Antibiotic resistance in bacteria is ever changing and adapting, as once-novel β-lactam antibiotics are losing their efficacy, primarily due to the production of β-lactamases. Metallo-β-lactamases (MBLs) efficiently inactivate a broad range of β-lactam antibiotics, including carbapenems, and are often coexpressed with other antibacterial resistance factors. The rapid dissemination of MBLs and lack of novel antibacterials pose an imminent threat to global health. In an effort to better counter these resistance-conferring β-lactamases, an investigation of their natural evolution and resulting substrate specificity was employed. In this study, we elucidated the effects of different amino acid substitutions at position 67 in IMP-type MBLs on the ability to hydrolyze and confer resistance to a range of β-lactam antibiotics. Wild-type β-lactamases IMP-1 and IMP-10 and mutants IMP-1-V67A and IMP-1-V67I were characterized biophysically and biochemically, and MICs for Escherichia coli cells expressing these enzymes were determined. We found that all variants exhibited catalytic efficiencies (kcat/Km) equal to or higher than that of IMP-1 against all tested β-lactams except penicillins, against which IMP-1 and IMP-1-V67I showed the highest kcat/Km values. The substrate-specific effects of the different amino acid substitutions at position 67 are discussed in light of their side chain structures and possible interactions with the substrates. Docking calculations were employed to investigate interactions between different side chains and an inhibitor used as a β-lactam surrogate. The differences in binding affinities determined experimentally and computationally seem to be governed by hydrophobic interactions between residue 67 and the inhibitor and, by inference, the β-lactam substrates.
Submitter: Paulie Dang
Submission Date: Sept. 9, 2019, 10:50 a.m.
Explanation of amino acid numbering: Residue 31 that was mutated in this study is called residue 67 according to the standard numbering scheme (PMID 15215079) and the title of the publication. The wild-type enzyme IMP-1 has the mutation V31F (V67F according to standard numbering). ND indicates not detectable.
|Number of data points||360|
|Proteins||Metallo-beta-lactamase type 2|
|Assays/Quantities/Protocols||Experimental Assay: Inhibition ; Experimental Assay: Melting temperature ; Experimental Assay: Solubility ; Experimental Assay: MIC relative ; Experimental Assay: Resistance ; Experimental Assay: MIC absolute ; Experimental Assay: kcat/Km ; Experimental Assay: Km ; Experimental Assay: kcat ; Derived Quantity: SD of Inhibition ; Derived Quantity: SD of kcat ; Derived Quantity: SD of kcat/Km ; Derived Quantity: SD of Km|
|Libraries||Inhibition ; Solubility ; Activity Data|
|Structure ID||Release Date||Resolution||Structure Title|
|6JED||2019-08-07||1.57||Crystal structure of IMP-1 metallo-beta-lactamase in a complex with MCR|
|5Y5B||2018-08-08||1.7||Crystal Structure Of IMP-1 Metallo-beta-lactamase|
|4C1G||2014-08-27||1.71||Crystal structure of the metallo-beta-lactamase IMP-1 with D-captopril|
|5EV6||2016-06-01||1.98||Crystal structure of the native, di-zinc metallo-beta-lactamase IMP-1|
|5HH4||2017-01-18||2.0||Crystal structure of metallo-beta-lactamase IMP-1 in complex with a phosphonate-based inhibitor|
|1DD6||2000-11-08||2.0||IMP-1 METALLO BETA-LACTAMASE FROM PSEUDOMONAS AERUGINOSA IN COMPLEX WITH A MERCAPTOCARBOXYLATE INHIBITOR|
|1WUO||2005-03-29||2.01||Crystal structure of metallo-beta-lactamase IMP-1 mutant (D81A)|
|4C1F||2014-08-27||2.01||Crystal structure of the metallo-beta-lactamase IMP-1 with L-captopril|
|5EV8||2016-06-01||2.3||Crystal structure of the metallo-beta-lactamase IMP-1 in complex with the bisthiazolidine inhibitor D-CS319|
|5EWA||2016-06-01||2.3||Crystal structure of the metallo-beta-lactamase IMP-1 in complex with the bisthiazolidine inhibitor L-VC26|
|2DOO||2006-11-28||2.43||The structure of IMP-1 complexed with the detecting reagent (DansylC4SH) by a fluorescent probe|
|1VGN||2005-06-21||2.63||Structure-based design of the irreversible inhibitors to metallo--lactamase (IMP-1)|
|1WUP||2005-03-29||3.0||Crystal structure of metallo-beta-lactamase IMP-1 mutant (D81E)|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|
|100.0||A||Metallo-beta-lactamase type 2||P52699||BLAB_SERMA|