Structural insights into the TLA-3 extended-spectrum {beta}-lactamase and its inhibition by avibactam and OP0595 [PublishAheadOfPrint]

Development of effective inhibitors that block extended-spectrum β-lactamases (ESBLs) and restore the action of β-lactams represents an effective strategy against ESBL-producing Enterobacteriaceae. We evaluated the inhibitory effect of the diazabicyclooctanes avibactam and OP0595 against TLA-3, an ESBL we identified previously. Avibactam and OP0595 inhibited TLA-3 with apparent K_{i app} of 1.71 ± 0.10 and 1.49 ± 0.05 μM, respectively, and could restore susceptibility to cephalosporins in TLA-3-producing Escherichia coli. The acylation rate constant [k₂/K, (3.25 ± 0.03) x 10³ M^-1s^-1] of avibactam was closer to those of class C and D β-lactamases (k₂/K, <10⁴ M^-1s^-1) than those of class A β-lactamases (k₂/K, >10⁴ M^-1s^-1). In addition, we determined the structure of TLA-3, and those of TLA-3 complexed with avibactam or OP0595, at resolutions of 1.6-, 1.6-, and 2.0-Å, respectively. TLA-3 contains an inverted -loop and an extended loop between the β5 and β6 strands (insertion after Ser237), which appear only in PER-type class A β-lactamases. These structures might favor the accommodation of cephalosporins harboring bulky R1 side chains. TLA-3 presented high catalytic efficiency (k_cat/K_m) against cephalosporins, including cephalothin, cefuroxime, and cefotaxime. Avibactam and OP0595 bound covalently to TLA-3 via the Ser70 residue, and made contacts with residues Ser130, Thr235, and Ser237, which are conserved in ESBLs. Additionally, the sulfate group of the inhibitors formed polar contacts with amino acid residues in a positively charged pocket of TLA-3. Our findings provide a structural template for designing improved diazabicyclooctane-based inhibitors that are effective against ESBL-producing Enterobacteriaceae.

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