The G132S Mutation Enhances the Resistance of Mycobacterium tuberculosis β-Lactamase against Sulbactam

Abstract

The current rise of antibiotic resistant forms of Mycobacterium tuberculosis is a global health threat that calls for new antibiotics. The β-lactamase BlaC of this pathogen prevents the use of β-lactam antibiotics, except in combination with a β-lactamase inhibitor. To understand if exposure to such inhibitors can easily result in resistance, a BlaC evolution experiment was performed, studying the evolutionary adaptability against the inhibitor sulbactam. Several amino acid substitutions in BlaC were shown to confer reduced sensitivity to sulbactam. The G132S mutation causes a reduction in the rate of nitrocefin and ampicillin hydrolysis and simultaneously reduces the sensitivity for sulbactam inhibition. Introduction of the side chain moiety of Ser132 causes the 104–105 peptide bond to assume the cis conformation and the side chain of Ser104 to be rotated toward the sulbactam adduct with which it forms a hydrogen bond not present in the wild-type enzyme. The gatekeeper residue Ile105 also moves. These changes in the entrance of the active site can explain the decreased affinity of G132S BlaC for both substrates and sulbactam. Our results show that BlaC can easily acquire a reduced sensitivity for sulbactam, with a single-amino acid mutation, which could hinder the use of combination therapies.

QM/MM Simulations Reveal the Determinants of Carbapenemase Activity in Class A β-Lactamases

“…The most significant difference between BlaC and other class A β-lactamase active sites is the lack of Asn132 in the former, in which it is replaced by Gly. , Asn132 may help keep the 6α-1R-hydroxyethyl dihedral in Position II (Figure ), thereby lowering the barriers to reaction in carbapenemases. Previous structural and computational studies have suggested that interactions of Asn132 with the 6α-1R-hydroxyethyl group are important in determining carbapenemase activity. ,,, Comparisons between enzymes here (e.g.…”

The roles of highly conserved, non‐catalytic residues in class A β‐lactamases

“…36,37 A few laboratory evolution studies on BlaC found mutations in the second-shell residue 132, which is a conserved Asn in most class A β-lactamases, but not in BlaC. 10,17 Deep mutational scanning was performed on TEM-1, yielding the fitness effect of substitutions of every amino acid residue to all other 19 amino acids. 38,39 These extensive studies showed that robustness and evolvability of TEM-1 are dependent on the strength of purifying selective pressure, 38 and that mutational effects on protein thermodynamic stability shape the distribution of fitness effects of mutations.…”

“…New β-lactam resistant strains are frequently reported, with sometimes only one point mutation in the enzyme leading to a new catalytic function. [10][11][12][13] This room for evolution in β-lactamases is reflected in the low amino acid conservation. A sequence alignment of the β-lactamase from Mycobacterium tuberculosis (Mtb), BlaC, the object of this study, with 493 other class A β-lactamases shows 49%-81% identity.…”

Asp179 in the class A β‐lactamase from Mycobacterium tuberculosis is a conserved yet not essential residue due to epistasis

“…Residues are numbered according to Ambler notation, and correspond to residue numbers 43-307 of BlaC Uniprot entry P9WKD3-1, 29-291 of CTX-M-14 Uniprot entry Q9L5C7, 25-293 of KPC-2 Uniprot entry Q9F663, 28-292 of NMC-A Uniprot entry P52663, and 24-286 of TEM-1 Uniprot entry P62593. The purple shading indicates the degree of sequence identity.For in cell studies, these sequences are preceded by a TAT-signal sequence, for in vitro studies a N-terminal TEV cleavable HIS-tag was added[19]. The alignment is generated using Clustal Omega[40] and visualized using Jalview[46].…”