beta-lactamases are the main molecules responsible for giving bacterial resistance against beta-lactam antibiotics. The study of beta-lactamases has allowed the development of antibiotics capable of inhibiting these enzymes. In this context, extended spectrum beta-lactamase (ESBL) TLA-1 has spread in Escherichia coll. and Enterobacter cloacae clinical isolates during the last 30 years in Mexico. In this research, the 3D structures of ESBL TLA-1 and TLA-1 S70G mutant, both ligand-free and in complex with clavulanic acid were determined by X-ray crystallography. Four clavulanic acid molecules were found in the structure of TLA-1, two of those were intermediaries of the acylation process and were localized covalently bound to two different amino acid residues, Ser70 and Ser237. The coordinates of TLA-1 in complex with clavulanic acid shows the existence of a second acylation site, additional to Ser70, which might be extendable to several members of the subclass A beta-lactamases family. This is the first time that two serines involved in binding clavulanic acid has been reported and described to an atomic level. (C) 2019 Elsevier Inc. All rights reserved.