Award details

Domain motion coupled to radical catalysis in ornithine aminomutase

Reference	BB/H000577/1
Principal Investigator / Supervisor	Professor David Leys
Co-Investigators / Co-Supervisors	Dr Stephen Rigby, Professor Nigel Scrutton
Institution	The University of Manchester
Department	Life Sciences
Funding type	Research
Value (£)	398,351
Status	Completed
Type	Research Grant
Start date	17/08/2009
End date	16/12/2012
Duration	40 months

Abstract

The number and types of enzymes that deploy radical-based chemistry continues to grow as methods for radical detection and characterisation improve. Most radicals are unstable, are highly reactive and short-lived, making their presence in the reaction cycle elusive. With improved methods for their rapid detection more radical-based enzymes are emerging, and with an explosion in the amount genome sequence data, entire families or superfamilies of radical-containing enzymes are being uncovered. Radicals are essential to biological catalysis, but have often evaded in-depth study due to high reactivity and short life time. B12 dependent isomerases use radical chemistry to swap a hydrogen atom with an electron withdrawing group, X, on vicinal carbon atoms. The challenge for enzymes of this type is to contain and control the reactive radical species, directing them towards productive catalysis, and minimizing aberrant side reactions with radical scavengers such as oxygen. Tight control of radical trajectories is essential for (i) obtaining the desired chemistry and (ii) suppressing aberrant side reactions deleterious to the enzyme and organism. Control is also key to harnessing B12 chemistry in biocatalysis. We are using the enzyme ornithine aminomutase as a model to study these issues. This enzyme is unique in using both B12 and B6 cofactors, using (substrate dependent) domain dynamics to bring them in close proximity during catalysis. Using an integrated combination of crystallography, solution spectroscopy methods and EPR we aim to gain deeper understanding of the mechanism of ornithine aminomutase. In particular the connection between domain motion and catalysis and the structure of the radical intermediate states.

Summary

Atoms and molecules contain small negatively charged particles called electrons. Typically, two electrons on an atom or molecule pair with one another, thereby lowering their overall energy and increasing their stability. An unpaired or lone electron on a molecule or atom is referred to as a radical. The increased energy associated with radicals cause them to be unstable and reactive towards other molecules, for example oxygen or water. Within a living organism, the targets for these aberrant radical reactions can damage cellular components like DNA or protein, leading to cancers, aging or even death. Humans, like all living things, have specialised molecules for carrying out specific biological functions. Some of these functions are executed by specifically designed enzymes. Interestingly, research is uncovering an ever increasing list of enzymes which use radicals to carry out unusual and very difficult chemical reactions. A certain class of these enzymes uses vitamin B12, which serves as a reservoir for radicals. The B12-molecule splits into two radicals when the substrate binds to the B12-enzyme. This permits a radical relay system to ensue; the lone electron will hop from one molecule to the next in the enzyme cavity during the course of the catalytic reaction. Two important challenges faced by B12-enzymes, as with all enzymes which use radicals, are (i) controlling the timing of radical production and (ii) controlling the reactivity and trajectory of radicals within the enzyme cavity. During the course of the catalytic cycle, the enzyme must direct the reactive radicals toward their intended targets in a series of highly synchronized events, whilst at the same time minimize or prevent the radical from extinguishing itself by collision with the wrong molecule or atom (i.e. water, oxygen, or the enzyme itself). To better understand how enzymes in general control the peregrination of radicals, we will focus on a particular enzyme, termed ornithine aminomutase. This enzyme is unique in that it not only contains vitamin B12, but also vitamin B6. Both B6 and B12 serve as sensitive probes for monitoring radical propagation during the catalytic cycle, making ornithine aminomutase an ideal system for detailed investigation of enzyme-mediate radical chemistry. We have recently determined the molecular architecture of the enzyme, (i.e. where each atom of the enzyme is located in a three dimensional space). From this information, we can examine the unique structural features of the enzyme that enables it to direct radicals towards their intended target. As part of this programme we will also investigate the distance and relative orientation of the radical pair, by a technique whereby we place the enzyme in a large magnetic field. Under this particular physical state, the radicals will act as miniature magnets, and we will be able to derive information on their immediate environment, (i.e. neighbouring atoms) as well as the distance and orientation to a second radical. We will also trap the enzyme at different stages of the catalytic cycle, and use the above techniques to determine how and to what extent the enzyme changes its conformation to enable productive radical propagation. From this research, we will better understand how enzymes control and harness the energy associated radicals, enabling chemically difficult and energetically challenging reactions to be performed.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Structural Biology
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

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search