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Title: NMR Investigation into the Role of Acyl Carrier Protein in Polyketide Biosynthesis
Author: Evans, Simon E.
ISNI:       0000 0001 3454 8662
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2007
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Acyl carrier proteins (ACPs) are essential to both the fatty acid (FAS) and polyketide (PKS) biosynthetic pathways, yet relatively little is known about how they function at a molecular level. Starting from the published solution NMR structure of actinorhodin (act) PKS ACP from Streptomyces coeHc%r, a program of work to extend the understanding of ACP's role in polyketide biosynthesis has been undertaken. To this end, nine different ACP derivatives have been synthesised and structurally characterised by NMR. Established methods for the growth, expression, and purification of ACP have been extended to produce reliably high yields of isotopically labelled protein for high-resolution NMR analysis. Holo ACP synthase (ACPS) has been used to prepare early stage ACP intermediates of polyketide biosynthesis (holo ACP, acetyl ACP, and malonyl ACP) from the respective coenzyme A derivatives. Following various unsuccessful attempts to isolate a sample of the next intermediate, acetoacetyl ACP, a novel route to stabilised thiolether ACPs has been developed. This route was then applied to the 'preparation of hydrolysis-resistant ACP-bound diketide and triketide analogues. Finally, NMR samples of three fatty-acyl ACPs with varying chain length were produced. Complete multidimensional-multinuclear NMR experiments were recorded for each of the nine derivatised ACP samples. The resulting data yielded an average of 31 restraints per residue, from which high-quality water-refined structure ensembles have been calculated, using the ARIA protocol for automatic distance restraint assignment. On the basis of a comparison of the structures for each ACP derivative, a number of conclusions have been possible. A molecular basis for the modulation of ACP:ACPS interaction is proposed, in which helix III switches between two states,initiating dissociation of the complex after the loading reaction is complete. The unique conformation of a conserved arginine residue (Arg72) revealed in the structure of malonyl ACP, may be important for binding to ketosynthase13 during chain initiation, and may also be involved in ACP-ACP malonyl transfer. Preliminary evidence that polyketide ACPs, like their FAS counterparts, are able to sequester biosynthetic intermediates is presented. From the structures of ACP with bound fatty-acyl chains, it is demonstrated that act PKS ACP has a substrate pocket distinct from that in FAS ACPs, which could be more suitable for binding polyketide, rather than fatty acid intermediates.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available