Asp179 in the class A ß-lactamase from Mycobacterium tuberculosis is a conserved yet not essential residue due to epistasis.

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 ß-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 ß-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 ß-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.

Two ß-Lactamase Variants with Reduced Clavulanic Acid Inhibition Display Different Millisecond Dynamics.

The ß-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 the most inhibitor resistance, K234R, as well as variant G132N that was found previously were characterized using X-ray crystallography and nuclear magnetic resonance (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 and Ser130) and a short loop (positions 214 to 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.