 Professor C.J. Schofield
Chemical Research Laboratory
Telephone: 44 (0) 1865 275 625 / Fax: 44 (0) 1865 275 674
Our research is driven by a desire to apply chemical principles
and techniques to understanding biology. We apply the knowledge
gained in the design and synthesis of enzyme inhibitors for therapeutic
use understanding biosynthetic and signaling pathways and in
developing new methods for the preparation of antibiotics. 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. All of our projects involve interesting
chemistry combined with biomedicinal applications
2-Oxoglutarate dependent non-haem iron 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). Several 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 together with researchers at the Wellcome Trust
Centre for Human Genetics are exploring the therapeutic potential
of this discovery for cardiovascular disease and cancer.
Mechanisms of antibiotic biosynthesis, mode of action and
resistance:
Clavulanic acid 1 is the most important clinically
used inhibitor of b-lactamases, which
mediate bacterial resistance to penicillin type antibiotics.
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.[1,2,3]
Carbapenems are some of the most clinically useful b-lactam
antibiotics due to their greater resistance to b-lactamases
which render many classic b-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 [4] a 2OG dependent oxygenase which
is responsible for an unprecedented and chemically very interesting
epimerisation step in carbapenem biosynthesis.
 
Proposed biosynthetic pathway of (R)-1-carbapen-2-em-3-carboxylate
and hexameric structure of CarC
Oxygen sensing and the hypoxic response (in collaboration
with Prof. P. Ratcliffe and Prof. C. Pugh):
Hypoxia-inducible factor (HIF) is
an a,b-heterodimeric
transcription factor that modulates the cellular response to
low oxygen tension (hypoxia). HIF-α levels are regulated
by dioxygen: under hypoxic conditions levels of HIF-α increase
enabling transcriptional activation of an array of genes including
erythropoietin, vascularendothelial growth factor and nitric
oxide synthase. Under normoxic conditions both the level of
HIF-a 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. Hydroxylation
of HIF-a at
either of two conserved proline residues is mediated by three
prolyl-hydroxylase isozymes (PHD1-3, for prolyl hydroxylase
domain enzymes) [5] whilst hydroxylation of an asparagine residue
in the C-terminal transactivation domain of HIF-a is
mediated by FIH (for factor inhibiting HIF) [6]. 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.
Recently we have solved the crystal structure of FIH complexed
with a segment of HIF [7], which will prove to be of significant
value in the design of specific FIH inhibitors, aimed at significant
modulation of the hypoxic response.
 
Views of the crystal structure of FIH with HIF-a substrate
bound (in red on left and yellow on right).
Flavonoid biosynthesis:
The biosynthesis of the medicinally
important plant secondary metabolites, the flavonoids involves
several steps catalysed by non-haem Fe(II), 2-oxoglutarate
dependent dioxygenases. 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 [8]. This substrate
work combined with our 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 continuing to investigate the mechanism
of ANS and related plant enzymes such as flavonol synthase
(FLS).
 
Structure of ANS on the cover of Structure
and two of the reactions catalysed.
The Chemical Biology of Refsums
Disease:
Phytanoyl-CoA hydroxylase (PhyH) is
one of the enzymes required for the metabolism of phytanic
acid, a dietary-acquired fatty acid derived from chlorophyll
that can accumulate in human tissue with deleterious effects.
Defects in the function of PhyH are responsible for an incurable
genetic disease known as Refsum's disease. We are investigating
the reaction catalysed by PhyH [10] and have investigated the
possibility of "rescuing" the activity of mutant forms of the
enzyme known to exist in patients with Refsum's disease [11].
Other enzymes involved in the degradation of phytanic acid
are being investigated.
DNA repair:
We are investigating the mechanism
of several enzymes involved in DNA repair including AlkB, a
non-haem Fe(II), 2-oxoglutarate dependent dioxygenase. AlkB
has been shown to repair 1-methyladenine and 3-methylcytosine
residues by oxidative demethylation, regenerating the original
base and producing formaldehyde. The mechanism of DNA repair
by AlkB is 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 [12].
Reaction catalysed by AlkB
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].
Metallo b-lactamases:
We are developing inhibitors, e.g.
peptidic trifluoromethylketones 2, of b-lactamases,
in particular the metal dependent enzymes, since these enzymes
accept almost all b-lactam antibiotics
as substrates [16,17] and represent a danger to the continued
use of all b-lactam antibiotics.
Selected recent publications
1. "Oligomeric structure of proclavaminic
acid amidino hydrolase: evolution of a hydrolytic enzyme in clavulanic
acid biosynthesis", Elkins, J. M., Clifton, I. J., Hernandez,
H., Doan, L. X., Robinson, C. V., Schofield, C. J., and Hewitson, K.
S. (2002) Biochem. J. 366, 423-434
2. "ORF6
from the clavulanic acid gene cluster of Streptomyces clavuligerus
has ornithine acetyltransferase activity", Kershaw, N.
J., McNaughton, H. J., Hewitson, K. S., Hernandez, H., Griffin,
J., Hughes, C., Greaves, P., Barton, B., Robinson, C. V., and
Schofield, C. J. (2002) Eur. J. Biochem. 269,
2052-2059
3. "Enzymatic
synthesis of monocyclic beta-lactams", Sleeman, M. C.,
MacKinnon, C. H., Hewitson, K. S., and Schofield, C. J. (2002) Bioorg. Med. Chem. Lett. 12,
597-599
4. "Crystal
structure of carbapenem synthase (CarC)", Clifton, I.
J., Doan, L. X., Sleeman, M. C., Topf, M., Suzuki, H., Wilmouth,
R. C., and Schofield, C. J. (2003) J.
Biol. Chem. 278,
20843-20850
5. "C-elegans
EGL-9 and mammalian homologs define a family of dioxygenases
that regulate HIF by prolyl hydroxylation", Epstein, A.
C. R., Gleadle, J. M., McNeill, L. A., Hewitson, K. S., O'Rourke,
J., Mole, D. R., Mukherji, M., Metzen, E., Wilson, M. I., Dhanda,
A., Tian, Y. M., Masson, N., Hamilton, D. L., Jaakkola, P.,
Barstead, R., Hodgkin, J., Maxwell, P. H., Pugh, C. W., Schofield,
C. J., and Ratcliffe, P. J. (2001) Cell 107,
43-54
6. "Hypoxia-inducible
factor (HIF) asparagine hydroxylase is identical to factor
inhibiting HIF (FIH) and is related to the cupin structural
family", Hewitson, K. S., McNeill, L. A., Riordan, M.
V., Tian, Y. M., Bullock, A. N., Welford, R. W., Elkins, J.
M., Oldham, N. J., Bhattacharya, S., Gleadle, J. M., Ratcliffe,
P. J., Pugh, C. W., and Schofield, C. J. (2002) J.
Biol. Chem. 277,
26351-26355
7. "Structure
of factor-inhibiting hypoxia-inducible factor (HIF) reveals
mechanism of oxidative modification of HIF-1 alpha", Elkins,
J. M., Hewitson, K. S., McNeill, L. A., Seibel, J. F., Schlemminger,
I., Pugh, C. W., Ratcliffe, P. J., and Schofield, C. J. (2003) J.
Biol. Chem. 278,
1802-1806
8. "Evidence
for oxidation at C-3 of the flavonoid C-ring during anthocyanin
biosynthesis", Welford, R. W. D., Turnbull, J. J., Claridge,
T. D. W., Prescott, A. G., and Schofield, C. J. (2001) Chem. Commun., 1828-1829
9. "Structure
and mechanism of anthocyanidin synthase from Arabidopsis thaliana",
Wilmouth, R. C., Turnbull, J. J., Welford, R. W. D., Clifton,
I. J., Prescott, A. G., and Schofield, C. J. (2002) Structure 10,
93-103
10. "Studies on phytanoyl-CoA
2-hydroxylase and synthesis of phytanoyl-coenzyme A",
Kershaw, N. J., Mukherji, M., MacKinnon, C. H., Claridge, T.
D. W., Odell, B., Wierzbicki, A. S., Lloyd, M. D., and Schofield,
C. J. (2001) Bioorg.
Med. Chem. Lett. 11,
2545-2548
11. "Structure-function
analysis of phytanoyl-CoA 2-hydroxylase mutations causing Refsum's
disease", Mukherji, M., Chien, W., Kershaw, N. J., Clifton,
I. J., Schofield, C. J., Wierzbicki, A. S., and Lloyd, M. D.
(2001) Hum. Mol. Genet. 10, 1971-1982
12. "The selectivity
and inhibition of AlkB", Welford, R. W. D., Schlemminger,
I., McNeill, L. A., Hewitson, K. S., and Schofield, C. J. (2003) J.
Biol. Chem. 278, 10157-10161
13. "Synthesis and
evaluation of delta-lactams (piperazones) as elastase inhibitors",
Seibel, E., Brown, D., Amour, A., MacDonald, S. J., Oldham,
N. J., and Schofield, C. J. (2003) Bioorg.
Med. Chem. Lett. 13,
387-389
14. "X-ray structure
of a serine protease acyl-enzyme complex at 0.95-angstrom resolution",
Katona, G., Wilmouth, R. C., Wright, P. A., Berglund, G. I.,
Hajdu, J., Neutze, R., and Schofield, C. J. (2002) J. Biol. Chem. 277, 21962-21970
15. "X-ray snapshots
of serine protease catalysis reveal a tetrahedral intermediate",
Wilmouth, R. C., Edman, K., Neutze, R., Wright, P. A., Clifton,
I. J., Schneider, T. R., Schofield, C. J., and Hajdu, J. (2001) Nat. Struct. Biol. 8,
689-694
16. "The inhibitor
thiomandelic acid binds to both metal ions in metallo-beta-lactamase
and induces positive cooperativity in metal binding",
Damblon, C., Jensen, M., Ababou, A., Barsukov, I., Papamicael,
C., Schofield, C. J., Olsen, L., Bauer, R., and Roberts, G.
C. K. (2003) J. Biol.
Chem. 278, 29240-29251
17. "Binding of D-
and L-captopril inhibitors to metallo-beta- lactamase studied
by polarizable molecular mechanics and quantum mechanics",
Antony, J., Gresh, N., Olsen, L., Hemmingsen, L., Schofield,
C. J., and Bauer, R. (2002) J.
Comput. Chem. 23, 1281-1296
|
