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Biochemical and Structural Studies of the Circadian Enzymes CLOCK and Casein Kinase 1, and Indolotryptoline Biosynthetic Enzymes

Zeringo, Nicholas A
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1404132328

Abstract Details

2014, Doctor of Philosophy, University of Toledo, College of Natural Sciences and Mathematics.
Circadian rhythms are cyclic changes in physiology and biochemistry with a ~24 hour period. On the cellular level circadian rhythms result in rhythmic gene expression regulated by the circadian oscillator, a transcription/translation negative feedback loop. A key component of the oscillator is the circadian transcription factor CLOCK. CLOCK is required for maintenance of circadian rhythms and is a reported protein/histone acetyltransferase (P/HAT). However there is little data in the literature beyond an initial report to support CLOCK as a P/HAT. Efforts to identify the minimal P/HAT domain of CLOCK have met with no success. Additionally, efforts to recapitulate the reported P/HAT activity of CLOCK have not replicated the previously observed P/HAT activity. Casein Kinase 1 (CK1) also plays a critical role in maintenance of circadian rhythms with mutations in either CK1 or its circadian substrate Per2 resulting in modulation of the circadian period length. To identify the biochemical affects of the clinically relevant tau mutation (R178C) in CK1e, a bisubstrate analogue has been synthesized and utilized to provide experimental evidence for the role of Arg178 in substrate recognition and to provide a rationale for conflicting reports in the literature for the effects of the tau mutation on CK1e activity. Additionally the structure of a new crystal polymorph of the catalytic domain of apo-CK1d has been determined. With the goal of lowering the barrier for in-depth kinetic studies of ATPases a simple, continuous, enzyme-coupled assay for ATPase activity has been proposed. The assay utilizes a prokaryotic nicotinamide nucleotide repair enzyme (NNR) to produce NADPH from NADPHX in an ADP-dependent manner with reaction progress monitored either by fluorescence or absorbance. A simple synthesis for NADHPX has been identified and initial studies to produce active prokaryotic NNR is reported. Recently identified indolotryptoline natural products represent a new class of biologically active compounds with promising antitumor properties. To understand the biosynthesis of indolotryptolines from indolocarbazole precursors and to characterize the enzymes responsible for indolotryptoline biosynsthesis, AbeX1, a putative flavin- monooxygenase necessary for in vivo indolotryptoline biosynthesis, has been cloned. Significant effort has been put forth to express recombinant AbeX1 in the soluble fraction of cells lysates with no success. However, limited success with resolublization has allowed for some preliminary experiments. These limited experiments tentatively suggest AbeX1 is a NADPH-dependent flavin-monooxygenase.
John Bellizzi, III (Advisor)
Max Funk (Committee Member)
Donald Ronning (Committee Member)
Brian Ashburner (Committee Member)
Biochemistry

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Citations

  • Zeringo, N. A. (2014). Biochemical and Structural Studies of the Circadian Enzymes CLOCK and Casein Kinase 1, and Indolotryptoline Biosynthetic Enzymes [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1404132328

    APA Style (7th edition)

  • Zeringo, Nicholas. Biochemical and Structural Studies of the Circadian Enzymes CLOCK and Casein Kinase 1, and Indolotryptoline Biosynthetic Enzymes. 2014. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1404132328.

    MLA Style (8th edition)

  • Zeringo, Nicholas. "Biochemical and Structural Studies of the Circadian Enzymes CLOCK and Casein Kinase 1, and Indolotryptoline Biosynthetic Enzymes." Doctoral dissertation, University of Toledo, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1404132328

    Chicago Manual of Style (17th edition)

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This open access ETD is published by University of Toledo and OhioLINK.