Bearne, Stephen L. - Dalhousie University - Department of Biochemistry & Molecular Biology

研究领域

DO YOU HAVE A PASSION FOR BIOLOGICAL CHEMISTRY, ENZYME MECHANISMS, & ENZYME CATALYSIS? We do! Our research program has three 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, (2) using this information to engineer new catalytic activities and develop tools for identification of specific enzymes in complex proteomes, and (3) to understand the chemical mechanism of enzymes that are of therapeutic interest. FUNDAMENTAL ENZYMOLOGY: Mechanism, Structure, Function, & Evolution Mandelate racemase (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, a common feature of many biological reactions. Using new transition state analogue inhibitors that we have developed, in combination with site-directed mutagenesis and biophysical techniques, we are studying 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, and we are conducting investigations to understand the evolution of complexity in enzyme structure. ENZYME TARGETS IN DISEASE: Neglected Tropical Diseases, Cancer, & Antibiotics The enzyme cytidine 5'-triphosphate synthase (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). PROTEOMICS Studies are focused on developing reagents for proteomic profiling of enzymatic activities. DEVELOPING YOUR TOOL BOX Graduate training in the Bearne Lab is designed to develop your critical and analytical thinking skills. Students have the opportunity to develop technical skills in a vast number of areas, including organic synthesis and protein chemistry, physical organic laboratory techniques, molecular modeling, enzyme kinetics, CD spectroscopy, microbiology, molecular biology, site-directed mutagenesis, isothermal titration calorimetry, macroion mobility spectrometry, NMR spectroscopy, chromatography (e.g.,HPLC & FPLC), 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.

近期论文

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Bearne, S.L.*, (2017) The interdigitating loop of the enolase superfamily as a specificity binding determinant or ‘flying buttress’ Biochim. Biophys. Acta 1865:619-630 [PubMed] Bhar, P., and Bearne, S.L.*, (2017) Unexpected side product formed during LDA-induced phosphonylation of uridine Chem. Lett. 46:609-611 Bearne, S.L.*, and St. Maurice, M.*, (2017) A paradigm for C–H bond cleavage: Structural and functional aspects of transition state stabilization by mandelate racemase Adv. Protein Chem. Struct. Biol. 109:113-160 Harty, M., and Bearne, S.L.*, (2016) Measuring benzohydroxamate complexation with Mg2+, Mn2+, Co2+, and Ni2+ using isothermal titration calorimetry J. Therm. Anal. Calorim. 123:2573–2582 Pal, M., Khanal, M., Marko, R., Thirumalairajan, S., and Bearne, S.L.*, (2016) Rational design and synthesis of substrate‐product analogue inhibitors of α‐methylacyl‐coenzyme A racemase from Mycobacterium tuberculosis Chem. Commun. 52:2740-2743 [PubMed] McCluskey, G.D., Mohamady, S., Taylor, S.D., Bearne, S.L.*, (2016) Exploring the potent inhibition of CTP synthase by gemcitabine-5'-triphosphate ChemBioChem 17:2240-2249 [PubMed] Nagar, M., Wyatt, B.N., St. Maurice, M., Bearne, S.L.*, (2015) Inactivation of mandelate racemase by 3-hydroxypyruvate reveals a potential mechanistic link between enzyme superfamilies Biochemistry 54:2747-2757 [PubMed] Nagar, M., Bearne, S.L.*, (2015) An additional role for the Brønsted acid-base catalysts of mandelate racemase in transition state stabilization Biochemistry 54:6743-6752 [PubMed] Bearne, S.L.*, (2015) Comment on “A better magnetic stir bar retriever” J. Chem. Educ. 92:1777-1777 [Article] Bearne, S.L.*, (2014) Illustrating the effect of pH on enzyme activity using Gibbs energy profiles J. Chem. Educ. 91:84-90 Harty, M., Nagar, M., Atkinson, L., LeGay, C.M., Derksen, D.J.*, Bearne, S.L.*, (2014) Inhibition of serine and proline racemases by substrate-product analogues Bioorg. Med. Chem. Lett. 24:390-393. [PubMed] Pal, M., and Bearne, S.L.*, (2014) Inhibition of glutamate racemase by substrate-product analogues Bioorg. Med. Chem. Lett. 24:1432-1436 [PubMed] Nagar, M., Lietzan, A.D., St. Maurice, M., Bearne, S.L.*, (2014) Potent inhibition of mandelate racemase by a fluorinated substrate-product analogue with a novel binding mode Biochemistry 53:1169-1178 [PubMed] Pal, M., and Bearne S.L.*, (2014) Synthesis of coenzyme A thioesters using methyl acyl phosphates in an aqueous medium Org. Biomol. Chem. 12:9760-9763 [PubMed] Bearne, S.L.*, (2012) Illustrating enzyme inhibition using free energy profiles J. Chem. Educ. 89:732-737. 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] 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] 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] 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] Narmandakh, A., and Bearne, S.L.*, (2010) Purification of recombinant mandelate racemase: Improved catalytic activity. Protein Expr. Purif. 69:39-46. [PubMed] 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] 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] 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] 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] 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]