The rise of multi- and even totally antibiotic resistant forms of Mycobacterium tuberculosis underlines the need for new antibiotics. The pathogen is resistant to β-lactam compounds due to its native serine β-lactamase, BlaC. This resistance can be circumvented by administration of a β-lactamase inhibitor. We studied the interaction between BlaC and the inhibitor clavulanic acid. Our data show hydrolysis of clavulanic acid and recovery of BlaC activity upon prolonged incubation. The rate of clavulanic acid hydrolysis is much higher in the presence of phosphate ions. A specific binding site for phosphate is identified in the active site pocket, both in the crystalline state and in solution. NMR spectroscopy experiments show that phosphate binds to this site with a dissociation constant of 30 mM in the free enzyme. We conclude that inhibition of BlaC by clavulanic acid is reversible and that phosphate ions can promote the hydrolysis of the inhibitor.

The Mycobacterium tuberculosis β-lactamase BlaC is a broad-spectrum β-lactamase that can convert a range of β-lactam antibiotics. Enzymes with low specificity are expected to exhibit active-site flexibility. To probe the motions in BlaC, we studied the dynamic behavior in solution using nuclear magnetic resonance (NMR) spectroscopy. 15N relaxation experiments show that BlaC is mostly rigid on the pico- to nanosecond timescale. Saturation transfer experiments indicate that also on the high-millisecond timescale BlaC is not dynamic. Using relaxation dispersion experiments, clear evidence was obtained for dynamics in the low-millisecond range, with an exchange rate of ca. 860 s−1. The dynamic amide groups are localized in the active site. Upon formation of an adduct with the inhibitor avibactam, extensive line broadening occurs, indicating an increase in magnitude of the active-site dynamics. Furthermore, the rate of the motions increases significantly. Upon reaction with the inhibitor clavulanic acid, similar line broadening is accompanied by duplication of NMR signals, indicative of at least one additional, slower exchange process (exchange rate, kex, of <100 s−1), while for this inhibitor also loss of pico- to nanosecond timescale rigidity is observed for some amides in the α domain. Possible sources of the observed dynamics, such as motions in the omega loop and rearrangements of active-site residues, are discussed. The increase in dynamics upon ligand binding argues against a model of inhibitor binding through conformational selection. Rather, the induced dynamics may serve to maximize the likelihood of sampling the optimal conformation for hydrolysis of the bound ligand.

The β-lactamase of Mycobacterium tuberculosis, BlaC, is susceptible to inhibition by clavulanic acid. The ability of this enzyme to escape inhibition through mutation was probed using error-prone PCR combined with functional screening in Escherichia coli. The variant that was found to confer most inhibitor resistance, K234R, as well as variant G132N that was found previously, were characterized using X-ray crystallography and NMR relaxation experiments to probe structural and dynamic properties. The G132N mutant exists in solution in two, almost equally populated conformations that exchange with a rate of ca. 88 s−1. The conformational change affects a broad region of the enzyme. The crystal structure reveals that the Asn132 side chain forces the peptide bond between Ser104 and Ile105 in a cis-conformation. The crystal structure suggests multiple conformations for several side chains (e.g. Ser104, Ser130) and a short loop (214-216). In the K234R mutant, the active site dynamics are significantly diminished with respect to the wild type enzyme. These results show that multiple evolutionary routes are available to increase inhibitor resistance in BlaC and that active site dynamics on the millisecond time scale are not required for catalytic function.

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