Smith, Helen Grace (2022) Enzymology of a split module in trans-acyltransferase polyketide synthases. PhD thesis, University of Warwick.

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Abstract

Polyketide (PK) natural products offer great potential as a source of novel antibiotics, helping to meet the clinical need posed by the impending antimicrobial resistance crisis. PKs are isolated from living organisms such as bacteria and fungi. Within these organisms there exists complex protein machinery; polyketide synthases (PKSs), which biosynthesise these molecules.

This work aims to increase our understanding of the protein-protein interactions underpinning these biosynthesis pathways and the chemistry the enzymes perform. It is hoped that by exploiting this knowledge we can create hybrid pathways producing novel analogues of natural product molecules with enhanced therapeutic potential. This work focuses on a previously uncharacterised ketoreductase / methyltransferase (KR/MT) domain junction within the gladiolin PKS. Gladiolin, isolated from the bacterium Burkholderia gladioli, shows potent activity against multidrug resistant strains of Mycobacterium tuberculosis. The activity of module 7 from the gladiolin PKS (containing the KR/MT interface) has been investigated in vitro using mass spectrometry-based assays. Mechanism-based crosslinking enabled covalent trapping of the multidomain complex giving initial evidence the protein constructs from this interface interact in vitro.

Figure: Partial biosynthetic scheme for the production of gladiolin. The KR/MT interface separating the GbnD2 and GbnD3 subunits is shown on the left and the chemical transformations across this boundary are highlighted in blue on both the biosynthetic intermediate and the structure of gladiolin.

Mass shifts monitored by intact protein mass spectrometry allowed the enzymatic activity of the domains of this module to be fully reconstituted. Substrate mimics to probe the stereochemical course of the a-methylation and b-ketoreduction reactions have been synthesised; the stereochemistry produced by the domains of this module has been probed for substrates tethered to the ACP domain and in trans. Carbene footprinting mass spectrometry experiments have identified specific regions involved in interaction across this interface. This information allowed crosstalk experiments to probe interaction between proteins at KR/MT interfaces from different trans-AT PKS pathways. KR/MT interfaces are prevalent in trans-AT PKSs, therefore insights from this work have the potential to guide future biosynthetic engineering efforts to create hybrid pathways that assemble novel PK products.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QD Chemistry Q Science > QH Natural history > QH301 Biology Q Science > QP Physiology
Library of Congress Subject Headings (LCSH):	Polyketides, Antibiotics, Drug resistance in microorganisms, Protein-protein interactions, Biosynthesis
Official Date:	January 2022
Dates:	Date Event January 2022 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	Warwick Medical School
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Challis, Gregory L. ; Lewandowski, Józef R. ; Jenner, Matthew
Format of File:	pdf
Extent:	xvii, 221 leaves : illustrations
Language:	eng

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