Metalloproteins: Structure Determination (HADH), Inhibition (P4H), and Biomimetic Systems (P-1[Ru(NO)(Cl)])

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Issue Date
2008-01-01Author
Reed, Timothy M.
Publisher
University of Kansas
Format
227 pages
Type
Dissertation
Degree Level
Ph.D.
Discipline
Chemistry
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This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
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Histamine dehydrogenase from Nocardioides simplex (HADH) is a flavoprotein that converts histamine to imidazole acetaldehyde and is highly specific for histamine. Chapter one describes the development of overexpression and purification a recombinant form of HADH (rHADH) and its basic biochemical characterization. Chapter two describes X-ray structure determination of rHADH. Diffraction data were collected to 2.7 Å resolution with 99.7% completeness. The histamine binding motif of HADH are very similar to those in the other histamine binding proteins. Prolyl-4-hydroxylase (P4H) belongs to a family of αketoglutarate-dependent non-heme iron oxygenases. Selective inhibitors of P4H can be potential therapeutics for fibrosis. Chapter three discusses the design of inhibitors that target P4H in the ER using the signal peptide KDEL, which is specific to the ER. Phenanthroline-GKDEL demonstrates a 100-fold increase in potency in inhibiting P4H produced in the cultured human fibroblast cells versus isolated enzyme. Fluorescent microscopy using a fluorescently tagged inhibitor demonstrates uptake of the phen-E(EDANS)VKDEL inhibitor into the ER. Nitric oxide (NO) is an important signaling molecule in the body, and the site-specific timed release NO could be utilized in the treatment of various medical conditions. Chapter four discusses the photorelease of NO from a ruthenium salen complex immobilized within a porous material. This material transfers NO to myoglobin within 20 minutes. NO is released from this material in the presence of light; however, during periods of darkness, the release of NO was not observed. This is the first system where NO is photoreleased from an immobilized polymer support.
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