Metallo-beta-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of beta-lactam antibiotics. The G262S point mutation distinguishing the metallo-beta-lactamase IMP-1 from IMP-6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single-nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y, and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin, and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R2 electron donors, which may stabilize the substrate intermediate in the binding pocket. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations), they are likely to evolve naturally.
Submitter: Peter Oelschlaeger
Submission Date: Dec. 11, 2018, 4:23 p.m.
|Number of data points||392|
|Proteins||Metallo-beta-lactamase type 2|
|Assays/Quantities/Protocols||Experimental Assay: KM ; Experimental Assay: MIC ; Experimental Assay: Soluble fraction ; Experimental Assay: kcat ; Experimental Assay: kcat/Km ; Experimental Assay: Concentration after purification ; Experimental Assay: Zn(II) ions/molecule ; Experimental Assay: Molecular mass (experimental) ; Derived Quantity: SD of kcat/Km ; Derived Quantity: SD of KM ; Derived Quantity: SD of kcat ; Derived Quantity: SD of Concentration after purification ; Derived Quantity: SD of Zn(II) ions/molecule ; Computational Protocol: Molecular mass (calculated)|
|Libraries||Expression and biophysical data for IMP-6 and variants (Table 1) ; Activity data for IMP-6 and variants with different beta-lactams|