AbstractUntil now, there has been no published study of a DH domain from an iterative polyketide synthase (iPKS). Previous work within the Cox group showed it is possible to isolate and characterise the selectivity of the enoyl reductase (ER) domain from squalestatin tetraketide synthase (SQTKS).
In order to further the understanding of the SQTKS enzyme, the isolated DH enzyme was produced and tested in vitro with substrates designed to test the enzymes’ stereoselectivity. The assays developed for this work utilised LCMS to measure the initial rate of the enzyme reaction in order to assess the selectivity of the DH domain. The results demonstrated that the DH domain is highly selective and the correct stereochemistry of the α and β positions is vital for efficient substrate turnover. A second set of assays investigated the potential for non-native substrates to act as inhibitors for the DH. None of the non-substrates inhibited the dehydration reaction, indicating that the substrates were unable to enter the active site. In the absence of a crystal structure, a model of the SQTKS DH domain was produced using crystal structures of isolated modular DH enzymes. Docking studies via a homologous model were undertaken to rationalise the selectivity of the SQTKS DH. Docking of the substrate into the active site of the model showed the programming of the DH domain arises from the position of the two catalytic amino acids, H44 and D235. The position of these amino acids dictates which substrates are tolerated and dehydrated. Through combining the results from the ER and DH assays, it is possible to elucidate the stereo selectivity of almost all of the steps of the SQTKS and show that these are identical to those of mammalian fatty acid synthase (FAS). The significance of these results and their relevance will be discussed.
|Date of Award||25 Sep 2018|
|Supervisor||Russell Cox (Supervisor)|
Kinetics and Selectivity of an Isolated Dehydratase Domain from a Fungal Polyketide Synthase
Liddle, E. (Author). 25 Sep 2018
Student thesis: Doctoral Thesis › Doctor of Philosophy (PhD)