Acyl-intermediate Structures of the Extended-spectrum Class A β-Lactamase, Toho-1, in Complex with Cefotaxime, Cephalothin, and Benzylpenicillin

Abstract

Bacterial resistance to ␤-lactam antibiotics is a serious problem limiting current clinical therapy. The most common form of resistance is the production of ␤-lactamases that inactivate ␤-lactam antibiotics. Toho-1 is an extended-spectrum ␤-lactamase that has acquired efficient activity not only to penicillins but also to cephalosporins including the expanded-spectrum cephalosporins that were developed to be stable in former ␤-lactamases. We present the acyl-intermediate structures of Toho-1 in complex with cefotaxime (expanded-spectrum cephalosporin), cephalothin (non-expanded-spectrum cephalosporin), and benzylpenicillin at 1.8-, 2.0-, and 2.1-Å resolutions, respectively. These structures reveal distinct features that can explain the ability of Toho-1 to hydrolyze expanded-spectrum cephalosporins. First, the ⍀-loop of Toho-1 is displaced to avoid the steric contacts with the bulky side chain of cefotaxime. Second, the conserved residues Asn 104 and Asp 240 form unique interactions with the bulky side chain of cefotaxime to fix it tightly. Finally, the unique interaction between the conserved Ser 237 and cephalosporins probably helps to bring the ␤-lactam carbonyl group to the suitable position in the oxyanion hole, thus increasing the cephalosporinase activity.

Atomic Resolution Structures of CTX-M β-Lactamases: Extended Spectrum Activities from Increased Mobility and Decreased Stability

“…In Toho-1, for instance, it has been proposed that the cefotaximase and ceftazidimase activities are partly explained by increased flexibility of the U loop and of the loop between B3 and B4, both part of the binding pocket. 15,19,20 Consistent with this view, there are fewer interactions between the U loop and the a/b domain of the CTX-M structures determined here, compared with TEM and SHV enzymes. Some of the hydrogen bonds connecting the N and C termini of the U loop are also lost in the CTX-M-like enzymes, such as the one between Thr160 (replaced by a phenylalanine) and Thr/Ser181 in non-ESBL b-lactamases.…”

“…32,33 Ser237 and Asn104 interact with the carboxylate group of cefotaxime and the carbonyl group of the acylamide side-chain, respectively, in the acyl intermediate structure of Toho-1. 20 The importance of these two residues for CTX-M-like enzymes make them suitable targets for inhibitor development against this new group of ESBLs.…”

“…An important step was the determination of the Toho-1 structure by X-ray crystallography, which significantly advanced our understanding of these enzymes. 15,19,20 A surprising feature of the Toho-1 structure was that its active site resembled that of the narrow spectrum TEM-1 enzyme, and did not feature the enlarged sites characteristic of ESBL mutants such as TEM-52 10 and TEM-64. 4 In these TEM-derived ESBLs, an increase in active site volume is thought to be key to their ability to recognize the relatively large third generation cephalosporins.…”

The continuing challenge of ESBLs

“…Overall, one appreciates that the first common theme to emerge is that the active site is selectively "remodeled and expanded" to accommodate the bulky R 1 side chain of extendedspectrum cephalosporins [6]. This remodeling is observed in the atomic structures of Toho-1, TEM-52, TEM-64, the Gly238Ala ESBL in TEM, SHV-2, PER-1 and OXA-10 betalactamases [46][47][48][49][50][51][52]. Recently, the crystal structure of KPC-2 was determined, demonstrating modifications in the active site that allow access to carbapenems [53].…”

Role of a Mutation at Position 167 of CTX-M-19 in Ceftazidime Hydrolysis

“…The interactions between ␤-lactamases and ␤-lactams have been previously studied by computer modeling, an approach that may provide details of plausible interactions between enzyme and substrates (7,13,21). In the present study, the CTX-M-type ␤-lactamase substrate specificity was elucidated by modeling complex structures of ␤-lactamases with ␤-lactams (10,20). Therefore, we constructed a model structure of CTX-M-18 and CTX-M-19.…”