Zn(II) Dependence of the Aeromonas hydrophila AE036 Metallo-β-lactamase Activity and Stability

Abstract

Two Zn2+ binding sites were found in the Aeromonas hydrophila AE036 metallo-beta-lactamase. The affinity of the first binding site for Zn2+ ions is so high that the dissociation constant could not be determined, but it is significantly lower than 20 nM. The mono-Zn2+ form of the enzyme exhibits a maximum activity against its carbapenem substrates. The presence of a Zn2+ ion in the second lower affinity binding site results in a loss of enzymatic activity with a Ki value of 46 microM at pH 6.5. The kinetic analysis is in agreement with a noncompetitive inhibition mechanism. The Zn content of the A. hydrophila enzyme is also strongly pH-dependent. With an external Zn2+ ion concentration of 0.4 microM, occupancy of the higher affinity site by metal ions is lower than 10% at pH 5 and 10. The affinity for the second binding site seems to increase from pH 6 to 7.5. Fluorescence emission and circular dichroism spectra revealed slight conformational changes upon titration of the apoenzyme by Zn2+ ions, resulting in the successive saturation of the first and second binding sites. Differential scanning calorimetry transitions and intrinsic fluorescence emission spectra in the presence of increasing concentrations of urea demonstrate that the catalytic zinc strongly stabilizes the conformation of the enzyme whereas the di-Zn enzyme is even more resistant to thermal and urea denaturation than the mono-Zn enzyme. The Zn2+ dependency of the activity of this metallo-beta-lactamase thus appears to be very different from that of the homologous Bacteroides fragilis enzyme for which the presence of two Zn2+ ions per molecule of protein appears to result in maximum activity.

Metallo-β-Lactamases: A Major Threat to Human Health

“…Competition-based assays rely on the direct competition of at least two compounds for binding to a target such as a metal ion. In order to study the binding of metal ions, in Figure 7); both compounds have high affinities for Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) [101]- [105]. For a detailed description of the data analysis refer to published literature [18] [60] [106] [107].…”

Catalytic Role of the Metal Ion in the Metallo-β-lactamase GOB

“…Subclass B1 includes several chromosomally encoded enzymes, such as BcII from Bacillus cereus (11)(12)(13), CcrA from Bacteroides fragilis (14 -17), BlaB from Elizabethkingia meningoseptica (18), and transferable VIM (19), IMP (20,21), SPM (22), and GIM (23) type enzymes. Subclass B2 includes the CphA (24,25) and ImiS (26) lactamases from Aeromonas species and Sfh-I from Serratia fonticola (27). Subclass B3, originally represented only by L1 from Stenotrophomonas maltophilia (28 -30), now includes enzymes from other opportunistic pathogens like FEZ-1 from Legionella gormanii (31) and GOB from E. meningoseptica (32,33) as well as from environmental bacteria, such as CAU-1 from Caulobacter crescentus (34) and THIN-B from Janthinobacterium lividum (35).…”

The 1.5-Å Structure of Chryseobacterium meningosepticum Zinc β-Lactamase in Complex with the Inhibitor, D-Captopril

“…MBLs have two metal binding sites (the His site, or first site, and the Cys site, or second site), which bind Zn 2ϩ ions required for enzyme activity. In general, both the mono-Zn 2ϩ (Zn 2ϩ in the His site) and di-Zn 2ϩ forms are active but have different kinetic properties (9,10); the exception is CphA from Aeromonas hydrophilia, which is inhibited by the presence of the second zinc ion (11). In the mono-Zn 2ϩ enzyme, a watermediated mechanism for the hydrolysis of the antibiotic ␤-lactam ring has been suggested in which a water molecule is activated by the presence of the Zn 2ϩ ion in the His site, then carries out nucleophilic attack against the carbonyl carbon of the ␤-lactam.…”