New Conformations of Acylation Adducts of Inhibitors of β-Lactamase from Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (TB), is naturally resistant to β-lactam antibiotics due to the production of the extended spectrum β-lactamase BlaC. β-Lactam/β-lactamase inhibitor combination therapies can circumvent the BlaC-mediated resistance of Mtb and are promising treatment options against TB. However, still little is known of the exact mechanism of BlaC inhibition by the β-lactamase inhibitors currently approved for clinical use, clavulanic acid, sulbactam, tazobactam, and avibactam. Here, we present the X-ray diffraction crystal structures of the acyl-enzyme adducts of wild-type BlaC with the four inhibitors. The +70 Da adduct derived from clavulanate and the trans-enamine acylation adducts of sulbactam and tazobactam are reported. BlaC in complex with avibactam revealed two inhibitor conformations. Preacylation binding could not be observed because inhibitor binding was not detected in BlaC variants carrying a substitution of the active site serine 70 to either alanine or cysteine, by crystallography, ITC or NMR. These results suggest that the catalytic serine 70 is necessary not only for enzyme acylation but also for increasing BlaC affinity for inhibitors in the preacylation state. The structure of BlaC with the serine to cysteine mutation showed a covalent linkage of the cysteine 70 Sγ atom to the nearby amino group of lysine 73. The differences of adduct conformations between BlaC and other β-lactamases are discussed.

Evaluating the covalent binding of carbapenems on BlaC using noncovalent interactions

“…[33][34][35][36] Some β-lactam BlaC inhibitors were not computed in this work since they don't possess the same core structure. [37] The covalent bond is built between the serine hydroxyl and the lactam carbonyl using a new residue SEP with force filed parameters only suitable for these specific carbapenem structures. Opportunely, these parameters gave proper binding free energy values in agreement with the experimental data and were successfully applied to the MD simulations of these carbapenems.…”

Crystal Structure of Phosphoserine BlaC from Mycobacterium tuberculosis Inactivated by Bis(Benzoyl) Phosphate

“…It is known that esterase enzymes can be phosphorylated at their nucleophilic serine residues upon exposure to organophosphates through a mechanism of inactivation followed by aging [31,32,33,34]. However, there was no precedence in the literature for the phosphorylation of the active site serine of BlaC, thus it was unknown whether the Ser-70 of BlaC could be irreversibly modified by a phosphorus ligand.…”

Direct Observation of the Mechanism of Antibiotic Resistance by Mix-and-Inject at the European XFEL

“…The diffusion time is fast enough that 66 ms after mixing all non-covalently bound SUB molecules in subunits B and D have reacted to the covalently bound trans -EN. This is quite unexpected as it was suggested that it would take minutes for the enamine to form after binding of SUB in the BlaC ( 32–34 ). Although the SUB binds non-covalently to the active sites in subunits A and C, the reaction with the Ser-70 did not occur within ΔT mi = 66 ms.…”

“…Most abundant is the so-called trans-enamine (trans-EN) species (Scheme S1, compound III) that inhibits β-lactamases and helps to eliminate β-lactamase induced antibiotic resistance. The static structures of trans -ENs with β-lactamases, including BlaC, were recently characterized ( 33, 34 ) but structures of the early species that form during SUB binding remain elusive.…”

“…Experiments exploring longer ΔT mi will clarify this situation. In subunits B and D Glu-112 is not near the active site, and Arg-173 displays a stretched, open conformation allowing the SUB to orient correctly towards the Ser-70, and to react further to the trans-EN that then non-competitively inhibits the BlaC ( 34 ). The near-by Pi, which is displaced when the much larger ligand CEF is present, stays in place in all subunits and likely adds to the stability of both complexes.…”