Faculty Collaboration Database

Metal content of metallo-beta-lactamase L1 is determined by the bioavailability of metal ions. Biochemistry 2008 Jul 29;47(30):7947-53

Date

07/04/2008

Pubmed ID

18597493

Pubmed Central ID

PMC2750885

DOI

10.1021/bi8004768

Scopus ID

2-s2.0-48249156597 (requires institutional sign-in at Scopus site)   37 Citations

Abstract

In an effort to probe whether the metal content of metallo-beta-lactamase L1 is affected by metal ion bioavailability, L1 was overexpressed as mature protein (M-L1) and full-length (FL-L1) analogues, and the analogues were characterized with metal analyses, kinetics, and EPR spectroscopy. FL-L1, containing the putative leader sequence, was localized in the periplasm of Escherichia coli and shown to bind Zn(II) preferentially. The metal content of FL-L1 could be altered if the enzyme was overexpressed in minimal medium containing Fe and Mn, and surprisingly, an Fe-binding analogue was obtained. On the other hand, M-L1, lacking the putative leader sequence, was localized in the cytoplasm of E. coli and shown to bind various amounts of Fe and Zn(II), and like FL-L1, the metal content of the resulting enzyme could be affected by the amount of metal ions in the growth medium. L1 was refolded in the presence of Fe, and a dinuclear Fe-containing analogue of L1 was obtained, although this analogue is catalytically inactive. EPR spectra demonstrate the presence of an antiferromagnetically coupled Fe(III)Fe(II) center in Fe-containing L1 and suggest the presence of a Fe(III)Zn(II) center in M-L1. Metal analyses on the cytoplasmic and periplasmic fractions of E. coli showed that the concentration of metal ions in the periplasm is not tightly controlled and increases as the concentration of metal ions in the growth medium increases. In contrast, the concentration of Zn(II) in the cytoplasm is tightly controlled while that of Fe is less so.

Author List

Hu Z, Gunasekera TS, Spadafora L, Bennett B, Crowder MW

Author

Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University

MESH terms used to index this publication - Major topics in bold

Cytoplasm
Electron Spin Resonance Spectroscopy
Escherichia coli
Escherichia coli Proteins
Ions
Iron
Kinetics
Manganese
Metals
Mutation
Periplasm
Protein Binding
Protein Folding
Zinc
beta-Lactamases