Structure and mechanism of a trans-acyltransferase polyketide synthase

Polyketides are among the most important compounds known to man. They perform many vital roles in nature acting as hormones, toxins, flavours, smells and pigments. These compounds are also the basis of numerous medically important drugs used to treat cancer, lower cholesterol, suppress the immune system and fight infection. Sales of polyketide based medicines total over £30 billion each year and there is enormous worldwide interest in identifying new polyketides and making new and improved versions of existing ones. Polyketides are made within microorganisms by clusters of proteins called polyketide synthases, usually shortened to PKSs. PKSs function like miniature factory assembly lines within cells. Each different protein within the assembly line is responsible for building or modifying a specific part of the carbon skeleton of the polyketide product. There are considerable differences in the structures and activities of different polyketides made by different PKSs from different microorganisms. This is despite the fact that all of these compounds are produced from the same initial chemical building blocks. Understanding how these differences are achieved relies on an in-depth knowledge of how PKSs work, essentially, what do each of the proteins in the assembly line do, how do they do it and how are they arranged relative to each other? This research project will focus on a new family of PKSs which generate highly unusual products used to treat a range of different diseases. By examining the structures of the components of the PKS in fine detail using a technique called X-ray crystallography, we will try to work out how each of different parts of the PKS works and how they fit together and interact with each other. These experiments will not only allow us to decipher how these systems make their important products, but will also provide us with a blue-print that we can use to construct new PKSs which make new medicines.

Modular polyketide synthases, non-ribosomal peptides synthases or hybrids thereof are giant (upto 5 MDa) multi-functional enzymes that assemble complex natural product scaffolds in a highly programmed linear fashion. Within the synthase complex, individual enzymatic domains are organised into associated extension modules, with each module responsible for the elongation of the product chain by a single functional unit. Extension modules are often elaborated to house multiple catalytic domains which act in concert with the minimal chain extension machinery to further modify the growing product chain. Recent work focused on the characterisation of modular systems from 'exotic' bacterial taxa has resulted in the identification of a new group of multi-modular synthases that do not adhere to the established colinearity rules of the polyketide biosynthetic pathway. These novel systems, termed trans-AT synthases, have evolved independently from other modular systems and comprise disparate enzymatic activities from apparently unrelated metabolic pathways, which have converged to yield a single functional synthase. The aim of this project is to provide a molecular description of this novel enzymatic machine. To achieve this goal we will deconstruct the individual components of the model trans-AT system bacillaene synthase and by marrying X-ray crystallography with in vitro kinetic, thermodynamic and spectroscopic methods, and organic synthesis, provide a detailed description of the trans-AT biosynthetic framework at the domain, module and multi-module level.

Around two thirds of all drugs in current clinical use are derived from or based upon molecules isolated from natural sources. This accounts for a multi-billion pound global market comprising antibiotics, antifungals, antiparasitics, anticancer agents and the cholesterol lowering statins. In 2009 four of the top twenty best selling pharmacological agents world-wide were molecules based on natural product scaffolds, with the statin derived cholesterol lowering agent Lipitor ranked number one. As the feasibility of high-throughput screening methodologies for target based drug development is increasingly being brought into question, there in now renewed interest in the identification and isolation of new natural product based medicines. This renewed interest has resulted in the re-establishment of many natural product drug discovery pipelines in the pharmaceutical industry and companies dedicated to the development of new therapeutic agents from natural sources are in operation in the UK, France, Spain, Germany, Switzerland, USA, China and Japan. Among new natural product targets the polyketides, non-ribosomal peptides and hybrids thereof remain one of if not the most attractive molecules for clinical development, with many polyketide and non-ribosomal peptide derived compounds in the latter stages of clinical trials. The enormous potential to modify and manipulate the polyketide and non-ribosomal peptide biosynthetic pathway offers a route to both the rational generation of tailor-made product analogues and the high-throughput generation of large compound libraries without the cost, complexity and environmental impact of synthetic chemical approaches. For this goal to be realised however, a detailed understanding of the synthetic process is essential. The work outlined within this application will provide this information, bridging the gap that currently exists between gene sequence and product chemistry. Our studies will not only offer insight into one of nature's most complex biosynthetic assemblies impacting significantly on both the academic and industrial biosciences community, but also provide a structural framework for the manipulation of synthase systems for the production of 'next-generation' natural product derived therapeutic agents. Such output will inevitably impact on the healthcare and well-being of the populous and the economic competitiveness of the UK. This program will offer those involved (PDRAs, students, etc.) experience and training at the chemistry-biology interface, providing them with the skills to succeed in a future career in academia or industry. The research output from this program will be reported to the wider scientific community through publication in leading peer reviewed journals, presentation at national and international conferences and at meetings with industrial and academic collaborators. Appropriate training will be given to the PDRAs in the preparation of papers, posters and oral presentations to ensure that, alongside their scientific knowledge and skills, they are developing a portfolio of widely transferable skills. Further, significant opportunities exist for the presentation of research findings (by the PI and PDRA) to a more general audience through public engagement activities organised by the PIs department (school talks, Bristol sciences festival, etc.) and through the PIs links with the Royal Society eg. the Summer 2010 Science Festival. Such activities will ensure that as broad an audience as is feasible will be informed of our ongoing research.