Bacterial resistance to beta-lactams is mainly due to the production of beta-lactamase. Especially through the production of extended-spectrum beta-lactamases (ESBLs), bacteria have acquired resistance not only to penicillins, but also to expanded-spectrum cephems. Here, we describe the crystal structure of the E166A mutant of class A beta-lactamase Toho-1 at 1.8 A resolution, the first reported tertiary structure of an ESBL. Instead of the wild-type enzyme, a mutant Toho-1, in which Glu166 was replaced with alanine, was used for this study, because of the strong tendency of the wild-type enzyme to form twinned crystals. The overall structure of Toho-1 is similar to the crystal structures of non-ESBLs, with no pronounced backbone rearrangement 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, was revealed and the role of this arginine residue is discussed. Moreover, changes in the hydrogen-bonding pattern and in the formation of the hydrophobic 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 extended substrate specificity of Toho-1. Through the generation of a model for the enzyme-substrate complex, a conformational change of Toho-1 occurring on complex formation is discussed based on the active-site cleft structure and the substrate profile.
Copyright 1998 Academic Press.