‘Trans AT’ polyketide synthases (PKSs): studies of the mode of interaction between catalytic domains and their carrier protein-bound substrates (in cis/trans) – PKS-PPIs

Our experimental strategy is firstly to clone and express the catalytic ‘workers’ in recombinant form in the bacterium Escherichia coli using standard molecular biological tools, and then to rigorously purify the resulting proteins. We then investigate how the proteins interact with each other using a variety of biophysical techniques including surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). In this way, we can obtain a detailed molecular-level description of the way in which the various partners in the polyketide assembly process communicate. By studying all of the interactions that occur during the construction of a polyketide, we will better understand how to modify these systems without perturbing this essential set of contacts.

Over the last seven months, we have succeeded in obtaining a number of partner proteins from our two model systems, and are beginning to investigate how they interact. We are also now interested in solving the three-dimensional structures of certain of these catalysts using X-ray crystallography, in order to understand how they realize their chemical tasks, how they recognize their substrates, etc.

We hope that the insights that we obtain into our two selected model systems will directly enable the engineering of the PKSs towards the generation of modified polyketide products. Indeed, we already understand in detail how these molecules interact with their shared target in bacteria, the ribosome, opening the way to altering certain portions of the structures to further optimize their antibiotic properties. Our aim is to carry out such applied experiments in collaboration with pharmaceutical companies.

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Complex polyketide natural products and their derivatives form the basis for a number of medicines, including antibiotics (erythromycin), immunosuppressants (rapamycin), anti-parasitic agents (avermectin), and anti-cancer drugs (epothilone). The unique structures of these secondary metabolites arise from the coordinated action of multiple enzymatic domains, which are organized into gigantic, multienzyme ‘assembly lines’ called polyketide synthases (PKS), alongside non-catalytic acyl carrier proteins (ACPs). This modularity of function has motivated efforts to reconfigure the systems by genetic engineering towards the generation of variant product structures with improved or wholly novel biological properties (a strategy referred to as ‘combinatorial biosynthesis’). Encouragingly, several hundred polyketides new to Nature have been created by such approaches. However, it is now widely accepted that realizing the true potential of combinatorial biosynthesis hinges on obtaining significantly deeper insights into all aspects of modular PKS function. Although PKS systems have been the object of study for the last 20 years, significant gaps remain in our knowledge concerning fundamental aspects of the biosynthesis: the basic mechanisms and molecular basis for the substrate specificity of the individual catalytic domains, the balance between protein-protein and enzyme-substrate recognition in programming chain extension, and the overall structures of the modules. These deficits are apparent for the largest known class of modular PKS (referred to as ‘cis AT’ PKS), but are even more pronounced for a more recently-discovered group of systems called the ‘trans AT’ PKS. Products of trans AT PKS include mupirocin, which is used clinically against methicillin-resistant Staphylococcus aureus (MRSA), and virginiamycin M, whose semi-synthetic derivative dalfopristin is a frontline treatment for vancomycin-resistant Enterococcus faecium. The trans AT PKS exhibit marked architectural differences with the cis AT PKS (e.g. the presence of a discrete AT domain which acts repeatedly in trans, tandemly duplicated domains, non-standard domain orderings, and modules whose component domains are split across two polypeptides), and appear to have distinct evolutionary origins.

The proposed research project aims to facilitate future engineering efforts of trans AT PKS by illuminating a central aspect of the polyketide chain-building process: the mode by which the enzymatic domains interact with their subtrates attached to the ACPs. As maintaining this interdomain communication is vital to the proper functioning of all modified PKS, there is an urgent need for information on this specific aspect of the biosynthesis. As potential model systems, we have selected two medically-relevant PKS: that responsible for virginiamycin, and a second for the antibiotic lankacidin, which is exploited in animal husbandry. In principle, this choice will allow us to address several unique functional and structural aspects of the trans AT PKS, but may additionally reveal general principles of interdomain communication in modular systems which are applicable to the cis AT assembly lines. The multi-disciplinary project will involve structure elucidation of ACP domains coupled with dynamical studies, as well as detailed investigation of how acylated ACPs interact with various partner domains from the PKSs which normally operate both in cis and in trans. The primary technique used in this research will be NMR, as it is particularly suited to studying weak interprotein interfaces and protein dynamics. We anticipate that insights obtained during this research should directly translate into new, more effective strategies to generate analogues of virginiamycin, lankacidin and other clinically-important polyketides, for biological evaluation.