Project No. – 2014

Primary Supervisor

Dr Andrew Lawrence – University of Southampton

Co-Supervisor

Prof Martin Warren – University of Kent

Summary

Enzymes from the B12-dependent radical SAM (rSAM) superfamily catalyse wide ranging and chemically challenging reactions, including the methylation of unactivated carbon centres.

They have emerged as significant players in the biosynthesis of many important natural products from bacteriochlorophyll to many antibiotics and anticancer agents such as pactamycin, mitomycin C, fosfomycin, polytheonamide and quinomycin. Despite their importance they remain poorly characterised.

The aim of this project is to purify and characterise a selection of B12-dependent rSAM enzymes involved in the biosynthesis of vitamins and antibiotics. We have currently developed systems to express and purify these proteins using E. coli as a host and are now in an ideal position to progress with the biochemical studies.

This is a highly interdisciplinary project and will involve the expression and purification of the target enzymes, biochemical studies to improve understanding of the mechanism and crystallographic studies of the enzymes with their substrates. The enzymes and the reactions they catalyse will be investigated using UV-visible, stopped-flow, HPLC-MS, EPR and NMR spectroscopy.

This research will not only provide molecular detail and mechanistic insight into a poorly characterised family of enzymes but, coupled with the use of cofactor analogues, will be used to produce novel natural product derivatives, potentially leading to new pharmaceutical agents.
The project spans synthetic biology and structural biology and a broad training will be provided in molecular biology, microbiology, protein purification, enzymology, structural biology and handling oxygen sensitive proteins and reagents. The successful candidate will be expected to spend time at both institutions, with the final 1-2 years of the project being based at the University of Southampton.