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Title: Enzymology of type I modular polyketide synthases
Author: Bali, Shilpa
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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Modular type I polyketide synthase (PKSs), for example the 6-deoxyerythronolide B synthase (DEBS) responsible for formation of the aglycone core of erythromycin A, are giant, multifunctional enzyme complexes that catalyse the orchestrated assembly line biosynthesis of antibiotic polyketide natural products. To analyse these multienzymes in detail, this research aimed to study the enzymology of individual catalytic domains, including the ketoreductase (KR), enoyl reductase (ER) and ketosynthase (KS) activities, by expressing them as discrete recombinant proteins. Ketoreductase domains derived from DEBS and the tylosin (TYL) PKS were evaluated for their intrinsic specificity and stereospecificity towards surrogate diketide substrates. The data, together with modelling of diketide binding to DEBS KR domains (B Popovic), demonstrate that the energetics of ketoacylthioester substrate presentation to KR active sites are delicately balanced. Extending this approach to KR domains from the mycolactone (MLS) PKS further supported this role of substrate tethering in achieving stereocontrol during ketoreduction, and led to a model for substrate recognition in this system. The KR domains from DEBS, TYL and the MLS PKS also show significant activity towards a wide range of non-polyketide substrates, particularly favouring those which incorporate cyclohexyl moieties. Site-directed mutagenesis of amino acid residues previously implicated in stereocontrol identified mutants with improved activity towards such compounds, suggesting that PKS KRs might in future be developed as useful biotransformation catalysts for the production of chiral alcohols. The ER domain from DEBS module 4 was also obtained as a recombinant protein, in collaboration with RJ Moorlen (Part II student). Analysis by circular dichroism showed the domain to be well-folded with significant α-helical secondary structure. The gel filtration chromatography indicated that the domain is homodimeric, a finding with the important implications for structural models of modular PKS. A number of conditions have been explored to reconstitute enoly reduction activity in vitro, and initial attempts to obtain diffracting crystals of the domain have shown promise.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available