Daunorubicin and its congener, doxorubicin, are two of the most important anti-tumor antibiotics used in cancer chemotherapy. Both agents exhibit a broad range of activity against solid tumors and hematologic malignancies, yet their clinical potential is limited by severe dosedependent cumulative cardiotoxicity. So far, over 2000 analogs have been synthesized comprising numerous chemical modifications, substitutions and conjugations to the tetracyclic ring, the side chain or the amino-sugar. However, only few of those have reached the stage of clinical development and approval. Furthermore no analog offers a definite advantage over daunorubicin and doxorubicin in terms of activity or toxicity. The search for a better anthracycline may consequently require a radically different approach. These drugs are synthesized by a type II polyketide synthase consisting of a number of fully dissociable enzymes. Although the structural, regulatory and resistance genes of the daunorubicinldoxorubicin biosynthetic clusters have been identified, their functions characterized and a general scheme for the biosynthetic pathway has been proposed, little is known about the structural organization of these enzymes within the PKS and how this affects catalysis. Furthermore, the exact mechanism of starter unit selection remains elusive. Knowledge of the above would help us modify the biosynthetic pathway in order to engineer new anthracycline natural products with improved activity and pharmacological properties. To accomplish these goals, all dps proteins were expressed and purified to homogeneity. Two enzymes, the ketosynthase subunits (DpsAlB) and the malonyl transferase (DpsD) were expressed for the first time. Functional interactions between the dps components were then identified in vitro by ITC and 1FT and, based on the interactions data, a model for the architectural organization of the PKS was proposed. The roles of DpsC and DpsD as the starter unit specifying enzymes were also examined in detail. Our analysis showed that, contrary to previous reports, DpsD seems to be the starter unit specifying enzyme. Finally, crystals of DpsC and DpsE were obtained and diffraction data collected that could aid in structure elucidation for these components.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:685414 |
| Date | January 2013 |
| Creators | Vasilakis, Konstantinos |
| Publisher | University of Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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