Unpicking the role of acyl carrier proteins from polyketide synthases with mass spectrometry

Bellamy-Carter, Jeddidiah S. G. (2020) Unpicking the role of acyl carrier proteins from polyketide synthases with mass spectrometry. PhD thesis, University of Nottingham.

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Polyketide synthases (PKS) are multi-domain enzymes responsible for the biosynthesis of structurally and functionally diverse natural products. A particular subfamily—trans-AT PKSs—have modular architecture and account for much of this diversity. These have attracted much attention as targets for engineering tailored PKSs to produce feed-stocks or synthetically challenging organic compounds. Phylogenetic and subsequent experimental studies have shown that ketosynthases (KS), the key domains for catalysing polyketide elongation, from trans-AT PKSs are highly substrate specific and may mediate polyketide structure. Until recently, the acyl carrier proteins (ACP) of trans-AT PKSs have been seen as passive carriers of the growing polyketide chain.

This thesis explores the role of ACPs through a series of mass spectrometry (MS) based and computational techniques. In Chapter 3, the interactions of acyl-ACPs from the psymberin PKS with cognate enzymes, including acyltransferase (AT) and acylhydrolase (AH) domains, show that ACPs are directly involved in controlling their acyl processing through protein interactions. In most cases, the observed specificities reflect the ACPs' native substrates and position within the PKS. The first ACP of the psymberin PKS was found to protect its native acetyl moiety and resist malonyl loading. Bioinformatics analysis of trans-AT ACPs through hidden Markov model profiling demonstrates the surprising relationship between ACPs and their downstream KSs, hinting at co-evolution and a mutual role in polyketide determination.

Native MS has been explored, in Chapter 4, as a prospective technique for studying PKS protein interactions in the gas-phase. In particular, collision-induced unfolding (CIU) shows great promise for understanding the structure of PKS proteins. A variety of PKS proteins—including whole modules—have been successfully analysed by native MS, however, determining acyl-state becomes impractical for larger ions.

Carbene footprinting is an emerging MS technique for the study of protein interactions, including those of PKSs. However, the data are often complex, making high-throughput analyses difficult. Chapter 5 describes the development of PepFoot, an open-source and user-interactive software, to streamline analysis and interpretation of footprinting data, paving the way for in-depth analysis and screening of PKS protein interactions. Benchmarking against experimental data-sets, showed highly comparable results to those published and additional insight—in a fraction of the time.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Oldham, Neil J.
Keywords: Polyketide synthases, Mass spectrometry, Native mass spectrometry, Software, Protein footprinting
Subjects: Q Science > QP Physiology
Faculties/Schools: UK Campuses > Faculty of Science > School of Chemistry
Item ID: 59797
Depositing User: BELLAMY-CARTER, Jeddidiah
Date Deposited: 15 Jul 2020 04:40
Last Modified: 15 Jul 2022 04:30
URI: https://eprints.nottingham.ac.uk/id/eprint/59797

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