Biophysical basis for the chain termination in the enacyloxin polyketide synthase

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.

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.

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.