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Antimicrobial Agents and Chemotherapy, April 1998, p. 921-926, Vol. 42, No. 4
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Overexpression, Purification, and Characterization of the Cloned Metallo-beta -Lactamase L1 from Stenotrophomonas maltophilia

Michael W. Crowder,1,* Timothy R. Walsh,2 Linda Banovic,1 Margaret Pettit,1 and James Spencer3

Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056,1 and Department of Pathology and Microbiology, University of Bristol, Bristol BS8 1TD,2 and Protein Structure, National Institute of Medical Research, Mill Hill, London NW7 1AA,3 United Kingdom

Received 17 July 1997/Returned for modification 7 January 1998/Accepted 26 January 1998

The metallo-beta -lactamase L1 from Stenotrophomonas maltophilia was cloned, overexpressed, and characterized by spectrometric and biochemical techniques. Results of metal analyses were consistent with the cloned enzyme having 2 mol of tightly bound Zn(II) per monomer. Gel filtration chromatography demonstrated that the cloned enzyme exists as a tightly held tetramer with a molecular mass of ca. 115 kDa, and matrix-assisted laser desorption ionization and time-of-flight mass spectrometry indicated a monomeric molecular mass of 28.8 kDa. Steady-state kinetic studies with a number of diverse penicillin and cephalosporin antibiotics demonstrated that L1 effectively hydrolyzes all tested compounds, with kcat/Km values ranging between 0.002 and 5.5 µM-1 s-1. These characteristics of the recombinant enzyme are contrasted to those previously reported for metallo-beta -lactamases isolated directly from S. maltophilia.

* Corresponding author. Mailing address: Department of Chemistry and Biochemistry, 112 Hughes Hall, Miami University, Oxford, OH 45056. Phone: (513) 529-7274. Fax: (513) 529-5715. E-mail: crowdemw{at}muohio.edu.

Antimicrobial Agents and Chemotherapy, April 1998, p. 921-926, Vol. 42, No. 4
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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