Biophysical basis for the chain termination in the enacyloxin polyketide synthase

Lead Research Organisation: University of Warwick
Department Name: Chemistry

Abstract

We face many health related challenges in our everyday life. One of the major challenges is the emergence of multidrug resistant bacteria, which progressively render our arsenal of antibiotics ineffective against them. This rapidly growing problem may eventually lead to a situation where even the smallest infections, e.g. from a scratch, can become lethal as it was common in the pre-antibiotic era.
In order to avoid such a situation, there is an urgent need to develop new antibiotics that are effective against disease-causing microorganisms with resistance to the currently available drugs.
Enacyloxin IIa has been shown to possess antibacterial activity against the multidrug resistant bacteria Acinetobacter baumannii that is an increasing cause for hospital-acquired infections around the world. Enacyloxin is not stable enough for direct clinical applications but with a number of modifications it could be possibly turned into an effective drug. However, due to its complex structure enacyloxin is difficult to synthesise from scratch. At the same time, the polyketide synthetic biology field has progressed over last 30 years to the point where producing and modifying enacyloxin biosynthetically is a viable alternative.
Synthetic biology strives to construct new molecules by exploiting and modifying the biosynthetic machineries available in nature. In particular, polyketide synthases (PKSs) are nature's very large modular enzymatic assembly lines for a wide range of natural products with medicinal properties, ranging from antitumor agents through cholesterol-lowering agents to antibiotics. Polyketide-derived molecules comprise 20% of the top-selling drugs, with the combined worldwide revenues of over £10 billion per year. Due to their modular nature PKSs can be effectively modified to synthesise new compounds. The approach based on mixing and matching components from different assembly lines is very successful with a few hundred new molecules being synthesised to date. Yet, in order to harness these systems for rational production of new compounds, such as enacyloxin analogues, we need to understand the molecular structures and dynamics responsible for specificity and directionality of biosynthesis.
In this project we shall obtain such insights about biosynthesis of enacyloxin. To achieve that we propose to study molecular details of enacyloxin PKS and in particular, atomic resolution structures, motions and interactions of the components involved in controlling the crucial step of chain release where two separately assembled molecules are joined together through an ester bond. To obtain the required structural and dynamical insights, we propose employing a combination of highly complementary solution and solid-state magic angle spinning NMR spectroscopies. The proposed approach will enable us, for the first time, to learn how the structure of the proteins evolve on the time scale in the full relevant range from picoseconds to milliseconds. It will also enable us to access direct structural and dynamical information on the large complex of the chain-releasing enzyme and substrate-carrying protein. The solid-state NMR studies on this type of system will be the first of its kind.
This project will result in better understanding of enacyloxin biosynthesis and will enable deployment of the studied molecular machinery as a general tool for synthetic biology and synthesis of other compounds. This proposed approach is highly complementary to other structural biology approaches, such as x-ray crystallography and cryo-EM.

Technical Summary

Enacyloxin IIa is an antibiotic with activity against Gram-positive and Gram-negative bacteria. In particular, it has been shown to have clinically relevant activity against Acinetobacter baumanii, which is a multidrug resistant Gram-negative pathogen responsible for an increasing number of hospital-derived infections. However, enacyloxin has not been used in a clinical setting, presumably due its lack of stability caused by an ester group that can be easily hydrolysed in vivo. It is difficult to synthesise enacyloxin and enacyloxin analogues, which could address the above issue, from scratch because of their structural complexity. However, synthetic biology approaches exploiting modularity of enacyloxin polyketide synthase (PKS) provide a viable way of biosynthesising the required molecules.
A key step in enacyloxin biosynthesis is unusual chain release involving intermolecular condensation of a polyketide chain bound to an acyl carrier protein (ACP17) and (1S, 3R, 4S)-3,4-dihydroxycyclohexane carboxylic acid (DHCCA). This reaction is catalysed by an enzyme (C15) similar to nonribosomal peptide synthase condensation domains. A detailed molecular level knowledge of structures, dynamics and interactions of proteins responsible for the specificity and directionality of this process would greatly facilitate both rational engineering of PKSs to produce more therapeutically suitable enacyloxin analogues and the incorporation of C15 into the synthetic biology toolbox.
In this project, we propose to use a synergistic approach combining solution and solid-state magic angle spinning NMR spectroscopies aided by molecular modelling to obtain structures and picosecond to millisecond dynamics of ACP17 alone and in a complex with C15. Direct structural and dynamical studies of ACP17:C15 complexes by solid-state NMR will be the first of their kind.

Planned Impact

Impact through outreach
Over the past 6 years, Warwick Chemistry has established an innovative and extensive programme for the engagement of children in science managed by Mr. Nick Barker (supported initially, 2007-08, as an RSC Outreach Fellow and subsequently by the Chemistry Department). The programme has involved all members of staff and researchers and many PhD students and has reached 3,500 children in the past year alone. Activities are run in the Department and in schools.
The PI and PDRA will work together with Mr Barker in organising activities to help educating children in local schools about the importance of antibiotics for human health, problem of antibiotic resistance and about protein motions and why they are important in biology. Such activities will help children to understand problems associated with excessive use of prescription drugs and possibly inspire some of them to pursue a scientific career.

Impact through training
The PDRA working on this project will obtain high quality training with a unique set of interdisciplinary skills including experimental NMR, modelling and molecular biology. The practical skills acquired by the PDRA during the project will be useful for his employment in either academic or industrial setting. The transferrable skills developed by PDRA, including planning and project management, working in a team, and engaging with the public, will also be very valuable for non-research based careers.

Impact through collaborations with industry
Polyketide-derived molecules comprise 20% of the top-selling drugs, with the combined worldwide revenues of over £10 billion per year. Enacyloxin serves as a useful starting point for development of novel antibiotics to combat multidrug resistant Gram-negative pathogens. Consequently, this work will benefit the biotechnology and pharmaceutical companies that search for new antibiotics. In particular, a decision by any such company to actively develop enacyloxin into a drug would result in easily perceivable economic benefits, such as creation of jobs for scientists involved in the development. Significant long-term benefits to the UK economy will be obtained if such research results in a marketable drug.
The PI has established an industrial collaboration with Pfizer that will be used as a platform to transfer the methodology developed in the context of fundamental biomolecular studies to practical industrial applications, including drug development.

Publications

10 25 50
publication icon
Ackerley DF (2018) Understanding biosynthetic protein-protein interactions. in Natural product reports

publication icon
Hoop CL (2016) Huntingtin exon 1 fibrils feature an interdigitated ß-hairpin-based polyglutamine core. in Proceedings of the National Academy of Sciences of the United States of America

publication icon
Kosol S (2018) Protein-protein interactions in trans-AT polyketide synthases. in Natural product reports

publication icon
Lamley JM (2015) Intermolecular Interactions and Protein Dynamics by Solid-State NMR Spectroscopy. in Angewandte Chemie (International ed. in English)

publication icon
Lamley JM (2015) Intermolecular Interactions and Protein Dynamics by Solid-State NMR Spectroscopy. in Angewandte Chemie (Weinheim an der Bergstrasse, Germany)

 
Description In this project we have uncovered molecular basis for how one of the important steps in biosynthesis of antibiotic enacyloxin, chain termination, is accomplished in a multienzymatic assembly line called polyketide synthase. We have characterised different proteins that take part in the project and discovered factors contribute to control of the biosynthetic process. Importantly, we have characterised portable parts of the proteins responsible for controlling the protein-protein interactions required for reactions to happen, so called docking domains. Using bioninformatic tools we have discovered that these kind of portable docking domains are a very wide spread phenomenon and provide fantastic opportunities for engineering new hybrid assembly lines for making new molecules. Using biochemical tools we have shown that we can make component parts from different assmebly lines communicate with each other and make molecules. These findings provide a venue to effective rational engineering strategies for sythetic biology to make new molecules such as anbtibiotics.
Exploitation Route The discovered principles of communication between different domains in molecular assembly lines provide basis for synthetic biologists to create new hybrid molecular assembly lines and make new molecules. We collaborate closely with such groups to ensure that this knowledge is carried forward to practical applications. We also intend at some point to approach pharmaceutical companies who are interested in synthetic biology approaches.
Sectors Agriculture, Food and Drink,Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The research findings from these project was used in a number of outreach activities to make young people aware of the growing problem of antibiotic resistance and what are the ways we can tackle it. We intend to disseminate the approaches developed as a part of this project to industry through different means. For example, we got involved into Medicines Discovery Catapult where we will provide to both academic and industrial users in the UK access to expertise acquired during this project to tackle the challenge of making new drugs.
First Year Of Impact 2017
Sector Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
 
Description An integrated solution and solid-state NMR roadmap to serve the Physical and Life Sciences community
Geographic Reach National 
Policy Influence Type Citation in other policy documents
 
Description BBSRC Responsive Mode
Amount £730,455 (GBP)
Funding ID BB/R010218/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2018 
End 02/2021
 
Description Bridging the Gaps between the Engineering and Physical Sciences and Antimicrobial Resistance
Amount £495,412 (GBP)
Funding ID EP/M027503/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 09/2015 
End 08/2017
 
Description ERC Starting Grant
Amount € 1,999,044 (EUR)
Funding ID 639907 
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 05/2015 
End 04/2020
 
Description Elucidating and exploiting docking domain-mediated carrier protein recognition in natural product megasynthetases
Amount £742,035 (GBP)
Funding ID BB/R010218/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2018 
End 02/2021
 
Description Gates Foundation Grant
Amount $750,000 (USD)
Funding ID OPP1160394 
Organisation Bill and Melinda Gates Foundation 
Sector Charity/Non Profit
Country United States
Start 10/2016 
End 12/2017
 
Description INTEGRATE AMR Pump Priming Fund
Amount £13,000 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 03/2017 
End 06/2017
 
Description Access to fast spinning methodology for SMEs in drug discovery 
Organisation Medicines Discovery Catapult
Country United Kingdom 
Sector Private 
PI Contribution We provide access and expertise in fast magic angle spinning solid-state NMR to SMEs in the context of drug discovery projects.
Collaborator Contribution Purchased a 0.7mm magic angle spinning NMR probe to be used for the projects. Provide project management.
Impact Pilot project on characterisation of a docking domain in non-ribosomal peptide synthesase involved in biosynthesis of antibiotic tyrocidine. https://md.catapult.org.uk/case-studies/protein-interactions-in-non-ribosomal-peptide-synthetases-nrpss/
Start Year 2017
 
Description Development of fast magic angle spinning instrumentation 
Organisation Tallinn University of Technology
Department Technomedicum
Country Estonia 
Sector Academic/University 
PI Contribution Evaluated and provided feedback about various generations of 0.8 mm MAS probe for application on biomolecules.
Collaborator Contribution Provide experimental 0.8mm MAS probe.
Impact (1) Lamley, J. M.; Iuga, D.; Öster, C.; Sass, H.-J.; Rogowski, M.; Oss, A.; Past, J.; Reinhold, A.; Grzesiek, S.; Samoson, A.; Lewandowski, J. R. J. Am. Chem. Soc. 2014, 136 (48), 16800.
Start Year 2012
 
Description Medicines Discovery Catapult 
Organisation Medicines Discovery Catapult
Country United Kingdom 
Sector Private 
PI Contribution Established a collaborative facility with Medicines Discovery Catapult providing expertise on solid-state NMR to facilitate R&D for SMEs.
Collaborator Contribution Purchased a 0.7 mm probe. Funded a facility manager partially embedded in Lewandowski group.
Impact Establishing the facility with one call for collaborations so far.
Start Year 2017
 
Description Solid-state NMR of multidomain proteins 
Organisation University of Patras
Department Department of Environmental & Natural Resources Management
Country Greece 
Sector Academic/University 
PI Contribution Solid-state NMR of a multidomain protein
Collaborator Contribution Supply samples.
Impact No outputs yet.
Start Year 2016
 
Description Structural investigations of PPM synthase 
Organisation University of Warwick
Department Department of Economics
Country United Kingdom 
Sector Academic/University 
PI Contribution Working towards structure determination of a PPM synthase
Collaborator Contribution Produce samples. Perform biochemical essays.
Impact No outputs so far.
Start Year 2016
 
Description Structural studies of RANTES:CCR5 complex 
Organisation University of Basel
Department Biozentrum Basel
Country Switzerland 
Sector Academic/University 
PI Contribution We applied the solid-state NMR methodology developed in our laboratory to study structure of RANTES in complex with CCR5.
Collaborator Contribution Our partner provided isotopically labeled samples for the study and assisted in optimising conditions for sample preparation.
Impact Update
Start Year 2015
 
Description Structures of antibiotic-lipid II complexes 
Organisation University of Warwick
Country United Kingdom 
Sector Academic/University 
PI Contribution We have used combination of solution and solid-state NMR (including 100 kHz spinning methodology) to solve structures of antibiotics in complexes with lipid II to inform rational drug development efforts.
Collaborator Contribution Synthesize lipid II.
Impact No outcomes yet. Publication in preparation.
Start Year 2015
 
Description Using of solvent Paramagnetic Relaxation Enhancements (PREs) in solid state 
Organisation Technical University of Munich
Country Germany 
Sector Academic/University 
PI Contribution Designed and performed NMR measurements on a range of systems.
Collaborator Contribution Provided expertise on computational protocols for using PREs for structure calculation.
Impact 10.1021/jacs.7b03875
Start Year 2015
 
Description ZapA 
Organisation University of Warwick
Department School of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Started investigation of protein involved in bacterial wall biosynthesis by solution and solid-state NMR.
Collaborator Contribution Help with protein production and background data and expertise.
Impact no outputs yet
Start Year 2016
 
Description Analytical Science Networking and Partnership 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact We were involved in a workshop involving representatives from several companies (e.g. AstraZeneca, JEOL, Bruker, Syngenta, Pfizer, Unilever, Lubrizol, Linear Diagnostics etc.) to explore potential collaborations. One of the purposes of these workshops was to familiarize the representatives of the industry with our scientific capabilities and for the representatives of the industry to present us with problems they would like to tackle.
Year(s) Of Engagement Activity 2015,2016
URL http://www2.warwick.ac.uk/fac/sci/mas/aboutmascdt/
 
Description Chemistry outreach for Year 6 children from Thornton Junior School, Birmingham 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Chemistry outreach for Year 6 children from Thornton Junior School, Birmingham. The 50 pupils visiting the chemistry department as part of the outreach program showed great interest and participated with enthusiasm in the program and especially the practical experiments, which inspired discussions and curiosity among the children.
Year(s) Of Engagement Activity 2015
 
Description Info session with Mahidol University representatives 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact A group of the representatives from Mahidol University, Bangkok, Thailand met with a number researchers to explore possibility of establishing links between Mahidol University and University of Warwick. We have presented our work and participated in a discussion about potential collaborations.
Year(s) Of Engagement Activity 2015
 
Description Organisation of an Alpine Conference on Magnetic Resonance in Solids 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact About 200 scientists at different stages of their careers participated in this conference leading to exchange of expertise.
Year(s) Of Engagement Activity 2019
URL https://alpine-conference.org/
 
Description Think Science - lecture 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 150 students participated in a lecture about science and encouraging them to go into science. 4 high school teachers participated in discussion on UCAS admissions process.
Year(s) Of Engagement Activity 2016