Dissertation
STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF CLASS III PLANT PEROXIDASE, JUNCTIN CALSEQUESTRIN INTERACTION, AND MYCOBACTERIUM TUBERCULOSIS -LACTAMASE
Doctor of Philosophy (PhD), Washington State University
01/2018
Handle:
https://hdl.handle.net/2376/111487
Abstract
This dissertation is a collective work consisting of the studies on the structural and biochemical characterization of three separate proteins. The first protein was a class III plant peroxidase, PviPRX9 from switchgrass. The crystal structure, kinetic experiments, molecular docking, as well as expression patterns of PviPRX9 across multiple tissues and treatments, and its levels of coexpression with the majority of genes in the monolignol biosynthesis pathway, revealed the function of PviPRX9 in lignification. Significantly, our study suggested that PviPRX9 has the ability to oxidize a broad range of phenylpropanoids with rather similar efficiencies, which reflects its role in fortification of cell walls during normal growth and root development, and in response to insect feeding. We discovered that PviPRX9 is expressed in conjunction with most of the enzymes of lignin biosynthesis pathway. Additionally, we found that PviPRX9 catalyzes the oxidation of the major components making up the lignin polymer. The second protein was on the Ca2+ binding protein calsequestrin. Skeletal calsequestrin, Casq1, is the major Ca2+ binding and storage protein in the sarcoplasmic reticulum of skeletal muscle cells. We investigated the varied oligomeric response of Casq1 for multiple species at different concentration of Ca2+. We determined high and low Ca2+ structures for bat and mouse Casq1. We also conducted multi-angle light scattering molecular weight determination and turbidity assays to establish a profile of oligomeric response for Casq1 in response to changes in Ca2+ concentrations. Additionally, isothermal titration calorimetry was carried out to establish the binding affinity between Casq1 and junction. The third protein was a class A serine -lactamase, BlaC conferring Mycobacterium tuberculosis resistance to -lactam antibiotic. The primary mechanism of bacterial resistance to -lactam antibiotics, is expression of a -lactamase capable of hydrolyzing the -lactam ring. We conducted protein X-ray crystallographic analysis of the inhibition of BlaC upon exposure to the inhibitor dibenzoyl phosphate. For the first time, we provided crystal structure evidences to show that serine -lactamases can be phosphorylated at the nucleophilic serine by a new potential -lactamase inhibitor. Additionally, we conducted phylogenetic and comparative structural analysis of BlaC and other -lactamases.
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Details
- Title
- STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF CLASS III PLANT PEROXIDASE, JUNCTIN CALSEQUESTRIN INTERACTION, AND MYCOBACTERIUM TUBERCULOSIS -LACTAMASE
- Creators
- Timothy Walter Moural
- Contributors
- ChulHee Kang (Advisor)Clifford E Berkman (Committee Member)James A Brozik (Committee Member)Jeffrey P Jones (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Department of Chemistry
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 129
- Identifiers
- 99900581510401842
- Language
- English
- Resource Type
- Dissertation