Department of Chemsitry

Dr Emily Flashman



 

Royal Society Dorothy Hodgkin Fellow

Organic Chemistry

Chemistry Research Laboratory,
12 Mansfield Road,
Oxford,
OX1 3TA

emily.flashman@chem.ox.ac.uk

Telephone: +44 (0)1865 285110 (office); +44 (0)1865 275677 (lab).

Research

Mechanisms of Oxygen Sensing by the HIF Hydroxylases

All animals can respond to conditions of low oxygen (hypoxia) via a transcription factor protein, the hypoxia-inducible factor (HIF). In hypoxia, HIF upregulates genes which allow the body to adapt to decreased oxygen availability (the hypoxic response). A hypoxic response is seen in many cancer cells, either because of hypoxic conditions in tumours, or because cellular metabolic changes affect the normal regulation of HIF levels. The HIF hydroxylases are enzymes which downregulate HIF in the presence of oxygen. Our work focuses on understanding how these enzymes react with oxygen at the molecular level to enable them to be efficient oxygen sensors. Much of this work is carried out in collaboration with Prof. Chris Schofield's group.

Mechanism of Oxygen Sensing

The HIF hydroxylases are part of a broad family of enzymes, the Fe(II)/2-oxoglutarate dependent oxygenases, which have a variety of functions in biology. Using stopped-flow transient kinetic techniques, and in collaboration with Prof.’s Carsten Krebs and J. Marty Bollinger at Penn State University, we have found that one of the HIF hydroxylases, PHD2, reacts particularly slowly with oxygen. This is most likely to be related to its specific role as an oxygen sensor. We are interested in understanding what makes PHD2 unusual amongst the Fe(II)/2-oxoglutarate oxygenases in its reaction with oxygen, and are currently investigating this by looking at how variants of PHD2 function under different oxygen conditions. We are also interested in looking at other Fe(II)/2-oxoglutarate dependent oxygenases to understand which are able to act as oxygen sensors and what molecular features define the oxygen sensors.

Influence of Reducing Agents

HIF levels are affected by oxidising and reducing agents such as ascorbate, especially in cancer cells. These effects are primarily mediated by the HIF hydroxylases, although the precise mechanism is unknown. We are investigating how reducing agents increase the activity of the HIF hydroxylases, and interestingly, why different HIF hydroxylase reactions seem to be differentially affected by different reducing agents.

Looking Into Cells

The contents of a test tube are very different to the contents of a cell, and it is therefore important to be able to translate in vitro findings to a cellular context. To this end, we are trying to find ways of characterising the mechanistic properties of the HIF hydroxylases in a cellular context. We are currently investigating whether in-cell EPR can be used to visualise the active site of these enzymes under different cellular stress conditions.

 

Selected Publications

 
Flashman E, Hoffart LM, Hamed RB, Bollinger Jr JM, Krebs C and Schofield CJ (2010). Evidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen. FEBS J. 277: 4089-4099.
 
Flashman E, Davies SL, Yeoh KK and Schofield CJ (2010). Investigating the dependence of the hypoxia-inducible factor hydroxylases (factor inhibiting HIF and prolyl hydroxylase domain 2) on ascorbate and other reducing agents. Biochem J. 427: 135-142.
 
Stubbs CJ, Loenarz C, Mecinović J, Yeoh KK, Hindley N, Liénard BM, Sobott F, Schofield CJ and Flashman E (2009). Application of a proteolysis/mass spectrometry method for investigating the effects of inhibitors on hydroxylase structure. J Med Chem. 52: 2799-2805.
 
Flashman E, Bagg EAL, Chowdhury R, Mecinović J, Loenarz C, McDonough MA, Hewitson KS and Schofield CJ (2008). Kinetic rationale for selectivity towards N- and C-terminal oxygen dependent degradation domain substrates mediated by a loop region of the HIF prolyl hydroxylases. J Biol Chem. 283: 3808-3815.
 
McDonough MA, Li V, Flashman E, Chowdhury R, Mohr C, Leinard BM, Zondlo J, Oldham NJ, Clifton IJ, Lewis J, McNeill LA, Kurjeza R, Hewitson KS, Yang E, Jordan S, Syed R and Schofield CJ (2006). Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2). Proc. Natl. Acad. Sci. U.S.A. 103: 9814-9819.
 

McNeill LA, Flashman E­, Buck MRG, Hewitson KS, Clifton IJ, Jeschke G, Claridge TDW, Ehrismann D, Oldham NJ and Schofield CJ (2005). Hypoxia-inducible factor prolyl hydroxylase 2 has a high affinity for ferrous iron and 2-oxoglutarate. Mol Biosys. 1: 321-324.

 

Site Map | Printable View | © 2008 - 2014 Department of Chemistry - University of Oxford

Powered by mojoPortal | HTML 5 | CSS | Design by styleshout