Structural and mechanistic understanding of polyketide synthases

Lead Research Organisation: University of Nottingham
Department Name: Sch of Chemistry

Abstract

Polyketides represent a highly diverse class of natural products, often possessing potent biological activity. Unrivalled as potential therapeutic agents, many members of this group are employed as antibiotics, immunosuppressants, antiparasitics, cholesterol-lowering agents and anticancer drugs. Given the logical arrangement of polyketide synthases (PKSs) - the molecular assemble lines responsible for polyketide biosynthesis in bacteria - there is considerable scope for enginnering new, high value, bioactive compounds. Success in this area is dependent upon an understanding of the rules governing substrate tolerance and catalytic domain compatibility. We have developed new mass spectrometry tools to study the enzymology PKS domains, resulting in a number of high impact publications. This project will comprise expression, purification and functional testing of a PKS catalytic domain. Although mostly based in Nottingham, there will be the opportunity to spend a week at the World-class Harwell research site to undertake additonal structural studies.

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M008770/1 01/10/2015 31/10/2024
1644216 Studentship BB/M008770/1 01/10/2015 30/09/2019
 
Title Front Cover of Analysis & Sensing, First Issue 
Description Front cover of Analysis & Sensing journal. Accompanies a publication in the same journal. Shows the concept presented in the journal article, namely using collision-induced-unfolding ion-mobility mass-spectrometry to interrogate the stabilising or destabilising effects of point mutations to acyl carrier proteins. The image shows a backdrop of feasible mutations in 3-letter code form. Overlaid are the mobility traces at increasing energy and 3-D models of the structures at selected energies (derived from molecular dynamic simulations). 
Type Of Art Artwork 
Year Produced 2021 
Impact Featured on the front cover of the first issue of a cutting edge journal. 
 
Description The acyl carrier proteins (ACPs) of trans-AT polyketide synthases have been previously overlooked as passive carriers within biosynthesis of polyketides. Studies of the interactions between isolated ACPs and other polyketide synthase enzymes has shown that different ACPs exhibit specific interactions which correlate with their natural position substrates.
Further, bioinformatic analysis of over 300 ACPs from varied polyketide synthases revealed that ACPs can be grouped by the specificity of the downstream ketosynthase and known interacting enzymes, suggesting that ACPs have greater control over the progress of polyketide synthases than previously thought and supports a change in the traditional module definitions.

Native mass spectrometry is a powerful group of techniques that enable protein interactions to be studied. Several isolated polyketide synthase proteins/domains (including ACPs) were investigated, preliminary studies show that many such proteins are amenable to native mass spectrometry. A conserved post-translational modification to ACPs appears to stabilise gas-phase ACPs. While larger polyketide synthase proteins, such as whole modules, are amenable to native mass spectrometry, some instrumental barriers currently prevent the use of this technology to monitor 'live' reactions.

New software (named PepFoot) has been developed to greatly improve the analysis and visualisation of protein footprinting data. This allows protein interactions to be rapidly screened using available protein footprinting techniques. The software is open-source and available for all major platforms with a user-friendly interface outputting reproducible and shareable data. This new software was benchmarked against published data to show highly comparable outcomes and improved analysis, removing the analysis stage as a major bottleneck.
Exploitation Route In order to engineer polyketide synthases, either for the production of novel polyketides or for feedstock production, protein-protein interactions are important considerations as they drive the efficiency of biosynthesis. The importance of ACPs in such engineering must now be taken into consideration, primarily be ensuring that ACPs are paired with matching ketosynthase enzymes and processing enzymes.

The use of modified instrumentation could allow the reactions of polyketide synthase proteins to be monitored 'live', which will greatly improve the ability to screen and characterise protein-protein interactions, with relevance to the above.

Increasing take-up of protein footprinting is enabled by new software, the usage is not restricted and thus may be used in both academic and industrial settings. The characterising of new protein-protein interactions, or protein-small molecule interactions, is vital to understanding biological systems. Adding this technology in an accessible form, increases the toolbox available to researchers across biological fields.
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Title PepFoot v1.1 
Description PepFoot allows rapid analysis and visualisation of protein footprinting data, in particular at the peptide-level. LC-MS data from all major instrument vendors can be processed once converted to the open source format '.mz5', allowing open and reproducible analysis. A key feature of the software is semi-automated batch processing -- where the user will manually process a single representative data file (aided by the software) and subsequent analysis of all remaining data files is handled by the software -- which greatly reduces processing times and tackles human error in analysis of large and complex data-sets. The processed data are then automatically visualised, using an open-source PDB viewer, to allow identification of sites of interest. The output of the software is itself human-readable and open-source, allowing reproducible sharing of the analyses. 
Type Of Technology Software 
Year Produced 2019 
Open Source License? Yes  
Impact The software has been taken up by those in the protein footprinting community to aid analysis of their data, particularly when MS/MS data are not available or proprietary software are expensive. 
URL https://pubs.acs.org/doi/10.1021/acs.jproteome.9b00238
 
Title jbellamycarter/chargePlacer: ChargePlacer archived with Zenodo 
Description Python implementation of a charge positioning algorithm for gas-phase molecular dynamics 
Type Of Technology Software 
Year Produced 2021 
Open Source License? Yes  
Impact ChargePlacer was developed and used to predict charge distribution in the gas-phase for preparing gas-phase molecular dynamics simulations of acyl carrier proteins. This was used to inform and supplement gas-phase unfolding observations of site-directed mutants of a polyketide synthase acyl carrier protein. 
URL https://zenodo.org/record/4594254