In vivo folding of recombinant metallo-beta-lactamase L1 requires the presence of Zn(II).
Metallo-beta-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 kcat 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.
Periyannan
Gopalraj
G
Miami University, Department of Chemistry and Biochemistry, Oxford, Ohio 45056, USA.
Shaw
Patrick J
PJ
Sigdel
Tara
T
Crowder
Michael W
MW
D000900:Anti-Bacterial Agents / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D020615:Stenotrophomonas maltophilia / Q000201:enzymology*; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001618:beta-Lactamases; C496277:metallo-beta-lactamase L1, Stenotrophomonas maltophilia; D015032:Zinc