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.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/77289 |
| Date | 30 December 2010 |
| Creators | Mehere, Prajwalini V. |
| Contributors | Biochemistry, Li, Jianyong, Bevan, David R., Larson, Timothy J., Zhu, Jinsong |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation, Text |
| Format | application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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