Faculty of Medicine
Department of Biochemistry & Molecular Biology
HALIFAX, NOVA SCOTIA | CANADA B3H 4R2 | 1 (902) 494-2480

Stephen L. Bearne, Ph.D. (Toronto), M.D.C.M. (McGill)


Professor
Cross-appointment with the Department of Chemistry

9-J1 Tupper
(902) 494-1974
Email
Lab Homepage

Research Areas

KEYWORDS: biological chemistry, enzymology, kinetics, enzyme inhibition, drug design, enzyme evolution, proteomics, organic synthesis

Enzymes are remarkable catalysts, bringing about huge rate enhancements. This rate enhancement depends on the enzyme's transition state, binding the latter species with greater affinity and reducing the activation barrier of the reaction. Our research program has two major goals: (1) to understand the nature of the protein-ligand interactions that contribute to stabilization of reactive intermediates and transition states, relative to the ground state, and (2) to understand the chemical mechanism of enzymes that are of therapeutic interest. Knowledge about the energetics of enzyme catalysis is essential for the preparation of synthetic catalysts through protein engineering and the design of drugs.

Enzyme Targets in Disease: Cytidine 5'-Triphosphate Synthase and Racemases
We are studying several different enzymes involved in nucelotide metabolism as targets for the development of chemotherapeutic agents. To-date, we have focused our efforts on studying the enzyme cytidine 5'-triphosphate synthase (CTPS). CTPS belongs to a family of enzymes known as the glutamine amidotransferases and catalyzes the conversion of UTP to CTP. Inhibition of CTPS activity could offer a means of ameliorating a variety of diseases, including cancer, viral infections, and protozoal infections. Our goals are to understand how the allosteric effector, GTP acts to promote glutamine hydrolysis, and to develop specific inhibitors of GTP-dependent activation of CTPS. We are also studying the enzymology of racemases with the goal of developing specific and potent inhibitors. Racemases under investigation include the prostate cancer biomarker alpha-methylacyl-CoA racemase, and those racemases involved in generating D-amino acids (antibacterial targets).

Fundamental Enzymology: Mechanism, Structure, Function, & Evolution of Mandelate Racemase (MR)
MR is a member of the enolase superfamily of enzymes and catalyzes the rapid carbon-hydrogen bond cleavage of a carbon acid substrate with a relatively high pKa value. We are studying MR as a paradigm for understanding how enzymes overcome the problem of carbon acidity. We are using transition state analogue inhibitors in combination with site-directed mutagenesis to probe the nature of the protein-ligand interactions that stabilize the altered substrate in the transition state. We are also conducting protein engineering and directed evolution to alter the substrate specificity of MR. Finally, we are conducting investigations to understand the evolution of enzymes that catalyze proton abstraction reactions.

Proteomics
Previously, we investigated the variation of the proteome of Fusobacterium species under different growth conditions to further develop our understanding of the metabolism of bacteria comprising the human microbiome. Ongoing studies are focused on developing reagents for proteomic profiling of enzymatic activities.

Learning Opportunities

Students working in my laboratory have the opportunity to develop skills in the areas of organic synthesis and protein chemistry, physical organic laboratory techniques, molecular modeling, enzyme kinetics, CD spectroscopy, microbiology, molecular biology, site-directed mutagenesis, and proteomics. This broad set of techniques is a great asset for students interested in exploring the exciting frontiers at the interface of modern biology and chemistry.

We thank the following agencies for past and present grants in aid of our research:

NSERC, CIHR, NSHRF, the Beatrice Hunter Cancer Research Institute, the Dalhousie Medical Research Foundation, and the Prostate Cancer Research Foundation

Keywords:
enzymology, enzyme kinetics and inhibition, site-directed mutagenesis, chemical biology, organic synthesis, protein purification, protein modification, proteomics, HPLC, FPLC, CD, UV-vis, NMR, 2D gel electrophoresis, cancer, periodontal disease

Graduate Students


Mandar Khanal M.Sc. (Biochemistry) Program
Mitesh Nagar Ph.D. (Biochemistry) Program
Alexander Roy M.Sc. (Chemistry) Program

Postdoctoral Fellows


Dr. Mohan Pal Jawaharlal Nehru University

Honours Students


Mathew Harty Biochemistry
Yuriy Khalak NSERC USRA/Biology/Physics
Tavia Raiche-Marsden Chemistry/Biochemistry
George Worthen NSERC USRA
Biochemistry/Microbiology

Lab Personnel


Ahmed Rashid NSERC USRA/Chemistry

Publications

  1. Bearne, S.L.*, (2012) Illustrating Enzyme Inhibition Using Free Energy Profiles J. Chem. Educ. 89:in press.
  2. Lietzan, A.D., Nagar, M., Pellmann, E.A., Bourque, J.R., Bearne, S.L., St. Maurice, M.*, (2012) Structure of mandelate racemase with bound intermediate analogues benzohydroxamate and cupferron. Biochemistry 51 (6):1160-1170 [PubMed]
  3. Steeves, C.H., Potrykus, J., Barnett, D.A., and Bearne, S.L.*, (2011) Oxidative stress response in the opportunistic oral pathogen Fusobacterium nucleatum Proteomics 11:2027-2037. [PubMed]
  4. Steeves, C.H., and Bearne, S.L.*, (2011) Activation and inhibition of CTP synthase from Trypanosoma brucei, the causative agent of African sleeping sickness Bioorg. Med. Chem. Lett. 21:5188-5190. [PubMed]
  5. Nagar, M., Narmandakh, A., Khalak, Y., Bearne, S.L.*, (2011) Redefining the minimal substrate tolerance of mandelate racemase. Racemization of trifluorolactate. Biochemistry 50(41):8846-8852 [PubMed]
  6. Narmandakh, A., and Bearne, S.L.*, (2010) Purification of recombinant mandelate racemase: Improved catalytic activity. Protein Expr. Purif. 69:39-46. [PubMed]
  7. Ouazia, D., and Bearne, S.L.*, (2010) A continuous assay for alpha-methylacyl-coenzyme A racemase using circular dichroism. Anal. Biochem. 398:45-51. [PubMed]
  8. Roy, A.C., Lunn, F.A., and Bearne, S.L., (2010) Inhibition of CTP synthase from Escherichia coli by xanthines and uric acids Bioorg. Med. Chem. Lett. 20:141-144. [PubMed]
  9. Thirumalairajan, S., Mahaney, B., and Bearne, S.L.*, (2010) Interrogation of the active site of OMP decarboxylase from Escherichia coli with a substrate analogue bearing an anionic group at C6 Chem. Comm. 46:3158-3160. [PubMed]
  10. Potrykus, J., Flemming, J., and Bearne, S.L.*, (2009) Kinetic Characterization and Quaternary Structure of Glutamate Racemase from the Periodontal Anaerobe Fusobacterium nucleatum Arch. Biochem. Biophys. 491:16-24. [PubMed]
  11. Bourque, J.R., and Bearne, S.L.*, (2008) Mutational Analysis of the Active Site Flap (20s Loop) of Mandelate Racemase. Biochemistry 47:566-578. [PubMed]
  12. Lunn, F.A., MacDonnell, J.E., and Bearne, S.L.*, (2008) Structural requirements for the activation of Escherichia coli CTP synthase by the allosteric effector GTP are stringent, but requirements for inhibition are lax. J. Biol. Chem. 283:2010-2020. [PubMed]
  13. Taylor, S.D.*, Mirzaei, F., and Bearne, S.L., (2008) Bismethylene Triphosphate Nucleotides of Uridine 4-Phosphate Analogues: A New Class of Anionic Pyrimidine Nucleotide Analogues J. Org. Chem. 73:1403-1412. [PubMed]
  14. Lunn, F.A., MacLeod, T.J., Bearne, S.L.*, (2008) Mutational analysis of conserved glycine residues 142, 143 and 146 reveals Gly-142 is critical for tetramerization of CTP synthase from Escherichia coli. Biochem. J. 412:113-121. [PubMed]
  15. Potrykus, J., White, R.L.*, and Bearne, S. L.*, (2008) Proteomic Investigation of Amino Acid Catabolism in the Indigenous Gut Anaerobe Fusobacterium varium. Proteomics 8:2691-2703. [PubMed]
  16. Taylor, S.D.*, Lunn, F.A., and Bearne, S.L.*, (2008) Ground State, Intermediate, and Multivalent Nucleotide Analogue Inhibitors of Cytidine 5'-Triphosphate Synthase ChemMedChem 3:1853-1857. [PubMed]
  17. Bourque, J.R., Burley, R.K.M., and Bearne, S.L.*, (2007) Intermediate analogue inhibitors of mandelate racemase: N-Hydroxyformanilide and cupferron. Bioorg. Med. Chem. Lett. 17:105-108. [PubMed]
  18. Potrykus, J., Mahaney, B., White, R.L.*, and Bearne, S.L.*, (2007) Proteomic investigation of glucose metabolism in the butyrate-producing gut anaerobe Fusobacterium varium. Proteomics 7:1839-1853. [PubMed]
  19. Taylor, S.D.*, Mirzaei, F., and Bearne, S.L., (2006) An unsymmetrical approach to the synthesis of bismethylene triphosphate analogues. Org. Lett. 8:4243-4246. [PubMed]
  20. MacLeod, T.J., Lunn, F.A., and Bearne, S.L.*, (2006) The role of lysine residues 297 and 306 in nucleoside triphosphate regulation of E. coli CTP synthase. Inactivation by 2',3'-dialdehyde ATP and mutational analyses. Biochim. Biophys. Acta 1764:199-210. [PubMed]
  21. Taylor, S.D.*, Mirzaei, F., Sharifi, S., and Bearne, S.L., (2006) Synthesis of Methylene- and Difluoromethylenephosphonate Analogs of Uridine-4-Phosphate and 3-Deazauridine-4-Phosphate. J. Org. Chem. 71:9420-9430. [PubMed]
  22. O'Malley, P.G.P., Sangster, S.M., Abdelmagid, S.A., Bearne, S.L. and Too, C.K.L.*, (2005) Characterizaion of a novel, cytokine-inducible carboxypeptidase-D isoform in hematopoietic tumor cells. Biochem J. 390:665-673. [PubMed]
  23. Bearne, S.L.*, and Spiteri, R.J., (2005) Reduction of intrinsic kinetic and thermodynamic barriers for enzyme-catalysed proton transfers from carbon acid substrates. J. Theor. Biol. 233:563-571. [PubMed]
  24. Siddiqi, F., Bourque, J.R., Jiang, H., Gardner, M., St. Maurice, M., Blouin, C., and Bearne, S.L.*, (2005) Perturbing the Hydrophobic Pocket of Mandelate Racemase To Probe Phenyl Motion during Catalysis. Biochemistry 44:9013-9021. [PubMed]
  25. Brosseau, C.L., St. Maurice, M., Bearne, S.L., and Roscoe, S.G.*, (2005) Electrochemical quartz crystal nanobalance (EQCN) studies of the adsorption behaviour of an enzyme, mandelate racemase, and its substrate, mandelic acid, on Pt. Electrochim. Acta 50:1289-1297.
  26. Burley, R.K.M., and Bearne, S.L.*, (2005) Inhibition of mandelate racemase by the substrate-intermediate-product analogue 1,1-diphenyl-1-hydroxymethylphosphonate. Bioorg. Med. Chem. Lett. 15:4342-4344. [PubMed]
  27. Lunn, F.A., and Bearne, S.L.*, (2004) Alternative Substrates for Wild-type and L109A E. coli CTP Synthases. Kinetic Evidence for a Constricted Ammonia Tunnel. Eur. J. Biochem. 271:4204-4212. [PubMed]
  28. MacDonnell, J.E., Lunn, F.A., and Bearne, S.L.*, (2004) Inhibition of E. coli CTP synthase by the positive allosteric effector GTP. Biochim. Biophys. Acta 1699:213-220. [PubMed]
  29. St. Maurice, M., and Bearne, S.L.*, (2004) Hydrophobic Nature of the Active Site of Mandelate Racemase. Biochemistry 43:2524-2532. [PubMed]
  30. Iyengar, A., and Bearne, S.L.*, (2003) Aspartate-107 and leucine-109 facilitate efficient coupling of glutamine hydrolysis to CTP synthesis by Escherichia coli CTP synthase. Biochem. J. 369:497-507. [PubMed]
  31. Simard. D., Hewitt, K.A., Lunn, F., Iyengar, A., and Bearne, S.L.*, (2003) Limited proteolysis of Escherichia coli cytidine 5'-triphosphate synthase. Identification of residues required for CTP formation and GTP-dependent activation of glutamine hydrolysis. Eur. J. Biochem. 270:2195-2206. [PubMed]
  32. St. Maurice, M., Bearne, S.L.*, Lu, W., and Taylor, S.D., (2003) Inhibition of mandelate racemase by alpha-fluorobenzylphosphonates. Bioorg. Med. Chem. Lett. 13:2041-2044. [PubMed]
  33. St. Maurice, M., and Bearne, S.L.*, (2002) Kinetics and Thermodynamics of Mandelate Racemase Catalysis. Biochemistry 41:4048-4058. [PubMed]
  34. Iyengar, A., and Bearne, S.L.*, (2002) An assay for cytidine 5'-triphosphate synthetase glutaminase activity using high performance liquid chromatography. Anal. Biochem. 308:396-400. [PubMed]
  35. Bearne, S.L.*, White, R.L.*, MacDonnell, J.E., Bahrami, S., and Gronlund, J., (2001) Purification and characterization of beta-methylaspartase from Fusobacterium varium. Mol. Cell. Biochem. 221:117-126. [PubMed]
  36. Bearne, S.L.*, Hekmat, O., and MacDonnell, J.E., (2001) Inhibition of Escherichia coli CTP synthase by glutamate gamma-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis. Biochem. J. 356:223-232. [PubMed]
  37. St. Maurice, M., and Bearne, S.L.*, (2000) Reaction intermediate analogues for mandelate racemase: interaction between Asn 197 and the alpha-hydroxyl of the substrate promotes catalysis. Biochemistry 39:13324-13335. [PubMed]
  38. Bearne, S.L.*, and Blouin, C., (2000) Inhibition of Escherichia coli glucosamine-6-phosphate synthase by reactive intermediate analogues. The role of the 2-amino function in catalysis. J. Biol. Chem. 275:135-140. [PubMed]
  39. Bearne, S.L.*, St. Maurice, M., and Vaughan, M.D., (1999) An assay for mandelate racemase using high-performance liquid chromatography. Anal. Biochem. 269:332-336. [PubMed]
Last modified: 2012-03-31