研究领域

Organic Chemistry

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.

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Biochemical characterization of New Delhi metallo-β-lactamase variants reveals differences in protein stability. Makena A, Brem J, Pfeffer I, Geffen RE, Wilkins SE, Tarhonskaya H, Flashman E, Phee LM, Wareham DW, Schofield CJ. J Antimicrob Chemother. 2014 Oct 16. doi:pii: dku403. [Epub ahead of print] Investigating the contribution of the active site environment to the slow reaction of hypoxia-inducible factor prolyl hydroxylase domain 2 with oxygen. Tarhonskaya H, Chowdhury R, Leung IK, Loik ND, McCullagh JS, Claridge TD, Schofield CJ, Flashman E. Biochem J. 2014 Nov 1;463(3):363-72. doi: 10.1042/BJ20140779. Studies on deacetoxycephalosporin C synthase support a consensus mechanism for 2-oxoglutarate dependent oxygenases.Tarhonskaya H, Szöllössi A, Leung IK, Bush JT, Henry L, Chowdhury R, Iqbal A, Claridge TD, Schofield CJ, Flashman E.Biochemistry. 2014 Apr 22;53(15):2483-93. doi: 10.1021/bi500086p. Epub 2014 Apr 11. Non-enzymatic chemistry enables 2-hydroxyglutarate-mediated activation of 2-oxoglutarate oxygenases.Tarhonskaya H, Rydzik AM, Leung IK, Loik ND, Chan MC, Kawamura A, McCullagh JS, Claridge TD, Flashman E, Schofield CJ.Nat Commun. 2014 Mar 5;5:3423. doi: 10.1038/ncomms4423. OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation.Singleton RS, Liu-Yi P, Formenti F, Ge W, Sekirnik R, Fischer R, Adam J, Pollard PJ, Wolf A, Thalhammer A, Loenarz C, Flashman E, Yamamoto A, Coleman ML, Kessler BM, Wappner P, Schofield CJ, Ratcliffe PJ, Cockman ME.Proc Natl Acad Sci U S A. 2014 Mar 18;111(11):4031-6. doi: 10.1073/pnas.1314482111. Epub 2014 Feb 18. Investigations on the oxygen dependence of a 2-oxoglutarate histone demethylase.Sánchez-Fernández EM, Tarhonskaya H, Al-Qahtani K, Hopkinson RJ, McCullagh JS, Schofield CJ, Flashman E.Biochem J. 2013 Jan 15;449(2):491-6. doi: 10.1042/BJ20121155. Studies on the reaction of nitric oxide with the hypoxia-inducible factor prolyl hydroxylase domain 2 (EGLN1).Chowdhury R, Flashman E, Mecinović J, Kramer HB, Kessler BM, Frapart YM, Boucher JL, Clifton IJ, McDonough MA, Schofield CJ.J Mol Biol. 2011 Jul 8;410(2):268-79. doi: 10.1016/j.jmb.2011.04.075. Epub 2011 May 13. Evidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen.Flashman E, Hoffart LM, Hamed RB, Bollinger JM Jr, Krebs C, Schofield CJ.FEBS J. 2010 Oct;277(19):4089-99. doi: 10.1111/j.1742-4658.2010.07804.x. Epub 2010 Aug 31. Investigating the dependence of the hypoxia-inducible factor hydroxylases (factor inhibiting HIF and prolyl hydroxylase domain 2) on ascorbate and other reducing agents.Flashman E, Davies SL, Yeoh KK, Schofield CJ.Biochem J. 2010 Mar 15;427(1):135-42. doi: 10.1042/BJ20091609. Using NMR solvent water relaxation to investigate metalloenzyme-ligand binding interactions.Leung IK, Flashman E, Yeoh KK, Schofield CJ, Claridge TD.J Med Chem.2010 Jan 28;53(2):867-75. doi: 10.1021/jm901537q. Structural basis for binding of hypoxia-inducible factor to the oxygen-sensing prolyl hydroxylases.Chowdhury R, McDonough MA, Mecinović J, Loenarz C, Flashman E, Hewitson KS, Domene C, Schofield CJ.Structure. 2009 Jul 15;17(7):981-9. doi: 10.1016/j.str.2009.06.002. Use of mass spectrometry to probe the nucleophilicity of cysteinyl residues of prolyl hydroxylase domain 2.Mecinović J, Chowdhury R, Flashman E, Schofield CJ.Anal Biochem. 2009 Oct 15;393(2):215-21. doi: 10.1016/j.ab.2009.06.029. Epub 2009 Jun 27. Direct analysis of enzyme-catalyzed DNA demethylation.Karkhanina AA, Mecinović J, Musheev MU, Krylova SM, Petrov AP, Hewitson KS, Flashman E, Schofield CJ, Krylov SN.Anal Chem. 2009 Jul 15;81(14):5871-5. doi: 10.1021/ac9010556. Application of a proteolysis/mass spectrometry method for investigating the effects of inhibitors on hydroxylase structure.Stubbs CJ, Loenarz C, Mecinović J, Yeoh KK, Hindley N, Liénard BM, Sobott F, Schofield CJ, Flashman E.J Med Chem. 2009 May 14;52(9):2799-805. doi: 10.1021/jm900285r. Evidence for a stereoelectronic effect in human oxygen sensing.Loenarz C, Mecinović J, Chowdhury R, McNeill LA, Flashman E, Schofield CJ.Angew Chem Int Ed Engl. 2009;48(10):1784-7. doi: 10.1002/anie.200805427. Dynamic states of the DNA repair enzyme AlkB regulate product release.Bleijlevens B, Shivarattan T, Flashman E, Yang Y, Simpson PJ, Koivisto P, Sedgwick B, Schofield CJ, Matthews SJ.EMBO Rep. 2008 Sep;9(9):872-7. doi: 10.1038/embor.2008.120. Epub 2008 Jul 11. Crystal structure of the C1 domain of cardiac myosin binding protein-C: implications for hypertrophic cardiomyopathy.Govada L, Carpenter L, da Fonseca PC, Helliwell JR, Rizkallah P, Flashman E, Chayen NE, Redwood C, Squire JM.J Mol Biol. 2008 Apr 25;378(2):387-97. doi: 10.1016/j.jmb.2008.02.044. Epub 2008 Mar 4. Support for a trimeric collar of myosin binding protein C in cardiac and fast skeletal muscle, but not in slow skeletal muscle.Flashman E, Korkie L, Watkins H, Redwood C, Moolman-Smook JC.FEBS Lett. 2008 Feb 6;582(3):434-8. doi: 10.1016/j.febslet.2008.01.004. Epub 2008 Jan 15. Kinetic rationale for selectivity toward N- and C-terminal oxygen-dependent degradation domain substrates mediated by a loop region of hypoxia-inducible factor prolyl hydroxylases.Flashman E, Bagg EA, Chowdhury R, Mecinović J, Loenarz C, McDonough MA, Hewitson KS, Schofield CJ.J Biol Chem. 2008 Feb 15;283(7):3808-15. Epub 2007 Dec 5. ESI-MS studies on prolyl hydroxylase domain 2 reveal a new metal binding site.Mecinović J, Chowdhury R, Liénard BM, Flashman E, Buck MR, Oldham NJ, Schofield CJ.ChemMedChem. 2008 Apr;3(4):569-72.