Gibson, Richard (2018) Biochemical and biophysical characterisation of the quartromicin polyketide synthase. PhD thesis, University of Warwick.

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Abstract

Quartromicin (QMN), derived from Amycolatopsis orientalis Q427-8, are a complex of novel antiviral antibiotics active against HSV-1, HIV and influenza. Their biosynthesis involves a type Ipolyketide synthase (PKS), proposed to implement a module skipping strategy that produces two different precursors (Figure 1). A series of Diels-Alderase reactions, followed by oxidation and glycosylation, leads to QMN biosynthesis. Determining how this module skipping mechanism works would greatly increase the understanding of how modules within PKSs can be relocated to facilitate PKS engineering.

It was shown that module 5 (M5) can accept substrates from the upstream modules’ acyl carrier protein (ACP) domains (M3 ACP and M4 ACP) and utilise malonyl-CoA to catalyse substrate extension. Substrate specificity did not appear to be a factor in maintaining pathway fidelity.M3 ACP was observed to have a quicker rate of acyl transfer to M5 than M4 ACP and stronger protein-protein interactions (KD: 1.3 v 4.0 μM). Removal of M3 ACP’s C-terminal docking domain resulted in slower transfer and weakening of protein-protein interactions (KD: 2.6 μM). There was no significant change in rates of transfer or strength of interactions when the docking domain was omitted from M4 ACP. Sequence analysis of the docking domains highlighted the omission of a widely conserved negative residue from M4’s ACP docking domain, perhaps explaining the reduced efficiency.

A loss of antimicrobial and functionality was noted in M5 constructs lacking an AT-ACP linker region. Comparison with other spirotetronate PKS modules identified similar regions in ChlA6and KijS5, predicted to be remnants of KR domains. Creation of truncated constructs allowed the identification of an α-helix essential in module dimerisation and suggested a dimerization motif was located towards the C-terminal of the linker. Cryo-EM of M5 showed a large dimer interface between the KS domains of two modules and a second contact region, likely to be the aforementioned dimerisation motif.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QD Chemistry
Library of Congress Subject Headings (LCSH):	Antibiotics -- Development, Biosynthesis -- Research, Anti-infective agents -- Research, Natural products
Official Date:	27 September 2018
Dates:	Date Event 27 September 2018 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	Department of Chemistry
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Challis, Gregory L. ; Lewandowski, Józef R. ; Roper, David I.
Sponsors:	Engineering and Physical Sciences Research Council ; Molecular Organisation and Assembly in Cells
Format of File:	pdf
Extent:	xviii, 182 leaves : illustrations, charts
Language:	eng

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