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Title page for ETD etd-04042011-165321

Type of Document Dissertation
Author Nannemann, David Patrick
URN etd-04042011-165321
Title Directed Biosynthesis of the Nucleoside Analog Drug Didanosine
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
Brian O. Bachmann Committee Chair
Jens Meiler Committee Co-Chair
F. Peter Guengerich Committee Member
Richard N. Armstrong Committee Member
  • Enzyme Engineering
  • Bioretrosynthesis
  • Computational Enzyme Design
  • Directed Evolution
  • Purine Nucleoside Phosphorylase
  • Phosphopentomutase
Date of Defense 2011-03-25
Availability unrestricted
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.
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