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    Biochemical Characterization and Structure Determination of a Prolyl 4-Hydroxylase-like Protein from Bacillus anthracis

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    Issue Date
    2009-06-05
    Author
    Culpepper, Megen A.
    Publisher
    University of Kansas
    Format
    210 pages
    Type
    Dissertation
    Degree Level
    Ph.D.
    Discipline
    Chemistry
    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
    Procollagen prolyl-4-hydroxylase (P4H) catalyzes the conversion of peptidyl proline to trans-4-hydroxyproline (Hyp). P4H is a member of the alpha-ketoglutarate-dependent mononuclear non-heme iron oxygenase (alphaKG/Fe(II)-oxygenase) family of enzymes, which catalyze a wide variety of reactions. The genome of Bacillus anthracis (B. anthracis), the causative agent of anthrax, contains a gene that is annotated as a p4h based on the predicted amino acid sequence. Recently the immunodominant protein of the B. anthracis exosporium has been identified as BclA. BclA has collagen-like repeat sequences and has a triple helical structure similar to that of animal collagens. We have detected Hyp from the protein extracts of spores of B. anthracis and this is the first report for Hyp detection in bacteria. These results strongly support that B. anthracis contains a P4H-like protein, anthrax-P4H. We have expressed and purified a recombinant form of the putative anthrax-P4H and found that it is a homodimer of 24.3 kDa subunit. The anthrax-P4H exhibits activity and UV-vis spectroscopic properties characteristic of an alphaKG/Fe(II)-oxygenase, and it can bind a collagen-like substrate, (Gly-Pro-Pro)10, with an affinity comparable to that of human type (I) collagen-P4H (human-P4H-1). This is the first report of any P4H-like protein from a bacterial source. We propose that anthrax-P4H can serve as a model for human-P4H-1 from its substrate specificity and kinetic parameters comparable to human-P4H-1. We also report the crystal structure of anthrax-P4H to 1.40 Å. The structure reveals the double stranded alpha-helix core fold (jellyroll) motif, characteristic of Fe(II)/alphaKG oxygenases. The oligomerization of the protein is a homodimeric, alpha2, structure, which makes the anthrax-P4H a new member to the P4H family of enzymes. A putative peptide-binding groove has been proposed based on that identified in the structure of P4H from Chlamydomonas reinhardtii (green alga) (Cr-P4H-1). The anthrax-P4H structure provides insight into the dimeric structure of the alpha-subunits of human-P4H-1, as well as to help understand the mode of substrate recognition that may aide in the development of selective inhibitors.
    URI
    http://hdl.handle.net/1808/5576
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    • Dissertations [4473]
    • Chemistry Dissertations and Theses [336]

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    785-864-8983
    KU Libraries
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    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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