Research Network for Metals in Medicine

 

 

Dr Elizabeth Gillam

Affiliation: University of Queensland, School of Biomedical Sciences

Postal Address:
School of Biomedical Sciences
Skerman Building
The University of Queensland
Queensland, 4072
AUSTRALIA

Phone: +61 (07) 3365 1410
Fax: +61 (07) 3365 1766
Email: e.gillam@uq.edu.au
Webpage: http://www.uq.edu.au/sbms/index.html?page=11194&pid=10867


Research Profile

Our particular fascination is the functional diversity of cytochrome P450 enzymes (CYPs) especially those responsible for xenobiotic metabolism in humans. These are enzyme catalysts of exceptional versatility, being principally responsible for the clearance of a practically unlimited variety of chemicals from the body. The capabilities of P450s are only just coming to be fully recognized and structural studies on P450s should yield critical insights into how enzyme structure determines function. Moreover, the biotechnological potential of P450s remains yet to be exploited. All of the specific research themes detailed below take advantage of our recognized expertise in the expression of recombinant human cytochrome P450 enzymes in bacteria.

Structure function studies of human CYP3A, 2D and 2C enzymes: Collectively P450s of the 3A, 2D and 2C subfamilies handle about 95% of all drugs and other chemicals to which humans are exposed. This substrate range is truly exceptional, even more so when it is realised that a single form P450 3A4 is the major catalyst of the metabolism of ~40-50% of all drugs and can handle about 80% of drugs at some level. Our research is directed towards characterising the active sites of these enzymes, so we can see how they can accommodate such substrate diversity yet retain in some cases quite specific regioselectivity. The approach we have taken is to combine directed mutagenesis and metabolic analysis of the recombinant enzymes with electron paramagnetic resonance (done in collaboration with Dr. Graeme Hanson of the Centre for Magnetic Resonance) and homology modelling.

Molecular breeding as a way of exploring sequence space and catalytic potential of P450 enzymes: The demonstrated catalytic diversity of P450 enzymes makes them the ideal starting material for engineering sophisticated chemical reagents to catalyse difficult chemical transformations. In collaboration with another centre member, Dr. James DeVoss, we are using the emerging technology of molecular breeding (also known as DNA shuffling) to generate libraries of mutants from naturally-occurring P450 forms with the aim of selecting for desired properties. Recently we demonstrated that a variety of P450s catalyse the oxidation of indole and its derivatives to form indigoid dyes. This gives us a facile initial screen for mutants with altered activities as well as an end-point that has commercial relevance.

The role of human P450s in the molecular toxicology of drugs: While P450s are responsible for the metabolic clearance of drugs from the human body, this is not always a benevolent process: sometimes metabolites are generated that are chemically reactive and may cause mutagenic or other toxic effects. We are interested in two specific examples of this "bioactivation" of drugs: the drug hypersensitivity syndrome that follows administration of phenytoin and certain other anticonvulsants to a small proportion of patients; and the carcinogenic effects of the breast cancer drug, tamoxifen, in the endometrium.

Selected Publications

  1. Gillam, E.M.J., Notley, L.M., Cai, H., DeVoss, J.J., and Guengerich, F.P.: Oxidation of Indole by Cytochrome P450 Enzymes, Biochemistry, 39, 13817-18824, 2000
  2. Parikh, A., Gillam, E.M.J., and Guengerich, F.P.: Drug metabolism by Escherichia coli expressing human cytochromes P450, Nature Biotechnology, 15, 784-788,1997
  3. Munns, A.J., DeVoss, J.J. Dickinson, R.G., Hooper, W.D. and Gillam, E.M.J.: Bioactivation of Phenytoin by Human Cytochrome P450: Characterization of the Mechanism and Targets of Covalent Adduct Formation, Chemical Research in Toxicology, 10, 1049-1058, 1997
  4. Cuttle, L., Munns, A.J., Hogg, N.A., Scott, J.R., Hooper, W.D., Dickinson, R.G. , and E.M.J. Gillam: Phenytoin Metabolism by Human Cytochrome P450: Involvement of P450 3A and 2C Forms in Secondary Metabolism and Drug-Protein Adduct Formation, Drug Metabolism and Disposition 28, 945-950, 2000
  5. Gillam, E.M.J., Wunsch, R.M., Ueng, Y.-F., Shimada, T., Reilly, P. E. B., Kamataki, T., and Guengerich, F.P.: Expression of Cytochrome P450 3A7 in Escherichia coli: Effects of 5’ Modification and Catalytic Characterization of Recombinant Enzyme Expressed in Bicistronic Format with NADPH-Cytochrome P450 Reductase, Archives of Biochemistry and Biophysics, 345, 81-90, 1997