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

by Crawford, Patrick Anthony

Abstract (Summary)
Zinc-containing metallo-?-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-?-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 ?-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-?-lactamases, can be integrated and used to guide further rational inhibitor design efforts.
Bibliographical Information:


School:Miami University

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:imis aeromonas antibiotic resistance metallo ? lactamase zinc zn ii


Date of Publication:01/01/2003

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