HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: ps.04742704v1 13/8/2236    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Periyannan, G. Articles by Crowder, M. W. Search for Related Content PubMed PubMed Citation Articles by Periyannan, G. Articles by Crowder, M. W. Social Bookmarking                   What's this?

In vivo folding of recombinant metallo--lactamase L1 requires the presence of Zn(II)

Miami University, Department of Chemistry and Biochemistry, Oxford, Ohio 45056, USA

(RECEIVED March 16, 2004; FINAL REVISION May 3, 2004; ACCEPTED May 8, 2004)

Metallo--lactamase L1, secreted by pathogenic Stenotrophomonas maltophilia, is a dinuclear Zn(II)-containing enzyme that hydrolyzes almost all known penicillins, cephalosporins, and carbapenems. The presence of Zn(II) ions in both metal binding sites is essential for full enzymatic activity; however, the mechanism of physiological metal incorporation is unknown. To probe metal incorporation, L1 was over-expressed in minimal media with (mmL1+Zn) and without (mmL1–Zn) Zn(II) added to the media, and the resulting proteins were purified and characterized. The mmL1+Zn sample was bound by a Q-Sepharose column, exhibited steady-state kinetic properties, bound Zn(II), existed as a tetramer, and yielded fluorescence emission and CD spectra similar to L1 overexpressed in rich media. On the other hand, the mmL1–Zn sample did not bind to a Q-Sepharose column, and gel filtration studies demonstrated that this protein was monomeric. The mmL1–Zn sample exhibited a lower k_cat value, bound less Zn(II), and yielded fluorescence emission and CD spectra consistent with this enzyme being folded improperly. Taken together, these data demonstrate that the proper folding of L1 requires the presence of Zn(II) and suggest that in vitro, thermodynamic metal binding studies do not accurately reflect physiological metal incorporation into L1.

Keywords: metallo--lactamase; Zn(II); folding; CD spectroscopy; fluorescence; in vivo metal binding

Abbreviations: CcrA, metallo--lactamase from Bacteroides fragilis • CD, circular dichroism • , extinction coefficient • HEPES, N-2-Hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid • ICP-AES, inductively coupled plasma–atomic emission spectroscopy • IPTG, isopropyl--thiogalactoside • L1, metallo--lactamase from Stenotrophomonas maltophilia • LB, Luria-Bertani • MALDI-TOF, matrix-assisted laser desorption ionization time of flight • mmL1, L1 overexpressed in minimal media • SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Reprint requests to: Michael W. Crowder, Miami University, Department of Chemistry and Biochemistry, 112 Hughes Hall, Oxford, OH 45056, USA; e-mail: crowdemw{at}muohio.edu; fax: (513) 529–5715.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04742704.

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				L. I. Llarrull, M. F. Tioni, J. Kowalski, B. Bennett, and A. J. Vila Evidence for a Dinuclear Active Site in the Metallo-beta-lactamase BcII with Substoichiometric Co(II): A NEW MODEL FOR METAL UPTAKE J. Biol. Chem., October 19, 2007; 282(42): 30586 - 30595. [Abstract] [Full Text] [PDF]