Insights into an evolutionary strategy leading to antibiotic resistance

Abstract

Metallo-β-lactamases (MBLs) with activity towards a broad-spectrum of β-lactam antibiotics have become a major threat to public health, not least due to their ability to rapidly adapt their substrate preference. In this study, the capability of the MBL AIM-1 to evade antibiotic pressure by introducing specific mutations was probed by two alternative methods, i.e. site-saturation mutagenesis (SSM) of active site residues and in vitro evolution. Both approaches demonstrated that a single mutation in AIM-1 can greatly enhance a pathogen’s resistance towards broad spectrum antibiotics without significantly compromising the catalytic efficiency of the enzyme. Importantly, the evolution experiments demonstrated that relevant amino acids are not necessarily in close proximity to the catalytic centre of the enzyme. This observation is a powerful demonstration that MBLs have a diverse array of possibilities to adapt to new selection pressures, avenues that cannot easily be predicted from a crystal structure alone.

Structure and mechanism of potent bifunctional β-lactam- and homoserine lactone-degrading enzymes from marine microorganisms

“…Overall structures of MIM-1 (yellow) and SAM-1 (cyan). Both enzymes contain the characteristic αβ/βα structural motif and are very similar to other MBLs from the B3 subgroup such as AIM-131 , L1 44 , SMB-145 and BJP46 . The catalytically relevant metal ion center is located in the middle.…”

“…The data were integrated, scaled and merged using HKL-2000 28 . Refinement and model building were carried out using PHENIX 1.8.4 29 and COOT 0.7 30 , respectively, using the previously published coordinates for the B3 MBL AIM-1 from P. aeruginosa (4P62) 31 . All atoms were subsequently refined with anisotropic B-factors; most hydrogen atoms were fitted as riding models, though the proton of the bridging hydroxide was added manually based on the electron density.…”

Broad spectrum antibiotic-degrading metallo-β-lactamases are phylogenetically diverse and widespread in the environment

“…A conspicuous difference between the four enzymes is the relative position 225 of their N-termini. In L1, the N-terminus protrudes from the structure thereby making the active site accessible (16), whereas in AIM-1, a disulphide bridge between Cys32 and Cys66 locks its Nterminus in a position away from the catalytic center (Figure 3) (23,25). Similar to L1, GOB-1 (Figure 3) has a protruding N-terminus exposing the active site (26).…”

“…NHH/DCH for the α/β site motifs), members of the B2 subgroup only require one Zn 2+ for 75 catalysis (located in the β-site); the presence of a second metal ion leads to inhibition (1,7,15). Only a small number of B3-type MBLs have been identified, including L1 (16)(17)(18)(19)(20), FEZ-1 (21,22), AIM-1 (23)(24)(25), GOB-1 (26,27), LRA-8 (28), MIM-1 and MIM-2 (29,30). They have a structurally similar active site motif to B1 MBLs (HHH/DHH), and also require a bimetallic Zn 2+ center for optimal catalytic efficiency (1,7,13,31).…”

Kinetic, mechanistic, structural and spectroscopic investigations of Bimetallic Metallohydrolases

“…(216,278) The native Zn 2+ ions could easily be replaced with paramagnetic Co 2+ , and similar to other MBLs, the replacement of Zn 2+ by Co 2+ leads to an active form of these enzymes with moderately reduced catalytic activity, a slight impact on substrate binding and reduced competitive inhibitory effect of Dcaptropril (26) . Irrespective of the catalytic changes, MIM-1, MIM-2 and LRA-8 were suitable candidates for spectroscopic investigation via our previously mentioned and reported methodology.…”