATCase) is an enzyme in the pyrimidine nucleotide biosynthetic pathway. It was once an attractive target for anti-proliferation drugs but has since become a teaching model due to kinetic properties such as cooperativity and allostery exhibited by the Escherichia coli form of the enzyme. ATCase from E. coli has been extensively studied over that last 60 years and is the textbook example of allosteric enzymes. Through this past research it is understood that ATCase is allosterically inhibited by CTP, the end product of pyrimidine biosynthesis, and allosterically activated by ATP, the end product of the parallel purine biosynthetic pathway. Part of the work discussed in this dissertation involves further understanding the catalytic properties of ATCase by examining an unregulated trimeric form from Bacillus subtilis, a bacterial ATCase that more closely resembles the mammalian form than E. coli ATCase. Through X-ray crystallography and molecular modeling, the complete catalytic cycle of B. subtilis ATCase was visualized, which provided new insights into the manifestation of properties such as cooperativity and allostery in forms of ATCase that are regulated. Most of the work described in the following chapters involves understanding allostery in E. coli ATCase. The work here progressively builds a new model of allostery through new X-ray structures of ATCase*NTP complexes. Throughout these studies it has been determined that the allosteric site is bigger than previously thought and that metal ions play a significant role in the kinetic response of the enzyme to nucleotide effectors. This work proves that what is known about ATCase regulation is inaccurate and that currently accepted, and taught, models of allostery are wrong. This new model of allostery for E. coli ATCase unifies all old and current data for ATCase regulation, and has clarified many previously unexplainable results. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Aspartate transcarbamoylase (ATCase; encoded by pyrBI genes) is one of the most studied regulatory enzymes in bacteria. It is feedback inhibited by cytidine triphosphate (CTP) and activated by adenosine triphosphate (ATP). Much is known about the catalytic site of the enzyme, not nearly as much about the regulatory site, to which CTP binds. Until now a positive selection for feedback-modified mutants was not available. The selection we have developed involves the use of a pyrA deletion in S. typhimurium. This strain lacks carbamoylphosphate and requires both a pyrimidine and arginine for growth. In this strain citrulline is used to satisfy the pyrimidine and arginine requirements. The minimal flow through the pyrimidine pathway from the citrulline-produced carbamoylphosphate is exquisitely sensitive to feedback control of ATCase by CTP. By elevating the CTP pool, via exogenous cytidine, in a strain that also contains a cytidine deaminase mutant (cdd) growth can be stopped completely, indicating 100% inhibition. It was therefore possible to measure in vivo feedback inhibition of ATCase among the citrulline users and to isolate a family of ATCase regulatory mutants with either modified or no response to effectors.

Aspartokinase is the first enzyme of the aspartate family amino acids biosynthetic pathway. Cephamycin C is a &amp / #946 / -lactam antibiotic produced as a secondary metabolite via the enzymatic reactions in the lysine branch of this pathway in Streptomyces clavuligerus. The aspartokinase activity of S. clavuligerus is under concerted feedback inhibition by two of the end product amino acids, lysine plus threonine. It is also known that carbon flow through the lysine branch of the aspartate pathway is rate limiting step in the formation of cephamycin C. Therefore, genetic alterations in the regulatory regions of the aspartokinase enzyme are expected to lead to an increased cephamycin C production. The aim of this study was to obtain S. clavuligerus mutants that possess aspartokinase enzyme insensitive to feedback inhibition by lysine and threonine, identification of the mutation(s) accounting for the resistance being the ultimate goal. For this aim, chemical mutagenesis was employed to increase random mutation rate and a population of lysine anti-metabolite resistant S. clavuligerus mutants that can grow in the presence of S-(2-aminoethyl)-L-cysteine was obtained. The mutants were screened for their aspartokinase insensitivity via enzyme assays and one mutant exhibiting the highest level of deregulation was assessed for its cephamycin C production. The results revealed a 2-fold increase in specific production of the antibiotic. As a member of &amp / #946 / -lactam class antibiotics, cephamycin C has an importance in medicine. Therefore, the mutant strain obtained might be a candidate for industrial production of the compound.

No description available.

Die Tetrapyrrolbiosynthese (TBS) führt zu wichtigen Endprodukten wie Häm und Chlorophyll. Das gemeinsame Vorstufenmolekül aller Tetrapyrrole ist die 5-Aminolävulinsäure (ALA), die in Pflanzen über den C5-Weg aus Glutamat synthetisiert wird. Das erste spezifische Enzym der ALA-Synthese und somit auch der TBS ist die Glutamyl-tRNA Reduktase (GluTR). Sie unterliegt als Schlüsselenzym einer strengen Regulation. Aufgrund der unterschiedlichen Expression der HEMA-Gene in Arabidopsis wird ein differenzieller Beitrag der GluTR-Isoformen zu den Endprodukten der TBS vermutet. Analysen von knockout-Mutanten gaben Aufschluss darüber, inwiefern die Isoformen den Verlust des jeweils anderen kompensieren können. Die knockout-Mutante von HEMA1 zeigte einen blassgrünen Phänotyp, war nicht mehr in der Lage photoautotroph zu wachsen und demonstrierte eine essentielle Rolle der GluTR1 gegenüber GluTR2, wohingegen hema2-Mutanten einen wildtypartigen Phänotyp aufzeigten. Die Bedeutung der N-terminalen GluTR-Domäne in der posttranslationalen Regulation der ALA-Synthese wurde durch BiFC-Analysen und Komplementationsversuche aufgeklärt. BiFC-Analysen zeigten eine Interaktion der N-terminalen Domäne der GluTR1 mit Proteinen der Clp Proteasen und dem GluTR-Bindeprotein (GBP). Veränderte GluTR-Stabilitäten in gbp-Mutanten lassen eine schützende Funktion des GBP gegenüber dem Abbau des Proteins postulieren. Die Expression einer N-terminal verkürzten GluTR1 komplementierte hema1-Mutanten vollständig. Die in diesen Pflanzen und in clp-Mutanten beobachteten erhöhten GluTR1-Proteinstabilitäten im Dunkeln lassen einen Abbau der GluTR durch Clp Proteasen vermuten, bei dem der N-Terminus des Enzyms für die Substraterkennung notwendig zu sein scheint. Die Detektion von erhöhten Pchlid-Mengen als Folge der erhöhten Proteinstabilität in Linien, die die verkürzte GluTR1 exprimierten, demonstriert erstmals die Bedeutung einer kontrollierten Proteolyse der GluTR in der Regulation der ALA-Synthese. / In plants 5-aminolevulinic acid (ALA) is the common precursor of all tetrapyrrols and formed from glutamate via the C5 pathway. Glutamyl-tRNA reductase (GluTR) is the initial enzyme of ALA synthesis and thus tetrapyrrole biosynthesis (TBS). The most important control point of the TBS is the synthesis of ALA and GluTR is the key enzyme, that is tightly regulated. Due to the different expression of HEMA genes in Arabidopsis, a differential contribution to endproducts of the TBS is proposed for GluTR isoforms. Analysis of knockout mutants gave some indications of how the isoforms can compensate each other. I introduced a new knockout mutant of HEMA1 that was pale-green and not able to grow photoautotrophically, indicating that the remaining GluTR2 does not sufficiently compensate ALA synthesis for the extensive needs of chlorophyll. In contrast, hema2 mutants were wild-type-like. The function of the N-terminal region of GluTR1 in posttranslational regulation has been analyzed by BiFC analysis and complementation experiments of hema1. BiFC analysis showed an interaction of the N-terminal region of GluTR1 with the GluTR binding protein (GBP) and with proteins of the Clp proteases. Mutants of GBP revealed a decreased GluTR1 stability during the dark period, indicating a protective role of GBP against proteolysis of GluTR1 in darkness. The expression of a GluTR1 lacking the N-terminal amino acid residues successfully complemented hema1. These plants as well as clp mutants revealed an increased GluTR1 stability in darkness, suggesting a degradation of the protein through Clp proteases. Thereby, the N-terminal region of GluTR1 seems to be necessary for the recognition by Clp proteins. The observed high amount of truncated GluTR1 in transformed hema1 mutants was caused by the increased GluTR1 stability and lead to an accumulation of Pchlide in prolonged dark periods, demonstrating the importance of a controlled proteolysis of GluTR in the regulation of ALA synthesis.