ABSTRACT
Combinations of β-lactams with clavulanate are currently being investigated for tuberculosis treatment. Since
Mycobacterium tuberculosis
produces a broad spectrum β-lactamase, BlaC, the success of this approach could be compromised by the emergence of clavulanate-resistant variants, as observed for inhibitor-resistant TEM variants in enterobacteria. Previous analyses based on site-directed mutagenesis of BlaC have led to the conclusion that this risk was limited. Here, we used a different approach based on determination of the crystal structure of β-lactamase Bla
MAb
of
Mycobacterium abscessus
, which efficiently hydrolyzes clavulanate. Comparison of Bla
MAb
and BlaC allowed for structure-assisted site-directed mutagenesis of BlaC and identification of the G
132
N substitution that was sufficient to switch the interaction of BlaC with clavulanate from irreversible inactivation to efficient hydrolysis. The substitution, which restored the canonical SDN motif (SDG→SDN), allowed for efficient hydrolysis of clavulanate, with a more than 10
4
-fold increase in
kcat
(0.41 s
−1
), without affecting the hydrolysis of other β-lactams. Mass spectrometry revealed that acylation of BlaC and of its G
132
N variant by clavulanate follows similar paths, involving sequential formation of two acylenzymes. Decarboxylation of the first acylenzyme results in a stable secondary acylenzyme in BlaC, whereas hydrolysis occurs in the G
132
N variant. The SDN/SDG polymorphism defines two mycobacterial lineages comprising rapidly and slowly growing species, respectively. Together, these results suggest that the efficacy of β-lactam–clavulanate combinations may be limited by the emergence of resistance. β-Lactams active without clavulanate, such as faropenem, should be prioritized for the development of new therapies.