Tyrosine is involved in many biological processes including protein synthesis. This dissertation is focused on two different aspects: tyrosine catabolism and tyrosine derivative metabolism. Tyrosine undergoes degradation via tyrosine aminotransferase (TAT). Deficiency of TAT leads to some disease conditions or tyrosinemia type II. TAT has been characterized in several species, including humans. Mouse tyrosine aminotransferase was used as a model protein for the tyrosine catabolism portion of this study. Characterization of TAT included its expression in a bacterial expression system, purification using various chromatographic techniques, crystallization under different conditions, and its kinetic analysis, and molecular dynamics simulations. Based on sequence, structure, and kinetic data we have shown that mouse TAT behaves like human TAT. Our crystallization studies added new insights into the mechanism of TAT by shedding light on involvement of a disulfide bond in the regulation of mTAT. Molecular dynamics analysis provided perspective on the differences (preferences) in the substrate specificities of mouse and Trypanosome cruzi TAT. Tyrosine is a precursor of several key neurotransmitters. These neurotransmitters must be regulated in order to function properly. The hypothetical N-acetyltransferases from Aedes aegypti were used as model proteins for investigation of tyrosine derivative metabolism. We found nine potential arylalkylamine N-acetyltransferase (AANAT) genes in Ae. aegypti. Phylogenetic analysis suggests that these Ae. aegypti AANATs (AeAANATs) can be further divided into three clusters. Phylogenetic analysis suggests that insect AANATs may have different functions as compared with the mammalian AANATs, for which function is specific to circadian rhythm regulation. PCR amplification indicates that eight of the nine putative AeAANATs are expressed in the mosquito. Expression of the eight putative AeAANATs and substrate screening of their recombinant proteins against dopamine, octopamine, tyramine, epinephrine, tryptamine, 5-hydroxytryptamine, and methoxytryptamine established that five of the eight putative AeAANATs are true AANATs. The discontinuous expression profiles of AeAANAT genes were studied in detail. Six of the AeAANATs were expressed in the head before and after blood feeding, suggesting their potential role in neurotransmission inactivation. Down-regulation of these genes after blood feeding suggests that blood feeding or factors related to blood feeding impact on the regulation of these genes. Kinetic studies determined that two AeAANAT proteins are highly efficient in mediating the acetylation of dopamine and 5-hydroxytryptamine. Substrate analysis of AeAANATs supports the notion that acetylation of arylalkylamines is vital to the biology of mosquito species, and that these genes emerged in response to specific pressures related to necessities for biogenic amine acetylation. / Ph. D.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 122-137).

Thesis (Ph. D.)--Cornell University, December, 1997. / Vita. Includes bibliographical references (leaves 96-113).

Proefschrift--Utrecht. / Title also in English. Summary in English. Vita. Includes bibliographical references.

Proefschrift--Utrecht. / Title also in English. Summary in English. Vita. Includes bibliographical references.

Thesis (Ph. D.)--Illinois State University, 2006. / Title from title page screen, viewed on June 8, 2007. Dissertation Committee: Kevin A. Edwards (chair), Craig Gatto, Wade A. Nichols, Samuel Galewsky, John C. Sedbrook. Includes bibliographical references (leaves 218-245) and abstract. Also available in print.

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A major pathway of tyramine and dopamine synthesis in insects is through the decarboxylation of tyrosine and DOPA, respectively. Although tyrosine decarboxylase (TDC) has been mentioned in some reports, it has never been critically analyzed. The high sequence identity shared by tyrosine decarboxylase and DOPA decarboxylase in insects, and the similar structures of the substrates, tyrosine and DOPA, raise the possibility that both tyrosine decarboxylase and DOPA decarboxylase (DDC) have activities to tyrosine and DOPA. In this study, after tyrosine decarboxylase and DOPA decarboxylase enzymes of Anopheles gambiae were expressed, their substrate specificities and biochemical properties were critically analyzed. My results provide clear biochemical evidence establishing that the mosquito tyrosine decarboxylase functions primarily on the production of tyramine with low activity to DOPA. In contrast, mosquito DOPA decarboxylase is highly specific to DOPA with essentially no activity to tyrosine. / Master of Science in Life Sciences

Les arrestines sont connues pour leurs rôles dans la désensibilisation et l'endocytose des récepteurs couplés aux protéines G (RCPGs). Au fil des ans, plusieurs partenaires de ces protéines adaptatrices ont été identifiés, notamment diverses molécules impliquées dans la signalisation, incluant les kinases ERK, JNK, Akt, Raf et Src. Ainsi, les arrestines interagissent avec plusieurs kinases, mais seulement avec deux phosphatases, PPA2 et MKP7. Récemment, notre laboratoire a démontré une nouvelle interaction entre les arrestines non-visuelles et la phosphatase Shp2. En effet, des essais in vitro et in cellulo ont montré une interaction directe entre les deux protéines peuvent interagir ensembles et ce directement. Or, nous en sommes venus à nous demander si les arrestines peuvent être phosphorylées sur leurs résidus tyrosine et si cette modification pourrait être régulée par des partenaires connus, soient Src et Shp2. Il a déjà été montré que l'arrestine 2 peut être phosphorylée sur un résidu tyrosine qui lui est unique et que cette modification expliquerait peut-être les différences de modulation entre les arrestines non-visuelles. Par contre, il n'existe encore aucune preuve que l'arrestine 3 puisse aussi être phosphorylée. D'abord, nous démontrons pour la première fois que l'arrestine 3, tout comme l'arrestine 2, est phosphorylée sur des résidus tyrosines et que cette modification peut être amenée par l'activité de Src. Ensuite, un double mutant ponctuel de l'arrestine 3 a été construit afin de cibler les tyrosines régulées. Il semble que les tyrosines 380 et 404 de l'arrestine 3, soient d'importants sites de phosphorylation. Ce nouveau mutant de l'arrestine 3 représente un excellent outil afin de déterminer le rôle de la phosphorylation des tyrosines de l'arrestine 3. Aussi, puisque ces deux tyrosines sont absentes de la séquence de l'arrestine 2, leur phosphorylation pourrait être à la base des différences fonctionnelles entre les arrestines 2 et 3. De plus, nous faisons la démonstration, en conditions de surexpression en cellules, que l'activité de Shp2 peut mener à la déphosphorylation des arrestines non-visuelles. Dès lors, nos études montrent d'abord que, tout comme son homologue, l'arrestine 3 peut être phosphorylée sur ses tyrosines. De plus, cette phosphorylation est non seulement régulée par la phosphatase Shp2, mais représente également un nouveau mécanisme potentiel de régulation des multiples fonctions des arrestines.