研究领域

Analytical Chemistry Bioanalytical Chemistry Bioinorganic Chemistry Biophysical Chemistry Chemical Biology Nano Chemistry Optics and Imaging Organic Chemistry RNA BioChemistry Sensor Science

Our goal is to understand the mechanisms used by biological catalysts, both proteins and nucleic acids, to achieve high efficiency, stringent specificity and rigorous control. An understanding of these principles is essential for: understanding biological catalysis in vivo, designing novel inhibitors for therapeutic use, and developing novel catalysts for a variety of tasks, including organic synthesis and quantitative analysis of complex mixtures. We are elucidating catalytic mechanisms and essential active site features of metalloenzymes and ribozymes, including protein farnesyltransferase, UDP-3-O-acyl-GlcNAC deacetylase, histone deacetylase and ribonuclease P. These studies should enhance our ability to design potent inhibitors of these enzymes useful for the treatment of cancer or bacterial infections. In particular, we are investigating the role of proteins in modulating the reactivity of bound Zn(II) and developing methods for identifying novel metal sites in proteins. We are also investigating the biological importance of protein prenylation and acetylation. Finally, we are elucidating the role of metal ions and protein/RNA interactions in ribonuclease P, a ribozyme/protein complex. These studies are increasing our understanding of the catalytic modes used by ribozymes in comparison to protein catalysts. We are testing our understanding of biological catalysis by the rational design or redesign of an enzyme. To this end, we are redesigning the zinc metalloenzyme, carbonic anhydrase II, to optimize a fluorescent biosensor for measuring and imaging metal ions in complex biological mixtures. Zinc ions are proposed to play important signaling roles in vivo, especially in neurobiology, which can investigated using novel imaging methods. Additionally, we are using "directed evolution" approaches to prepare and identify aldolase variants with novel substrate specificities. These aldolase variants will be used to improve the utility of these enzymes as biocatalysts for organic synthetic reactions. Characterization of the structure and function of these novel proteins will provide insights into catalysis, molecular recognition and molecular evolution.

近期论文

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Wang D, Fierke CA. The BaeSR regulon is involved in defense against zinc toxicity in E. coli. Metallomics. 2013 Apr 27;5(4):372-83. doi: 10.1039/c3mt20217h. PubMed PMID: 23446818. Wolfson NA, Pitcairn CA, Fierke CA. HDAC8 substrates: Histones and beyond. Biopolymers. 2013 Feb;99(2):112-26. doi: 10.1002/bip.22135. PubMed PMID: 23175386; PubMed Central PMCID: PMC3507420. Zeng HH, Matveeva EG, Stoddard AK, Fierke CA, Thompson RB. Long Wavelength Fluorescence Ratiometric Zinc Biosensor. J Fluoresc. 2013 Jan 24. [Epub ahead of print] PubMed PMID: 23345045. Zverina EA, Lamphear CL, Wright EN, Fierke CA. Recent advances in protein prenyltransferases: substrate identification, regulation, and disease interventions. Curr Opin Chem Biol. 2012 Dec;16(5-6):544-52. doi: 10.1016/j.cbpa.2012.10.015. Epub 2012 Nov 8. PubMed PMID: 23141597; PubMed Central PMCID: PMC3518607. Hougland JL, Gangopadhyay SA, Fierke CA. Expansion of protein farnesyltransferase specificity using "tunable" active site interactions: development of bioengineered prenylation pathways. J Biol Chem. 2012 Nov 2;287(45):38090-100. doi: 10.1074/jbc.M112.404954. Epub 2012 Sep 19. PubMed PMID: 22992747; PubMed Central PMCID: PMC3488079. Subramanian T, Pais JE, Liu S, Troutman JM, Suzuki Y, Leela Subramanian K, Fierke CA, Andres DA, Spielmann HP. Farnesyl diphosphate analogues with aryl moieties are efficient alternate substrates for protein farnesyltransferase. Biochemistry. 2012 Oct 16;51(41):8307-19. doi: 10.1021/bi3011362. Epub 2012 Oct 2. PubMed PMID: 22989235; PubMed Central PMCID: PMC3562418. Howard MJ, Lim WH, Fierke CA, Koutmos M. Mitochondrial ribonuclease P structure provides insight into the evolution of catalytic strategies for precursor-tRNA 5' processing. Proc Natl Acad Sci U S A. 2012 Oct 2;109(40):16149-54. doi: 10.1073/pnas.1209062109. Epub 2012 Sep 18. PubMed PMID: 22991464; PubMed Central PMCID: PMC3479547. Wang D, Hosteen O, Fierke CA. ZntR-mediated transcription of zntA responds to nanomolar intracellular free zinc. J Inorg Biochem. 2012 Jun;111:173-81. doi: 10.1016/j.jinorgbio.2012.02.008. Epub 2012 Feb 22. PubMed PMID: 22459916; PubMed Central PMCID: PMC3408962. McCranor BJ, Bozym RA, Vitolo MI, Fierke CA, Bambrick L, Polster BM, Fiskum G, Thompson RB. Quantitative imaging of mitochondrial and cytosolic free zinc levels in an in vitro model of ischemia/reperfusion. J Bioenerg Biomembr. 2012 Apr;44(2):253-63. doi: 10.1007/s10863-012-9427-2. Epub 2012 Mar 20. PubMed PMID: 22430627; PubMed Central PMCID: PMC3539817. Cheriyan M, Toone EJ, Fierke CA. Improving upon nature: active site remodeling produces highly efficient aldolase activity toward hydrophobic electrophilic substrates. Biochemistry. 2012 Feb 28;51(8):1658-68. doi: 10.1021/bi201899b. Epub 2012 Feb 16. PubMed PMID: 22316217; PubMed Central PMCID: PMC3315183. Wang Y, Liu X, Schneider B, Zverina EA, Russ K, Wijeyesakere SJ, Fierke CA, Richardson RJ, Philbert MA. Mixed inhibition of adenosine deaminase activity by 1,3-dinitrobenzene: a model for understanding cell-selective neurotoxicity in chemically-induced energy deprivation syndromes in brain. Toxicol Sci. 2012 Feb;125(2):509-21. doi: 10.1093/toxsci/kfr317. Epub 2011 Nov 21. PubMed PMID: 22106038; PubMed Central PMCID: PMC3262860. Yang Y, Wang B, Ucisik MN, Cui G, Fierke CA, Merz KM Jr. Insights into the mechanistic dichotomy of the protein farnesyltransferase peptide substrates CVIM and CVLS. J Am Chem Soc. 2012 Jan 18;134(2):820-3. doi: 10.1021/ja209650h. Epub 2012 Jan 6. PubMed PMID: 22206225; PubMed Central PMCID: PMC3277741. Cheriyan M, Walters MJ, Kang BD, Anzaldi LL, Toone EJ, Fierke CA. Directed evolution of a pyruvate aldolase to recognize a long chain acyl substrate. Bioorg Med Chem. 2011 Nov 1;19(21):6447-53. doi: 10.1016/j.bmc.2011.08.056. Epub 2011 Aug 30. PubMed PMID: 21944547; PubMed Central PMCID: PMC3209416. London N, Lamphear CL, Hougland JL, Fierke CA, Schueler-Furman O. Identification of a novel class of farnesylation targets by structure-based modeling of binding specificity. PLoS Comput Biol. 2011 Oct;7(10):e1002170. doi: 10.1371/journal.pcbi.1002170. Epub 2011 Oct 6. PubMed PMID: 21998565; PubMed Central PMCID: PMC3188499. Wang D, Hurst TK, Thompson RB, Fierke CA. Genetically encoded ratiometric biosensors to measure intracellular exchangeable zinc in Escherichia coli. J Biomed Opt. 2011 Aug;16(8):087011. doi: 10.1117/1.3613926. PubMed PMID: 21895338; PubMed Central PMCID: PMC3166341.