Crystal Structure of Extended-Spectrum β-Lactamase Toho-1:  Insights into the Molecular Mechanism for Catalytic Reaction and Substrate Specificity Expansion^,

Abstract

The crystallographic structure of the class A beta-lactamase Toho-1, an extended-spectrum beta-lactamase with potent activity against expanded-spectrum cephems, has been determined at 1.65 A resolution. The result reveals that the Lys73 side chain can adopt two alternative conformations. The predominant conformation of Lys73 is different from that observed in the E166A mutant, indicating that removal of the Glu166 side chain changes the conformation of the Lys73 side chain and thus the interaction between Lys73 and Glu166. The Lys73 side chain would play an important role in proton relay, switching its conformation from one to the other depending on the circumstances. The electron density map also implies possible rotation of Ser237. Comparison of the Toho-1 structure with the structure of other class A beta-lactamases shows that the hydroxyl group of Ser237 is likely to rotate through interaction with the carboxyl group of the substrate. Another peculiarity is the existence of three sulfate ions positioned in or near the substrate-binding cavity. One of these sulfate ions is tightly bound to the active center, while the other two are held by a region of positive charge formed by two arginine residues, Arg274 and Arg276. This positively charged region is speculated to represent a pseudo-binding site of the beta-lactam antibiotics, presumably catching the methoxyimino group of the third-generation cephems prior to proper binding in the substrate-binding cleft for hydrolysis. This high-resolution structure, together with detailed kinetic analysis of Toho-1, provides a new hypothesis for the catalytic mechanism and substrate specificity of Toho-1.

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.…”

“…Toho-1 has a lower hydrolytic activity against penicillins and higher hydrolytic activity against ceftazidime than the "typical" CTX-M enzymes. 15 Three other atypical CTX-M enzymes have also been reported that exhibit an unusually high level of resistance to ceftazidime. The improved activity versus ceftazidime for these three enzymes, CTX-M-15, 16 CTX-M-16 17 and CTX-M-27 18 is correlated with the substitution Asp240Gly, which has never been observed in naturally occurring TEM and SHV-type 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].…”

Structure of PBP-A from Thermosynechococcus elongatus, a Penicillin-Binding Protein Closely Related to Class A β-Lactamases

“…Finally, a hydrogen bond is formed between the Ser61 hydroxyl group O γ and the Ser122 hydroxyl group O γ (3.02 Ǻ). For class A β-lactamases, distances of 3.17 Å for PER-1 (1e25), 15 3.15 A for TEM-1 in 1.55 Å resolution structure (1zg4), 16 but 3.55 Å in 1.8 Å resolution structure (1btl), 17 and 2.89 Å in Toho-1 (1iys) 18 are found. In class A β-lactamases, the Lys73 δ-NH 2 group is hydrogen bonded to the carboxylate group of Glu166.…”