Radical S-adenosyl-L-methionine (SAM) proteins use radical chemistry to perform numerous reactions which would be impossible for “classical” two-electron organic chemistry, and which remain challenging for chemists. This vast family of metalloenzymes, found in all living organisms, includes more than 110 000 members identified to date and catalyses over 70 different chemical reactions on a very broad variety of substrates. Their extensive variety combined with the power of radical chemistry makes them very attractive for applications in synthetic biology, and as a result the study of radical SAM proteins is a internationally competitive field.
Our project is to study the structure and function of the tryptophan lyase radical SAM protein (NosL) involved in the synthesis of the antibiotic nosiheptide. This antibiotic is effective in treating infections with multi-resistant Gram (+) pathogens. NosL converts tryptophan into 3-methylindolic acid (MIA), a component of nosiheptide. It precisely controls which C-C bond is broken, despite a remarkable capacity for promiscuity both in terms of substrates and reactions catalysed. The study of NosL is ideal to understand the precise mechanisms through which substrates and reactions are selected. We have already started structural (crystallography) and functional (electron paramagnetic resonance spectroscopy (EPR) and liquid chromatography combined with mass spectrometry) studies to kinetically characterise the different steps in the NosL reaction and identify reaction intermediates. We wish to pursue these studies to examine how the reaction is affected when tryptophan analogues are used. Finally, we have identified NosL mutants with unexpected properties. These mutants can be used to examine the role played by the protein matrix in controlling the reaction. We now wish to develop tools to monitor the reaction in real-time by EPR, which is the best method to track and characterise radical species.
The PhD research will be carried out in the two laboratories and the candidate selected will have to prepare the protein samples in anaerobic conditions, create the molecular biology constructs and perform the experiments to graft photo-activable chemical systems onto the proteins, as well as preparing samples for EPR spectroscopy analysis. The candidate will be fully integrated into the two teams, where they will have access to complementary fields of expertise, as a result they will benefit from a scientific environment providing PhD training at the highest level.