• About
    • Login
    View Item 
    •   Institutional Repository Home
    • Electronic Theses and Dissertations
    • Electronic Theses and Dissertations
    • View Item
    •   Institutional Repository Home
    • Electronic Theses and Dissertations
    • Electronic Theses and Dissertations
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Browse

    All of Institutional RepositoryCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsDepartmentThis CollectionBy Issue DateAuthorsTitlesSubjectsDepartment

    My Account

    LoginRegister

    Directed Biosynthesis of the Nucleoside Analog Drug Didanosine

    Nannemann, David Patrick
    : https://etd.library.vanderbilt.edu/etd-04042011-165321
    http://hdl.handle.net/1803/11998
    : 2011-04-06

    Abstract

    Nucleoside analogs comprise a large therapeutic class applied to the treatment of HIV, hepatitis, and other diseases. Their broad use and applicability are in contrast to their high manufacturing cost. Given the similarity of nucleoside analogs to natural compounds, it was hypothesized that biosynthetic pathways for their production could be formed through judicious selection of progenitor enzymes and enzyme engineering. Toward this aim, a prototype pathway has been generated for the directed biosynthesis of the antiretroviral drug didanosine (2´,3´-dideoxyinosine, ddI, Videx®) from 2,3-dideoxyribose, which in turn can be chemically synthesized from glutamic acid. Using new and extant structural and functional data, progenitor enzymes for this pathway have been identified and include human purine nucleoside phosphorylase (hPNP), Bacillus cereus phosphopentomutase (PPM) and Escherichia coli ribokinase. Enzymes of the pathway display low turnover for the targeted substrates; therefore, enzyme engineering methods have been utilized to improve turnover of each enzyme. RosettaLigand, a computational protein-small molecule docking algorithm, was used to predict transition state binding energies for active site mutants of hPNP at a single site, Tyr-88. Predicted transition state binding energies were correlated to experimentally-derived activation energies. Directed evolution of the best mutant, hPNP-Y88F, verified to have a 26-fold greater ddI turnover efficiency than wild type, to select for improved variants in E. coli resulted in a further 3-fold improvement. Basic characterization of PPM was necessary to enable enzyme engineering. The structure of PPM in the absence and presence of substrates and cofactors was determined through X-ray crystallography. PPM is a member of the alkaline phosphatase superfamily and exhibits intermolecular phosphate transfer, contrary to other superfamily members. Structures of PPM with substrate have allowed for identification of residues important in sugar orientation for targeted mutagenesis. Viability of the prototype pathway was demonstrated by directed biosynthesis of ddI from 2,3-dideoxyribose and hypoxanthine. Methods developed in this work could be applied to the synthesis of other nucleoside analogs facilitating large-scale, affordable treatment for HIV, hepatitis, or other diseases.
    Show full item record

    Files in this item

    Icon
    Name:
    Nannemann_PhD-Dissertation_Mar ...
    Size:
    10.11Mb
    Format:
    PDF
    View/Open

    This item appears in the following collection(s):

    • Electronic Theses and Dissertations

    Connect with Vanderbilt Libraries

    Your Vanderbilt

    • Alumni
    • Current Students
    • Faculty & Staff
    • International Students
    • Media
    • Parents & Family
    • Prospective Students
    • Researchers
    • Sports Fans
    • Visitors & Neighbors

    Support the Jean and Alexander Heard Libraries

    Support the Library...Give Now

    Gifts to the Libraries support the learning and research needs of the entire Vanderbilt community. Learn more about giving to the Libraries.

    Become a Friend of the Libraries

    Quick Links

    • Hours
    • About
    • Employment
    • Staff Directory
    • Accessibility Services
    • Contact
    • Vanderbilt Home
    • Privacy Policy