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Characterization of ImiS, the Metallo-Β-Lactamase from Aeromonas veronii bv. sobria

Crawford, Patrick Anthony
http://rave.ohiolink.edu/etdc/view?acc_num=miami1069106986

Abstract Details

2003, Doctor of Philosophy, Miami University, Chemistry and Biochemistry.
Zinc-containing metallo-Β-lactamases are an emerging class of enzymes that render bacteria resistant to b-lactam-containing antibiotics. In an effort to better understand the structure and function of the metallo-Β-lactamase ImiS from Aeromonas veronii bv. sobria, spectroscopic and mechanistic studies were performed. ImiS was over-expressed in E. coli and purified as a 25.2 kDa monomer, containing 0.48 equivalents of Zn(II). The purified enzyme exhibited substrate selectivity toward carbapenems, hydrolyzing imipenem with a kcat of 233 s-1 and KM of 154 mM. The presence of a second equivalent of Zn(II) resulted in the loss of enzymatic activity. For spectroscopic characterization the native and spectroscopically silent Zn(II) was replaced with Co(II). UV/Vis, NMR, and EPR spectroscopies were all gathered on the Co(II)-substituted ImiS samples and EXAFS data were collected on the Zn(II)-ImiS. The Co(II) in Co(II)-ImiS is 4-coordinate, with 1 cysteine, 1 histidine, and presumably 1 aspartic acid and 1 water serving as metal ligands. Proton inventory studies were inconclusive, not clearly indicating one or more than one proton being transferred during the rate-liming step. pH Dependence studies revealed the presence of a single pKa of 5.6, which was assigned to a Zn(II)-bound water. Rapid scanning and stopped-flow experiments revealed a possible reaction mechanism consistent with that seen for Β-lactamase II. Taken together, this dissertation offers, for the first time, models for the metal binding site and for the reaction mechanism of ImiS. These data, along with previous results on the other metallo-Β-lactamases, can be integrated and used to guide further rational inhibitor design efforts.
Michael Crowder (Advisor)
163 p.
Chemistry, Biochemistry
ImiS; Aeromonas; Antibiotic Resistance; Metallo-&914; -Lactamase; Zinc; Zn(II)

Recommended Citations

Citations

  • Crawford, P. A. (2003). Characterization of ImiS, the Metallo-Β-Lactamase from Aeromonas veronii bv. sobria [Doctoral dissertation, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1069106986

    APA Style (7th edition)

  • Crawford, Patrick. Characterization of ImiS, the Metallo-Β-Lactamase from Aeromonas veronii bv. sobria. 2003. Miami University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1069106986.

    MLA Style (8th edition)

  • Crawford, Patrick. "Characterization of ImiS, the Metallo-Β-Lactamase from Aeromonas veronii bv. sobria." Doctoral dissertation, Miami University, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=miami1069106986

    Chicago Manual of Style (17th edition)

miami1069106986
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© 2003, all rights reserved.
This open access ETD is published by Miami University and OhioLINK.
Release 3.2.12