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

Abstract

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.

Figure 1.

SDS-PAGE gels of the elution profiles of mmL1−Zn (A) and mmL1+Zn from Q-Sepharose column (B).

Figure 2.

Fluorescence emission spectra of L1 samples and the effect of added Zn(II). The spectra were obtained using 0.25 μM L1 samples (monomer) in 50 mM HEPES (pH 7.0) at 25°C. The emission spectrum of the buffer was subtracted from each of the spectra. The excitation wavelength used in these studies was 295 nm.

Figure 3.

CD spectra of L1 samples. The CD spectra were collected on 75 μg ml⁻¹ enzyme samples (monomer) in 5 mM phosphate (pH 7.0) and at 25°C.