Crystal structure of the E166A mutant of extended-spectrum beta-lactamase Toho-1 at 1.8 angstrom resolution

Bacterial resistance to beta-lactams is mainly due to the production of bet a-lactamase. Especially through the production of extended-spectrum beta-la ctamases (ESBLs), bacteria have acquired resistance not only to penicillins , but also to expanded-spectrum cephems. Here, we describe the crystal stru cture of the E166A mutant of class A beta-lactamase Toho-1 at 1.8 Angstrom resolution, the first reported tertiary structure of an ESBL. Instead of th e wild-type enzyme, a mutant Toho-1, in which Glu166 was replaced with alan ine, was used for this study, because of the strong tendency of the wild-ty pe enzyme to form twinned crystals. The overall structure of Toho-1 is simi lar to the crystal structures of non-ESBLs, with no pronounced backbone rea rrangement of the framework. However, there are some notable local changes. First, a difference in the disposition of an arginine residue, which is at position 244 in non-ESBLs but at position 276 in Toho-1 and other ESBLs, w as revealed and the role of this arginine residue is discussed. Moreover, c hanges in the hydrogen-bonding pattern and in the formation of the hydropho bic core were also observed near the Omega loop. In particular, the lack of hydrogen bonds in the vicinity of the Omega loop could be a cause of the e xtended substrate specificity of Toho-1. Through the generation of a model for the enzyme-substrate complex, a conformational change of Toho-1 occurri ng on complex formation is discussed based on the active-site cleft structu re and the substrate profile. (C) 1999 Academic Press.