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.

Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme that catalyzes the oxidation of proline to Δ1-pyrroline-5-carboxylate. Thermus thermophilus ProDH (TtProDH) contains in addition to its flavin-binding domain an N-terminal arm, consisting of helices αA, αB, and αC. Here, we report the biochemical properties of the helical arm truncated TtProDH variants ΔA, ΔAB, and ΔABC, produced with maltose-binding protein as solubility tag. All three truncated variants show similar spectral properties as TtProDH, indicative of a conserved flavin-binding pocket. ΔA and ΔAB are highly active tetramers that rapidly react with the suicide inhibitor N-propargylglycine. Removal of the entire N-terminal arm (ΔABC) results in barely active dimers that are incapable of forming a flavin adduct with N-propargylglycine. Characterization of V32D, Y35F, and V36D variants of ΔAB established that a hydrophobic patch between helix αC and helix α8 is critical for TtProDH catalysis and tetramer stabilization.

Conserved residues are often considered essential for function, and substitutions in such residues are expected to have a negative influence on the properties of a protein. However, mutations in a few highly conserved residues of the β‐lactamase from Mycobacterium tuberculosis, BlaC, were shown to have no or only limited negative effect on the enzyme. One such mutant, D179N, even conveyed increased ceftazidime resistance upon bacterial cells, while displaying good activity against penicillins. The crystal structures of BlaC D179N in resting state and in complex with sulbactam reveal subtle structural changes in the Ω‐loop as compared to the structure of wild‐type BlaC. Introducing this mutation in four other β‐lactamases, CTX‐M‐14, KPC‐2, NMC‐A and TEM‐1, resulted in decreased antibiotic resistance for penicillins and meropenem. The results demonstrate that the Asp in position 179 is generally essential for class A β‐lactamases but not for BlaC, which can be explained by the importance of the interaction with the side chain of Arg164 that is absent in BlaC. It is concluded that Asp179 though conserved is not essential in BlaC, as a consequence of epistasis.

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