Deciphering complex machineries that produce ribosomally synthesised natural products

Lead Research Organisation: University of Glasgow
Department Name: School of Chemistry


Human society is in constant need of new drugs, for example to win the arms race with emerging antibiotic resistant superbugs. Historically, natural products have been our best source of novel, bioactive compounds (for example, penicillin which was first isolated from a fungal mold). In fact, in the important fields of cancer and microbiology, almost 75% of all drugs on the market are natural products isolated from plants, marine organisms or microorganisms, such as bacteria. The ability of bacteria to make these sought-after natural products is encoded in their genomes. Over 300,000 bacterial genomes have been sequenced and made publicly available. They provide data for millions of unknown natural products, some of which will prove essential to curing diseases. The problem we face is that it is impossible to work on all of them, so we need to make smart choices.
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are natural products made by many different types of cells. RiPPs display a wide variety of promising bioactivities, including anti-viral, anti-tumor and antibiotic. They have also been linked to anti-fungal and painkilling activities. As a result of their manifold bioactivities, a variety of RiPP derivatives are currently undergoing therapeutic evaluation and RiPPs in general are the focus of many biotech start-up companies whose aim is to try to harness them as medicines of the future. RiPPs are made by the cell's ribosomes - the molecular machines that make proteins and shorter chains of amino acids. RiPPs start off as short chains of amino acids that are then modified by a cascade of enzymes within the cell to produce the final bioactive product. The chemical and structural diversity introduced by the modifying enzymes expands the available repertoire of products far beyond the 20 amino acids that would otherwise be available from the ribosome.
A result of this remarkable chemical and structural diversity is that the prediction of RiPP structures from the bacterial genome sequence alone is largely impossible. We need a much better understanding of the enzymes involved in their biosynthesis to make robust predictions and thus prioritize pathways to work on. A detailed understanding of the enzymes involved in RiPP biosynthesis will address this issue, and will allow us and others to improve the bioengineering of enzymes in order to improve their function and to induce them to make an even wider variety of useful medically valuable products. It will also address problems with the supply of natural products, inspire new methods for their production and, crucially, inform the process of rational compound modifications for drug development.
We have selected two protein complexes involved in the biosynthesis of RiPPs that share mechanistic but not chemical commonalities. These two complexes will be investigated using state-of-the art techniques that have never been combined to study RiPP complexes before. This approach and the results generated will provide a step-change in our understanding of the selected RiPP biosynthetic complexes and be instrumental in unlocking their full potential. We expect that the results of this work will enable research laboratories and biopharmaceutical companies to produce more of the drug compounds that the world needs and expects.

Technical Summary

Ribosomally synthesised and post-translationally modified peptides represent a rapidly growing natural product superfamily with exciting bioactivities and unprecedented enzymatic transformations. Their biosynthetic logic makes them very appealing to biotechnology companies: the starting material, a precursor peptide, is genetically encoded and expressed as a short structural gene. The precursor peptide is bipartite and consists of a leader, important for recognition by some of the modifying enzymes, and a core peptide, which ultimately becomes the natural product. The spatial separation of substrate recognition (leader) and catalysis (core) results in enzymes that can process a large variety of substrates. Additionally, simple genetic manipulations can be used to generate large libraries genes for related precursor peptides and thus natural products. A severe impediment to industrial (and academic) efforts is the lack of structural information about the biosynthetic complexes formed in the synthesis of these products. More importantly, very little is known about the dynamics of these complexes, the understanding of which is vital for rational enzyme, and thus compound, engineering.

We have selected two biosynthetic RiPP complexes that represent a (i) highly unusual class of bacterial alkaloids and (ii) the large family of thiopeptides (potent antibiotics). Both complexes represent a biosynthetic "black box" with neither their structures nor their dynamics known. We will combine and integrate data from several very powerful techniques to obtain insights with unprecedented detail: hydrogen-deuterium exchange mass spectrometry, small-angle X-ray scattering, analytical ultracentrifugation, pulsed electron-electron double resonance, X-ray crystallography and cryogenic electron microscopy. The complete and dynamic picture so generated will inform a range of research projects ranging from the development of novel synthetic methods to drug discovery.


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