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SoCoBio (Universities of Southampton, Kent, Sussex, Portsmouth and NIAB EMR)

Biosynthetic Potential of the Radical SAM Enzyme Lipoyl Synthase

Primary Supervisor: Prof. Peter L. Roach (University of Southampton)

Second Supervisor: Prof. John Spencer (University of Sussex)

Project Summary

Using enzymes to catalyse reactions has enormous potential to revolutionise the production of a wide range of valuable chemicals, lowering costs, achieving difficult biosynthetic reactions at low temperatures and in an environmentally benign manner. In this project, we will explore the biosynthetic potential of Lipoyl Synthase, a member of the ‘radical SAM’ family of enzymes, which catalyses the insertion of two sulfur atoms into an octanoyl substrate to yield lipoyl product.

Naturally, this reaction is essential for the functional assembly of active pyruvate dehydrogenase, but the key question for our project is what other products can be formed from structurally modified substrates? Several approaches will be used as part of this chemical biology project:
1. The heterologous expression and purification to yield active Lipoyl Synthase.
2. Measure Lipoyl Synthase activity using HPLC analysis of reactions.
3. Exploration of the substrate specificity of Lipoyl Synthase, starting with longer and shorter (nonanoyl and heptanoyl) substrates and spectroscopically characterise the novel products. Explore modification of pharmaceuticals.
4. Site directed mutagenesis of the active site sequences to permit the turnover of bulkier substrates.
5. Use of synthetic chemical ‘radical clock’ probes to investigate the mechanism of Lipoyl Synthase.
6. Use of X-ray crystallography to determine the interaction of substrates with Lipoyl Synthase.
7. Elaboration of the lipoyl products to form molecules of high value in e.g. medicinal chemistry.
This combination of approaches will provide insight into the biosynthetic potential of Lipoyl Synthase and other enzymes of the ‘radical SAM’ superfamily and permit the development of a clear hypothesis for the mechanism of Lipoyl Synthase catalysis. The training provided in these chemical biology approaches have wide application in academic and industrial research. The excitement of our project lies in the sustainable and green biotransformations catalysed by Lipoyl Synthase that will yield high impact conclusions/publications.