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A mechanism for HemN - a radical SAM (S-adenosylmethionine) enzyme

Reference	BB/C504686/1
Principal Investigator / Supervisor	Professor Peter Heathcote
Co-Investigators / Co-Supervisors	Dr Stephen Rigby, Professor Martin Warren
Institution	Queen Mary University of London
Department	Sch of Biological and Chemical Sciences
Funding type	Research
Value (£)	210,293
Status	Completed
Type	Research Grant
Start date	01/04/2005
End date	31/03/2008
Duration	36 months

Abstract

We aim to elucidate the catalytic mechanism of the oxygen-independent coproporphyrinogen III oxidase HemN of E. coli, for which a high-resolution structure is available, utilizing paramagnetic resonance spectroscopy to identify and characterise radical intermediates in catalysis in this member of the radical S-adenosylmethionine (SAM) family of enzymes. Electron paramagnetic resonance (EPR) and Electron Nuclear Double Resonance (ENDOR) spectroscopies will be used to identify and characterise radical intermediates in catalysis, in conjunction with regiospecifically labelled (2H, 13C, 15N and 17O) substrate and SAM, and site-directed mutagenesis of conserved residues in the active site cleft of HemN. We have already demonstrated in preliminary studies that we can observe the reduced Fe-S cluster and a substrate radical on coproporphyrinogen III using EPR and ENDOR spectroscopy. Using a non-reactive substitute for the substrate in conjunction with stopped flow EPR we will trap the remaining radical intermediate in catalysis, the deoxyadensoyl radical. The combination of detailed spectroscopic investigation together with a high-resolution structure will enable interpretation of existing spectroscopic data from other enzymes of this family.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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