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    The Biochemistry of Siderophore Biosynthesis

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    Issue Date
    2008-01-25
    Author
    Meneely, Kathleen M.
    Publisher
    University of Kansas
    Format
    203 pages
    Type
    Dissertation
    Degree Level
    PH.D.
    Discipline
    Biochemistry & Molecular Biology
    Rights
    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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    Abstract
    Pathogenic bacteria are becoming increasingly antibiotic resistant. For this reason, the development of novel antibiotics is extremely important. A potential new target for antimicrobial drugs is the production of siderophores. Pseudomonas aeruginosa produces two siderophores under iron-limiting conditions, pyoverdin and pyochelin. Pyoverdin contains ornithine derivatives as part of the peptide backbone important for iron chelation. PvdA, an ornithine hydroxylase, performs the first step in derivation of the ornithine followed with formylation by PvdF, a formyl transferase. Biochemical characterization of PvdA reveals that PvdA is specific for the coenzymes, FAD and NADPH, as well as for the substrate, L-ornithine. The enzyme follows Michaelis-Menten kinetics measuring NADPH oxidation, but substrate inhibition is detected when measuring the formation of hydroxylated product. Lysine is determined as a nonsubstrate effector and mixed inhibitor of PvdA with respect to ornithine. Chloride is a competitive inhibitor of the enzyme in relation to NADPH while a mixed inhibitor with respect to substrate. A mercurial compound, p-chloromercuribenzoate, is also a mixed inhibitor in relation to substrate. Steady state experiments reveal a ternary complex of PvdA:FAD with NADPH and ornithine during catalysis. PvdA was further characterized with transient state kinetics to develop a catalytic mechanism. The flavin in complex with PvdA can be reduced in the absence of substrate. Oxidation of the reduced flavin in the presence of substrate indicates the formation of two transient intermediates, hydroperoxyflavin and hydroxyflavin. However, in the absence of substrate, only the hydroxyflavin intermediate is detected and oxidation of the flavin is not through the production of hydrogen peroxide. A biochemical comparison of PvdA to two homologues, para-hydroxybenzoate hydroxylase (PHBH from Pseudomonas fluorescens) and flavin-containing monooxygenases (FMOs from Schizosaccharomyces pombe and hog liver microsomes) indicates that PvdA proceeds by a novel reaction mechanism. Structural characterization of PvdA and PvdF by x-ray crystallography is underway. Crystallization studies of the NADPH reductases involved in the synthesis of pyochelin from P. aeruginosa (PchG) and yersiniabactin from Yersinia enterocolitica (Irp3) are also being performed. The structures of these enzymes are a first step towards the rational design of new inhibitors for use as new antimicrobial agents.
    URI
    http://hdl.handle.net/1808/4027
    Collections
    • Dissertations [4473]
    • Molecular Biosciences Dissertations and Theses [273]

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    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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