• Login
    View Item 
    •   Home
    • UA Graduate and Undergraduate Research
    • UA Theses and Dissertations
    • Dissertations
    • View Item
    •   Home
    • UA Graduate and Undergraduate Research
    • UA Theses and Dissertations
    • Dissertations
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Browse

    All of UA Campus RepositoryCommunitiesTitleAuthorsIssue DateSubmit DateSubjectsPublisherJournalThis CollectionTitleAuthorsIssue DateSubmit DateSubjectsPublisherJournal

    My Account

    LoginRegister

    About

    AboutUA Faculty PublicationsUA DissertationsUA Master's ThesesUA Honors ThesesUA PressUA YearbooksUA CatalogsUA Libraries

    Statistics

    Most Popular ItemsStatistics by CountryMost Popular Authors

    Discovery and Biochemical Studies of Enzymes Involved in the Queuosine Biosynthetic Pathway

    • CSV
    • RefMan
    • EndNote
    • BibTex
    • RefWorks
    Thumbnail
    Name:
    etd_13572_sip1_m.pdf
    Size:
    10.53Mb
    Format:
    PDF
    Download
    Author
    Miles, Zachary David
    Issue Date
    2014
    Keywords
    Biochemistry
    Advisor
    Bandarian, Vahe
    
    Metadata
    Show full item record
    Publisher
    The University of Arizona.
    Rights
    Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
    Embargo
    Release 03-Nov-2015
    Abstract
    Queuosine (Q) is a hypermodified nucleoside present at the wobble position in the 5'-GUN-3' anticodon loop of asparagine, aspartic acid, histidine, and tyrosine encoding tRNAs. This hypermodified base contains a 7-deazapurine structure common to many antibiotics, antivirals, and antineoplastic secondary metabolites. It is synthesized de novo in prokaryotes from GTP, whereas in eukaryotes it is ingested from dietary sources as the free-base queuine and exchanged for guanine in mature tRNA. Queuosine has been associated with many physiological phenomena such as cancer pathology, neoplasia, and virulence; although a discrete physiological relevance of this modification remains to be determined due to the lack of observable phenotypes associated with its respective loss. However, conservation of this modification across almost all domains of life suggests that it confers a selective advantage to its host. CPH₄ synthase (QueD) catalyzes the second step in the queuosine biosynthetic pathway entailing conversion of 7,8-dihydroneopterin triphosphate to 6-carboxy-5,6,7,8-tetrahydropterin. By contrast, the almost structurally identical mammalian homolog catalyzes the conversion of the same substrate to 6-pyruvoyltetrahydropterin, which is an intermediate in the tetrahydrobiopterin biosynthetic pathway. Herein, we present multiple X-ray crystal structures coupled with biochemical studies that reveal an additional active site catalytic dyad in QueD responsible for the differing activity. Prior to the studies detailed in this dissertation, the enzyme responsible for catalyzing the final step in the pathway, conversion of epoxyqueuosine to queuosine, had yet to be elucidated. To search for this enzyme, we screened a library of isogenic variants of Escherichia coli where all of the nonessential genes are sequentially inactivated. RNA was extracted from each strain and analyzed using LC-MS methods, which led to the identification of a mutant strain that accumulates epoxyqueuosine and contains no queuosine. The enzyme, epoxyqueuosine reductase (QueG), has been subjected to extensive biochemical analyses both in vitro and in vivo. From these studies, we have shown QueG to contain two [4Fe-4S] clusters and cobalamin as cofactors that are absolutely required for catalysis. In addition, we have identified conserved residues that affect activity and modulate the coordination sphere around the cobalamin cofactor.
    Type
    text
    Electronic Dissertation
    Degree Name
    Ph.D.
    Degree Level
    doctoral
    Degree Program
    Graduate College
    Biochemistry
    Degree Grantor
    University of Arizona
    Collections
    Dissertations

    entitlement

     
    The University of Arizona Libraries | 1510 E. University Blvd. | Tucson, AZ 85721-0055
    Tel 520-621-6442 | repository@u.library.arizona.edu
    DSpace software copyright © 2002-2017  DuraSpace
    Quick Guide | Contact Us | Send Feedback
    Open Repository is a service operated by 
    Atmire NV
     

    Export search results

    The export option will allow you to export the current search results of the entered query to a file. Different formats are available for download. To export the items, click on the button corresponding with the preferred download format.

    By default, clicking on the export buttons will result in a download of the allowed maximum amount of items.

    To select a subset of the search results, click "Selective Export" button and make a selection of the items you want to export. The amount of items that can be exported at once is similarly restricted as the full export.

    After making a selection, click one of the export format buttons. The amount of items that will be exported is indicated in the bubble next to export format.