Crystal Structure and Activity Studies of the Mycobacterium tuberculosis β-Lactamase Reveal Its Critical Role in Resistance to β-Lactam Antibiotics

Abstract

␤-Lactam antibiotics are extremely effective in disrupting the synthesis of the bacterial cell wall in both gram-positive and gram-negative bacteria. However, they are ineffective against Mycobacterium tuberculosis, due to the production of a ␤-lactamase enzyme encoded on the chromosome of M. tuberculosis that degrades these antibiotics. Indeed, recent studies have demonstrated that deletion of the blaC gene, the only gene encoding a ␤-lactamase in M. tuberculosis, or inhibition of the encoded enzyme resulted in significantly increased sensitivity to ␤-lactam antibiotics. In this paper we present a biochemical and structural characterization of M. tuberculosis BlaC. Recombinant BlaC shows a broad range of specificity with almost equal penicillinase and cepholothinase activity. While clavulanate is a mechanism-based inhibitor to class A ␤-lactamase with high potency (typically K i < 0.1 M), it is a relatively poor inhibitor of the M. tuberculosis BlaC (K i ‫؍‬ 2.4 M). The crystal structure of the enzyme, determined at a resolution of 1.7 Å, shows that the overall fold of the M. tuberculosis enzyme is similar to other class A ␤-lactamases. There are, however, several distinct features of the active site, such as the amino acid substitutions N132G, R164A, R244A, and R276E, that explain the broad specificity of the enzyme, relatively low penicillinase activity, and resistance to clavulanate.

A Structure-Based Classification of Class A β-Lactamases, a Broadly Diverse Family of Enzymes

“…Many enzymatic studies have been carried out on Mycobacterium tuberculosis (BlaC), Mycobacterium abscessus (MAB-1), and Mycobacterium fortuitum (BlaF/MFO-1) (Table 1) (4,22,23,113). The enzymes produced by these species have a broader spectrum of activity, also degrading cephalosporins, for example, and they are less sensitive to clavulanic acid; nevertheless, they are classified as functional group 2b enzymes.…”

Bacterial Growth and Cell Division: a Mycobacterial Perspective

“…A mycobacterial AmpG homologue has not yet been identified and characterized, but some transport of the PG material back into the cytosol would be needed. It should be noted that mycobacteria do have ␤-lactamases, enzymes associated with cell wall monitoring and remodeling (136,137,271,434).…”

High tolerance to simultaneous active‐site mutations in TEM‐1 β‐lactamase: Distinct mutational paths provide more generalized β‐lactam recognition

“…Because residues 104 and 238 are involved in loop positioning and because residue 240 is the immediate neighbor of 238 (TEM-1 has no residue 239), their mutation may also contribute to active-site expansion, favoring binding of bulkier substrates as observed in BlaC b-lactamase from Mycobacterium tuberculosis. 33 We propose that replacements at positions 104 and 240 mainly fulfill steric requirements that support productive binding of CTX, rather than to provide specific contacts with the substrate. Our results are consistent with active-site expansion both through displacement of the b3-strand (mutations G238S or G238N) and with flexibility of residues 104 and 240, which could open the active site from opposite faces of the cavity and allow expansion.…”