Characterization of the metallo-[beta]-lactamase L1 from Stenotrophomonas maltophilia

by Periyannan, Gopal Raj.

Abstract (Summary)
CHARACTERIZATION OF METALLO-?-LACTAMASE L1 FROM STENOTROPHOMONAS MALTOPHILIA by Gopal Raj Periyannan Zinc-containing metallo-?-lactamases (m?Ls) are an emerging class of enzymes that render bacteria resistant to ?-lactam-containing antibiotics. In an effort to better understand the structure and function of the m?L L1 from Stenotrophomonas maltophilia, spectroscopic characterization of native and Co(II)-substituted L1 was performed. Co(II)-substituted L1 was characterized by using UV-Vis, EPR, EXAFS, and 1H paramagnetic NMR spectroscopic techniques. Our data show that Co(II) and Zn(II) bind L1 sequentially and preferentially. A model for Co(II) and Zn(II) binding to L1 is presented. Attempts were made to prepare mixed-metal analogs (CoZn or ZnCo) of L1, and our data show that only the ZnCo-analog can be prepared. In the concluding remarks, an ingenious way to use the ZnCo-analog of L1 to probe the reaction mechanism using spectroscopic techniques is provided. Metal-binding histidine ligands were mutated to cysteines in order to prepare the CoZn analog of L1; however, the binding preference of Co(II) and Zn(II) prevents the preparation of the CoZn analog of L1. The Co(II) and Zn(II) binding modes of the subgroup 3a m?L CcrA from Bacteroides fragilis was also studied using EPR, EXAFS, and 1H NMR spectroscopic techniques. In contrast to L1, Co(II) and Zn(II) both bind to the 3 His Zn1 site in CcrA first before binding to the Zn2 site. To probe metal incorporation in m?L, L1 was over-expressed in a minimal media with and without Zn(II), and the resulting proteins were purified and characterized by steadystate kinetics and CD, and fluorescence spectroscopies. Our data demonstrate that the proper in vivo folding of L1 requires the presence of Zn(II); however, in vitro folding of L1 does not require Zn(II). Our results suggest that in vitro refolding experiments do not necessarily reflect in vivo folding conditions.
Bibliographical Information:


School:Miami University

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:ccra spectroscopy beta lactamases lactam antibiotics stenotrophomonas maltophilia


Date of Publication:

© 2009 All Rights Reserved.