Research Guides

Our research is driven by a desire to apply chemical principles and techniques to understanding biology. A recent focus of our current work is contributing to a chemical understanding of genetics including how information can be transferred between generations without changes in the 'ATCG' DNA sequence (epigenetics). We have also worked closely with clinical researchers to uncover the mechanism by which humans sense and respond to changes in oxygen levels; This work is important for understanding how tumours grow and how animals adapt to limiting oxygen, e.g. on going to altitude. We apply the knowledge gained in our basic research to the design and synthesis of enzyme inhibitors for proof of principle' therapeutic use, in understanding biosynthetic and signaling pathways and in pioneering new methods for medicine. A variety of techniques are used in our research, including synthetic chemistry, enzyme purification and characterisation, cloning/mutagenesis, and biophysical techniques including X-ray crystallography, NMR and mass spectrometry. We intend that all of our projects involve interesting chemistry combined with biomedicinal applications and are of basic science interest.

2-Oxoglutarate dependent oxygenases

We are interested in enzymes which catalyse synthetically difficult or 'impossible' reactions, e.g. the stereoselective hydroxylation of unactivated carbon-hydrogen bonds. Of particular interest to us are the extended family of non-haem iron dependent oxygenases, most of which use 2-oxoglutarate (2OG) as a cosubstrate. We aim to develop our structural-mechanistic understanding of these enzymes in order to engineer a template catalyst (which may or may not be protein based) for stereo-selective oxidations. Enzymes under study are either of synthetic utility (amino acid oxygenases, e.g. proline hydroxylase), or of therapeutic importance (antibiotic biosynthesis, oxygen sensing). Crystal structures of enzymes from this family have been solved by us and we are using the information to alter substrate selectivities in order to modify in vivo biosynthetic pathways. We have shown that these enzymes are involved in a  novel oxygen sensing mechanism in humans and range of human diseases, including obesity. Together with researchers at the Wellcome Trust Centre for Human Genetics are exploring the therapeutic potential of this discovery for cardiovascular disease and cancer.

 

Oxygen sensing and the hypoxic response

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that modulates the cellular response to low oxygen tension (hypoxia). HIF levels are directly regulated by dioxygen: Under hypoxic conditions levels of HIF increase enabling transcriptional activation of an array of genes including erythropoietin, vascularendothelial growth factor and nitric oxide synthase. We have shown that under normoxic conditions both the level of HIF and its ability to enable transcription are directly controlled by its post-translational hydroxylation by members of the iron (II) and 2-oxoglutarate dependent oxygenase family (in collaboration with Peter Ratcliffe and coworkers, Dept. of Medicine). Hydroxylation of HIF at conserved prolyl-residues is mediated by three prolyl-hydroxylase isozymes (PHD1-3, for prolyl hydroxylase domain enzymes) whilst hydroxylation of an asparaginyl-residue in the C-terminal transactivation domain of HIF is mediated by FIH (for factor inhibiting HIF). Modulation of the HIF mediated hypoxic response is of potential use in a wide range of disease states including cardiovascular disease and cancer.

This laboratory is concerned with the biochemical characterization of both HIF and its hydroxylases. We have solved crystal structures of both the HIF prolyl and asparaginyl hydroxylases, which are significant value in the design of specific inhibitors, aimed at therapeutic  modulation of the hypoxic response for cardiovascular and other diseases.

Views of the crystal structure of FIH with a HIF substrate bound (in red on left and yellow on right).

 Recently, we have shown that the human HIF system is also present in the simplest known animals and are pursuing the paths by which animals evolved their oxygen sensing mechanisms.

 

 

Demethylases, DNA repair and obesity

We are investigating the mechanism of several enzymes involved in DNA repair including AlkB and related 2-oxoglutarate oxygenases. AlkB repairs 1-methyladenine and 3-methylcytosine residues by oxidative demethylation, regenerating the original base and producing formaldehyde. The mechanism of DNA repair by AlkB and related histone modifying enzymes are under further investigation in the group. Many chemotherapy treatments employ DNA alkylation as a means of destroying cancerous cells. Inhibition of enzymes involved in DNA repair may enable a reduction in the severity of such treatments. We have identified several inhibitors of AlkB and more are under investigation. Remarkably we have found that the Fat Mass and Obesity protein (FTO), mutations to which are associated with obesity, is related to AlkB and catalyses modifications to nucleic acids. We are working to understand how the biochemical activity of FTO is linked to obesity.

 

Mechanisms of antibiotic biosynthesis, mode of action and resistance

Penicillin type antibiotics are inactivated by hydrolysis as catalysed by beta-lactamase enzymes. We are developing inhibitors, e.g. peptidic trifluoromethylketones, of beta-lactamases, in particular the metal dependent enzymes, since these enzymes accept almost all β-lactam antibiotics as substrates and represent a danger to the continued use of all penicillin type antibiotics.

Clavulanic acid is the most important clinically used inhibitor of beta-lactamases. Despite its small size no asymmetric synthesis of 1 has been reported and it is produced by fermentation. We are studying the highly unusual biosynthesis of clavulanic acid 1 with a view to improving its production and modifying the pathway for the production of novel antibiotics. Functions have been assigned for a number of the enzymes in the pathway, and crystallographic studies have resulted in structure determination of several enzymes from the pathway.

Carbapenems are some of the most clinically useful beta-lactam antibiotics due to their greater resistance to beta-lactamases which render many classic beta-lactam antibiotics ineffective for the treatment of resistant strains of bacteria. (R)-1-carbapen-2-em-3-carboxylate 3 is the simplest of this family of compounds and is produced by several bacteria as part of their natural defenses. Recently we have reported the crystal structure of CarC a 2OG dependent oxygenase which is responsible for an unprecedented and chemically very interesting epimerisation step in carbapenem biosynthesis.

 

 

 

 

Flavonoid and ethylene biosynthesis:

Like that of ethylene, which is a plant signalling molecule, the biosynthesis of the medicinally important plant secondary metabolites, the flavonoids involves steps catalysed by non-haem Fe(II), 2-oxoglutarate oxygenases. We have undertaken extensive analysis of one such enzyme, anthocyanidin synthase (ANS). ANS was found to catalyse conversion of a number of unnatural substrates producing multiple products with the outcome being highly dependent on the stereochemistry of the starting material. This substrate work combined with crystal structure of ANS [9] has given a detailed mechanistic insight into ANS catalysis. Recently, other 2-oxoglutarate dependent dioxygenases of flavonoid biosynthesis have also been shown to have diverse substrate selectivities. We are  investigating the mechanism of ANS and related plant enzymes such as flavonol synthase and the ethylene biosynthesis enzyme.

View of ANS from the cover of Structure and two of the reactions it catalyses.

 

Proteases

Hydrolytic enzymes are involved in most metabolic processes and include enzymes involved in many disease states, including emphysema, malaria and viral infections. Genomic studies indicate proteases are one of the most abundant enzyme families. We are interested in discovering 'atomic resolution' mechanisms for hydrolytic enzymes. A combination of synthetic probes and biophysical techniques are used to investigate the stereoelectronics of protease catalysed reactions. The results are useful for the design of inhibitors. The work has resulted in the trapping of an acyl-enzyme complex catalysis by the serine protease elastase. Time resolved crystallographic studies in conjunction with 'pH' jump experiments are in progress. A combination of synthetic and crystallographic studies have been used to rationalise the mechanism of elastase inhibition by b-lactams and to design simple monocyclic g-lactam inhibitors. We are extending these studies to other hydrolytic enzymes [13, 14, 15].

 

1. Crystal-structure of isopenicillin N-synthase is the first from a new structural family of enzymes

Roach PL, Clifton IJ, Fulop V, Harlos K, Barton G J, Hajdu J, Andersson I, Schofield CJ, Baldwin JE

Nature 375: 700-704, 1995

 

2. Inhibition of TEM-2 beta-lactamase from Escherichia-coli by clavulanic acid - observation of intermediates by electrospray-ionization mass-spectrometry

Brown RPA, Aplin RT, Schofield CJ

Biochemistry 35: 12421-12432, 1996

 

3. Structure of a specific acyl-enzyme complex formed between beta-casomorphin-7 and porcine pancreatic elastase

Wilmouth RC, Clifton IJ, Robinson CV, Roach PL, Aplin RT, Westwood NJ, Hajdu J, Schofield CJ Nature Structural Biology 4: 456-4627, 1997

 

4. Structural origins of the selectivity of the trifunctional oxygenase clavaminic acid synthase

Zhang ZH, Ren JS, Stammers DK, Baldwin JE, Harlos K, Schofield CJ

Nature Structural Biology 7: 127-133, 2000

 

5. C. elegans EGL-9 and mammalian SM-20 define a conserved family of dioxygenases that regulate hypoxia inducible factor (HIF) through prolyl hydroxylation

Epstein ACR, Gleadle JM, McNeil LA, Hewitson KS, O'Rourke J, Mole DR, Mukherji M, Metzen E, Wilson MI, Dhanda A, Tian Y-M, Masson N, Hamilton DL, Jaakkola P, Barstead R, Hodgkin J, Maxwell PH, Pugh CW, Schofield CJ, Ratcliffe PJ,

Cell 107: 43-54, 2001

 

6. Structure and mechanism of anthocyanidin synthase from Arabidopsis thaliana

Wilmouth RC, Turnbull JJ, Clifton IJ, Prescott AG; Schofield CJ;

Structure, 10: 1-20, 2002

 

7. Hypoxia-inducible factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural family

Hewitson KS, McNeill LA, Riordan MV, Tian YM, Bullock AN, Welford RW, Elkins JM, Oldham NJ, Bhattacharya S, Gleadle JM, Ratcliffe PJ, Pugh CW, Schofield CJ, J. Biol. Chem 277: 26351-26355, 2002

 

8. Structure of factor-inhibiting hypoxia-inducible factor (HIF) reveals mechanism of oxidative modification of HIF-1 alpha

Elkins JM, Hewitson KS, McNeill LA, Seibel JF, Schlemminger I, Pugh CW, Ratcliffe PJ, Schofield CJ

J Biol Chem 278: 1802-1806, 2003

 

9. Carboxymethylproline synthase (CarB), an unusual C-C bond-forming enzyme of the crotonase superfamily involved in carbapenem biosynthesis

Sleeman MC, Schofield CJ

J Biol Chem 279: 6730-6736, 2004

 

10. Structure of human phytanoyl-CoA 2-hydroxylase identifies molecular mechanisms of Refsum disease

McDonough MA, Kavanagh KL, Butler D, Searls T, Oppermann U, Schofield CJ

J Biol Chem 280: 41101-41110, 2005

 

11. Posttranslational hydroxylation of ankyrin repeats in IkappaB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH)

Cockman ME, Lancaster DE, Stolze IP, Hewitson KS, McDonough MA, Coleman ML, Coles CH, Yu XH, Hay RT, Ley SC, Pugh CW Oldham NJ, Schofield CJ, Ratcliffe PJ

PNAS 103: 14767-14772, 2006

 

12. Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2)

McDonough MA, Li V, Flashman E, Chowdhury R, Mohr C, Lienard BMR, Zondlo J, Oldham NJ, Clifton IJ, Lewis J, McNeill LA, Kurzeja RJM, Hewitson KS, Yang E, Jordan S, Syed RS, Schofield CJ,

PNAS 103: 9814-9819, 2006

 

13. Crystal structures of histone demethylase JMJD2A reveal basis for substrate specificity

Ng SS, Kavanagh KL, McDonough MA, Butler D, Pilka ES, Lienard BMR, Bray JE, Savitsky P, Gileadi O von Delft F Rose NR, Offer J Scheinost JC, Borowski T Sundstrom M Schofield CJ, Oppermann U,

Nature 448: 87-91, 2007

 

14. Asparaginyl Hydroxylation of the Notch Ankyrin Repeat Domain by Factor Inhibiting Hypoxia-inducible Factor

Coleman ML, McDonough MA, Hewitson KS, Coles C, Mecinovic J, Edelmann M, Cook KM, Cockman ME, Lancaster DE, Kessler BM, Oldham NJ, Ratcliffe PJ, and Schofield CJ

J. Biol. Chem., 282: 24027-24038, 2007

 

15. The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase

Gerken T, Girard CA, Tung Y. C. L., Webby CJ, Saudek V, Hewitson KS, Yeo GSH, McDonough MA, Cunliffe S, McNeill LA, Galvanovskis J, Rorsman P, Robins P, Prieur X, Coll AP, Ma M, Jovanovic Z, Farooqi IS, Sedgwick B, Barroso I, Lindahl T, Ponting CP, Ashcroft, FM, O'Rahilly S, Schofield CJ

Science 318: 1469-1472, 2007

 

16. Structural and mechanistic basis of penicillin-binding protein inhibition by lactivicins

Macheboeuf P, Fischer DS, Brown T, Zervosen A, Luxen A, Joris B, Andréa Dessen A and Christopher J Schofield

Nature Chemical Biology 3: 565-569, 2007

 

17. Evidence for a Stereoelectronic Effect in Human Oxygen Sensing

Loenarz C, Mecinovic J, Chowdhury R, McNeill LA, Flashman E, Schofield CJ

Angewandte Chemie-International Edition 48: 1784-1787, 2009

 

18. Jmjd6 Catalyses Lysyl-Hydroxylation of U2AF65, a Protein Associated with RNA Splicing

Webby CJ, Wolf A, Gromak N, Dreger M, Kramer H, Kessler B, Michael L. Nielsen ML, Corinna Schmitz C, Butler DS, Yates, III JR, Delahunty CM, Hahn P, Lengeling A, Mann M, Proudfoot NJ, Schofield CJ, Böttger A

Science 325: 90-93, 2009

 

19.  Structural Basis for Binding of Hypoxia-Inducible Factor to the Oxygen-Sensing Prolyl Hydroxylases

Chowdhury R, McDonough MA, Mecinovic J, Loenarz C, Flashman E, Hewitson KS, Domene C, Schofield CJ

Structure, 17: 981-989, 2009

 

20. PHF8, a gene associated with cleft lip/palate and mental retardation, encodes for an N-epsilon-dimethyl lysine demethylase

Loenarz C, Ge W, Coleman ML, Rose NR, Cooper CDO, Klose RJ, Ratcliffe PJ, Schofield CJ

Human Molecular Genetics. 19: 217-222, 2010.