Biophysical basis for the chain termination in the enacyloxin polyketide synthase

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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.