title: Metal content of metallo-beta-lactamase L1 is determined by the bioavailability of metal ions.
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.
authors.affiliation: Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University, Oxford, Ohio 45056, USA.
authors.forename: Zhenxin
authors.initials: Z
authors.lastname: Hu
authors.forename: Thusitha S
authors.initials: TS
authors.lastname: Gunasekera
authors.forename: Lauren
authors.initials: L
authors.lastname: Spadafora
authors.forename: Brian
authors.initials: B
authors.lastname: Bennett
authors.forename: Michael W
authors.initials: MW
authors.lastname: Crowder
mesh_terms: D003593:Cytoplasm / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007477:Ions / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D009154:Mutation; D019897:Periplasm / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D015032:Zinc / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D007477:Ions; D008670:Metals; D008345:Manganese; D007501:Iron; C088032:beta-lactamase L1; D001618:beta-Lactamases; D015032:Zinc