Acyl-intermediate Structures of the Extended-spectrum Class A (cid:1) -Lactamase, Toho-1, in Complex with Cefotaxime, Cephalothin, and Benzylpenicillin*

Bacterial resistance to (cid:1) -lactam antibiotics is a seri-ous problem limiting current clinical therapy. The most common form of resistance is the production of (cid:1) -lacta-mases that inactivate (cid:1) -lactam antibiotics. Toho-1 is an extended-spectrum (cid:1) -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 (cid:1) -lac-tamases. 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-,

␤-Lactam antibiotics are effectively used against a wide range of bacterial infectious diseases (1). The antibiotics form stable acyl-enzymes with penicillin-binding proteins (PBPs) 1 in the membrane of the bacterial cell. PBPs function in the biosynthesis and repair of the peptidoglycan of the cell wall; thus, the inhibition of these enzymes induces cell death. However, resistant bacteria escape from the lethal action of ␤-lactam antibiotics mainly by producing ␤-lactamases that hydrolyze the antibiotics. ␤-Lactamases are acylated by ␤-lactam antibiotics in a similar manner as PBPs; however, they are rapidly deacylated. ␤-Lactamases are classified into four groups (classes A, B, C, and D) according to their amino acid sequences and substrate profiles (1). Classes A, C, and D ␤-lactamases are serine ␤-lactamases, whereas class B ␤-lactamases are zinccontaining ␤-lactamases. Among these four classes of ␤-lactamases, class A ␤-lactamases are especially important, because they exhibit highly variable substrate profiles and in general are encoded by plasmids and are easily transferable between cells, thereby threatening clinical antibiotic therapy.
The expanded-spectrum cephalosporins (oxyimino-cephalosporins) including cefotaxime were developed to be stable in the former class A ␤-lactamases such as TEM-1 and SHV-1 (1,2). These compounds are characterized by a bulky acylamide side chain containing an oxyimino group. Many of these compounds also contain an aminothiazole ring. However, after extensive and sometimes abusive clinical use of these antibiotics, resistant bacteria began to produce new class A ␤-lactamases capable of hydrolyzing oxyimino-cephalosporins called extendedspectrum ␤-lactamases (ESBLs) (1,2). ESBLs are classified into two groups. The first of these groups (type I) consists of variants of TEM-1 or SHV-1 that differ by a few amino acid substitutions. The second group (type II) includes enzymes that are not related to TEM-1 or SHV-1 (2). The plasmid encoded CTX-M-type ESBLs are the most widespread family of type II ESBLs and are increasingly on the rise. Contrary to what was originally thought of class A ␤-lactamases, CTX-M-type ESBLs have an efficient hydrolytic activity toward oxyimino-cephalosporins but exhibit lower activity toward penicillins than non-ESBLs (3).
Toho-1 belongs to the CTX-M-type ESBLs (4). We have previously reported the structure of the Toho-1 mutant E166A whose overall fold was shown to be similar to non-ESBLs (5). Toho-1 has some variations in hydrogen-bonding patterns and an increase in flexibility of the ␤-strand B3 as well as the ⍀-loop. Yet, it was still unclear how these differences increase oxyimino-cephalosporinase activity of Toho-1, because a complex structure with an oxyimino-cephalosporin was not determined. In addition to Toho-1, there have been seven other x-ray structures of oxyimino-cephalosporin-hydrolyzing ␤-lactamases solved (TEM-52 (6), TEM-64 (7), TEM-1 G238A mutant (7), PER-1 (8), a ␤-lactamase from Proteus vulgaris K1 (9), NMC-A (10), and a class C ␤-lactamase from Enterobacter cloacae GC1 (11)), but none of these ␤-lactamase structures has been determined in complex with an oxyimino-cephalosporin. Here, we report the acyl-intermediate structures of a Toho-1 mutant E166A in complex with the substrates cefotaxime, cephalothin, and benzylpenicillin. These are the first acyl-intermediate structures of an ESBL with the substrate. These structures present the mechanism by which CTX-M-type ESBLs can efficiently hydrolyze oxyimino-cephalosporins. These findings may prove to be essential for the development of stable compounds in ESBLs.

EXPERIMENTAL PROCEDURES
Crystallization and Data Collection-Toho-1 E166A mutant was expressed and purified as described previously (5). Crystals were grown by the vapor diffusion technique using a 2.1 M ammonium sulfate solution as precipitant. Acyl-intermediates were prepared by soaking crystals in 2.5 M ammonium sulfate containing 0.5 mM substrate (cefotaxime, cephalothin, or benzylpenicillin) and 10% sucrose as the cryoprotectant. Concentrations of the substrate and sucrose were increased stepwise to 1.5 mM and 30%, respectively. X-ray diffraction data were collected at 100 K on beamline 6A of Photon Factory, the High Energy Acceleration Research Organization (Tsukuba, Japan).
Structure Solution and Refinement-Images were processed with Denzo and Scalepack (12). The soaked crystals were isomorphous with the unbound crystal. Phases were calculated by molecular replacement with the program CNS (13) using the structure of Toho-1 E166A (5) as a search model. The refinement and model building were performed with programs CNS and O (14). The chemical topologies of the acylated form of substrates were estimated from the structures covalently bound to a PBP from Streptomyces sp. R61 (15), Staphylococcus aureus ␤-lactamase (16), and TEM-1 (17). All of the residues with the exception of one N-terminal residue in the benzylpenicillin-intermediate and two N-terminal residues in the cephalosporin-intermediates were included in the final models. Data collection and refinement statistics are shown in Table I.  (16,17). In a class A ␤-lactamase from Bacillus licheniformis, the structure of E166A is known to display few differences with the wild type enzyme (19).

Acyl-intermediate Structures of Toho-1-␤-Lactamases
The acyl-intermediate structures of the mutant E166A with cefotaxime, cephalothin, and benzylpenicillin were determined at 1.8-, 2.0-, and 2.1-Å resolutions, respectively ( Table I) Fig. 1, A-C, with 2F o Ϫ F c electron density maps around the bound substrates. In the cephalosporin-intermediate structures (Fig.  1, A and B), the C3Ј-leaving group has been removed as observed in other acyl-enzyme structures in complex with cephalosporins (15, 16, 20 -22). In the cefotaxime-intermediate structure, residues Pro 167 , Asn 170 , Ser 237 , Asp 240 , and Arg 274 surround the bulky side chain of cefotaxime. In addition, both side chain oxygens of Asp 240 interact with the amino group in the aminothiazole ring, which may be involved in the binding of cefotaxime (Fig. 1A). The thiophene substituent of cephalothin in the cephalothin-intermediate and the benzyl group of benzylpenicillin in the benzylpenicillin-intermediate are seen in a nearly identical position with the aminothiazole ring and the methoxyimino group of cefotaxime, respectively (Fig. 1, B and C).
Differences among Three Acyl-intermediate and Unbound Toho-1 Structures-Substrate binding does not induce conformational changes in the ⍀-loop and the ␤-strand B3 but rather causes a structural rearrangement of residues Arg 274 , Ser 237 , and Asn 104 (Fig. 2, A and B). Arg 274 is a unique residue in Toho-1 not found in any other CTX-M-type ESBLs (Fig. 3). In the unbound Toho-1 structure, Arg 274 is obstructing the substrate binding pocket, but upon substrate binding, Arg 274 is forced out of the active site by the side chain of the substrate ( Fig. 2A).
A comparison of the acyl-intermediate structures with the unbound Toho-1 structure shows the side chain of Ser 237 in two where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. c R free ϭ ⌺͉FoϪFc͉/⌺͉Fo͉, calculated using a test data set, 5% of total data randomly selected from the observed reflections.
different conformations ( Fig. 2A). In the cephalothin-intermediate structure, Ser 237 has rotated ϳ30°, whereas in the cefotaxime-intermediate and benzylpenicillin-intermediate, there  is an ϳ150°rotation of the side chain. This rotation prevents steric clashes with the methoxyimino (cefotaxime) and the methyl group (benzylpenicillin) of the substrate. Asn 104 is positioned at a bend in the binding site formed by Val 103 -Asn 104 -Tyr 105 -Asn 106 (Fig. 2B). This VNYN sequence is conserved in the CTX-M-type ESBLs, whereas a VXYS sequence is most common in the non-ESBLs in this region (Fig. 3). In the unbound structure, the side chain of Asn 106 makes hydrogen bonds with the backbone groups of Val 103 to maintain this bent conformation. However, in the acyl-intermediate structures, the N ␦ of Asn 106 changes its hydrogen bond acceptor from backbone oxygen of Val 103 to that of Asn 106 by causing a slight rotation of the peptide bond between Asn 104 and Tyr 105 , which forces Val 103 and Asn 106 to be held in unfavorable conformations. This exchange is induced by the side chain movement of Asn 104 to interact with the substrate. The movement of Asn 104 also induces the movement of Asn 132 whose side chain interacts with Asn 104 and the substrate (Fig. 2B).
The main differences between the benzylpenicillin-intermediate and the cephalosporin-intermediates are observed around Ser 70 and the oxyanion hole. In the benzylpenicillin-intermediate, the carbonyl group of the ␤-lactam ring does not position suitably for the hydrolysis in the oxyanion hole (Fig. 2C). This is caused by van der Waals contact between the methyl group of the thiazolidine ring, and the C ␤ and the O ␥ of Ser 237 . An oxyanion hole formed by the backbone amides of Ser 70 and residue 237 interacts with the carbonyl oxygen of the ␤-lactam ring in the substrate (1). These interactions play essential roles for the hydrolysis by stabilizing the negative charge appeared on the ␤-lactam carbonyl oxygen when the tetrahedral intermediates are formed during both acylation and deacylation. These interactions also help to polarize the carbonyl group of the ␤-lactam ring, thus favoring the nucleophilic attack to the carbonyl carbon by Ser 70 in acylation and by the hydrolytic water in deacylation. In the cephalosporin-intermediate structures of Toho-1, the carbonyl oxygens of the ␤-lactam ring are located at similar distances from both backbone nitrogens (2.6 -2.9 Å) to those in other class A ␤-lactamase acyl-intermediate structures with substrate (2.7-3.0 Å) (16,17,20). In the benzylpenicillin-intermediate structure, however, the carbonyl oxygen exists at a distance of 2.6 Å from the backbone nitrogen of Ser 70 , but 3.2 Å from the backbone nitrogen of Ser 237 , meaning that the polarization of the carbonyl group of benzylpenicillin is less efficient for deacylation than that of cephalosporins. This finding indicates that the lower penicillinase activity of CTX-M-type ESBLs compared with non-ESBLs is due to the conserved residue Ser 237 (Fig. 3). Actually, in CTX-M-4 and a chromosomal ␤-lactamase from P. vulgaris K1 sharing high sequence homologies with CTX-M-type ESBLs, the S237A mutants exhibited increased k cat /K m Ϫ value toward benzylpenicillin (23,24 (Figs. 1, A-C, and 4, black dotted lines), but there are some key differences that can explain their specificities. First, Toho-1 lacks two hydrogen bonds formed by Arg 244 in non-ESBLs, because CTX-M-type ESBLs do not contain Arg 244 present in most non-ESBLs (Fig. 3). In the acyl-intermediate structures of non-ESBLs, Arg 244 interacts with the carboxylate oxygen of the substrate and the backbone oxygen of Gly 236 (Fig. 4). Sitedirected mutagenesis analyses indicate that Arg 244 is critical for the catalysis of TEM-1 (25,26). In Toho-1, Arg 276 was predicted to be a substitute for Arg 244 (4); however, this residue has no interactions with the substrates (Figs. 1, A-C, and 2A). Instead, in Toho-1, Ser 237 forms a hydrogen bond with the carboxylate oxygen of the substrate (Fig. 4). This unique interaction induces the rotation of the carboxylate group in the acylintermediate structures of Toho-1 compared with those of non-ESBLs. The residue Ser 237 is conserved in all CTX-M-type ESBLs (Fig. 3), suggesting that this interaction plays a key role in the hydrolysis of oxyimino-cephalosporins. Interestingly, in the cefotaxime-intermediate structure of a PBP from Streptomyces sp. R61, Thr 301 , the corresponding residue to Ser 237 of Toho-1, interacts with the amide group of acylamide side chain in cefotaxime to stabilize substrate binding, whereas in the cephalothin-intermediate structure (15), Thr 301 interacts with the carboxylate oxygen as is seen in Toho-1 acyl-intermediate structures. This difference is brought about by steric hindrance between the bulky side chain of the substrates and the active site residues causing a tilt in cefotaxime binding compared with that of cephalothin.
Other differences are observed on the interactions with the acylamide side chain of the substrate in Toho-1. In the acylintermediate structures of class A ␤-lactamases, the acylamide side chain of the substrate interacts with the N ␦ of Asn 132 and the backbone oxygen of residue 237 (Fig. 4). The residue Asn 132 is the third residue of the conserved Ser 130 -Asp 131 -Asn 132 motif in class A ␤-lactamases, and the hydrogen bond between the N ␦ of Asn 132 and the carbonyl oxygen of the substrate is proposed to be important for the recognition of cephalosporins (27). The residue 237 exists just after the conserved Lys 234 -Thr 235 -Gly 236 motif on the ␤-strand B3 in class A ␤-lactamases. These residues are found at slightly different positions among these three acyl-intermediate structures in Fig. 4. The differences between two non-ESBLs caused by the narrower binding cavity of TEM-1 are thought to produce the divergence of substrate specificities between these non-ESBLs (28). Considering that the oxyimino group and the aminothiazole ring are introduced in the acylamide side chain of oxyimino-cephalosporins, the different positions of Asn 132 and Ser 237 would contribute to the oxyimino-cephalosporinase activity of Toho-1. In non-ESBLs, the position of Asn 132 is fixed by the interactions between Asp 131 and Thr/Ser 109 as well as the interaction between the N ␦ of Asn 132 and the backbone oxygen of residue 104 (Fig. 4) (28,29). However, CTX-M-type ESBLs lacks these interactions because these enzymes have an alanine at position 109 (Fig. 3), and the peptide bond between residues 104 and 105 is flipped compared with non-ESBLs (Fig. 4). Instead, the O ␦ of Asn 104 interacts with the N ␦ of Asn 132 in Toho-1. This unique peptide bond conformation in the vicinity of Asn 104 is attained by the conserved VNYN sequence in Toho-1 as mentioned above. As for residue 237, the position of this residue is affected by van der Waals contact between the ␤-strand B3 and the side chain of residue 69 in class A ␤-lactamases (28,30). Although residue 69 is variable in class A ␤-lactamases, a cysteine is conserved at this position in CTX-M-type ESBLs (Fig. 3), which may place Ser 237 at a unique position. The lack of an interaction between Gly 236 and Arg 244 present in non-ESBLs and the flexibility of the ␤-strand B3 as mentioned previously (5) also affect the position of Ser 237 in Toho-1. In fact, in Toho-1, the distance between the backbone amide groups of Ser 70 and Ser 237 constructing an oxyanion hole (4.3 Å) is shorter than that in TEM-1 (4.9 Å) (28) and the average in class A ␤-lactamases (4.7 Å) (30).
Moreover, in Toho-1, a unique hydrogen bond is formed between the N ␦ of Asn 104 and the carbonyl oxygen of the acylamide side chain of the substrate in addition to the hydrogen bonds seen in non-ESBLs (Fig. 4). Furthermore, in the cefotaxime-intermediate structure, Asp 240 interacts with the amino group in the aminothiazole ring of the acylamide side chain (Fig. 4). Thus, Toho-1 can fix the bulky side chain of cefotaxime more tightly in the binding site. Asn 104 and Asp 240 are conserved in CTX-M-type ESBLs (Fig. 3), indicating that these interactions promote oxyimino-cephalosporinase activity.
Role of the ⍀-loop-Non-ESBLs have a low oxyimino-cephalosporinase activity (1). It is thought that steric hindrance between the bulky side chains of oxyimino-cephalosporins and the ⍀-loop of non-ESBLs reduces substrate affinity and consequently decreases activity (2,(31)(32)(33)(34). Models of the TEM-1 Michaelis-Menten complex and the acyl-enzyme complex with oxyimino-cephalosporins predicted unfavorable steric interactions between the bulky side chain of the substrate and residues Pro 167 and Asn 170 on the ⍀-loop (31,32). Thereby, TEM-1 exhibits a very high K m value (6 mM) against cefotaxime (31). TEM-derived ESBLs such as TEM-7, TEM-11, and TEM-64 have decreased K m values to attain higher k cat /K m Ϫ values against oxyimino-cephalosporins (7, 31). These TEM derivatives lose important salt bridges that stabilize the ⍀-loop via a mutation on the loop, which may perturb the ⍀-loop and remove the steric hindrance. This improves the affinities and decreases K m values against oxyimino-cephalosporins. The structure of TEM-64 has revealed that the R164S mutation allows Asn 170 to flip almost 180°, which unwinds a short helix in the region and leaves a large cavity in the binding site. A type II ESBL PER-1 also has changed the conformation of the ⍀-loop to enlarge the binding site by the unique sequence of the loop (8). Furthermore, it has been shown that an ⍀-loop deletion mutant of the ␤-lactamase from S. aureus can bind oxyimino-cephalosporins (34).
A comparison between non-ESBLs and Toho-1 shows the ⍀-loop in a slightly different position (Fig. 5A). In contrast to the other ESBLs mentioned above, the ⍀-loop in Toho-1 has shifted to the helix H5. As a result, the positions of Pro 167 and Asn 170 are ϳ1 Å closer to the active site of Ser 70 in Toho-1 than TEM-1. This shift shortens the depth of the binding site, and thereby, the binding position of the aminothiazole ring of cefotaxime is more exposed to solvents. In Fig. 5A, the aminothiazole ring of cefotaxime is positioned within van der Waals distances from the ⍀-loops of non-ESBLs. For example, in TEM-1, the sulfur atom in the aminothiazole ring is located at distances of 3.0 Å from the backbone oxygen and the C ␤ of Pro 167 . In contrast, in Toho-1, the closest distance is observed between a carbon atom in the aminothiazole ring of cefotaxime and the backbone oxygen of Pro 167 (3.3 Å) (Fig. 1A). This means that the shift of the ⍀-loop in Toho-1 keeps the loop away from the aminothiazole ring of cefotaxime and prevents the unfavorable contacts. Thus, Toho-1 has high affinities for oxyiminocephalosporins. We believe that the different position of the ⍀-loop is the main reason why Toho-1 can hydrolyze oxyiminocephalosporins and non-ESBLs cannot.
We investigated the probable causes of this shift of the ⍀-loop by comparing Toho-1 with TEM-1. Van der Waals contacts between Trp 165 on the ⍀-loop and residues Leu 139 , Thr 140 , and Pro 145 on the ␣-helix H5 restrict the position of the ⍀-loop in TEM-1 (Fig. 5B). In Toho-1, these restrictions are removed by replacing Trp 165 with a threonine (Fig. 3). Therefore, the ⍀-loop of Toho-1 is positioned closer to the ␣-helix H5 relative to TEM-1 (Fig. 5, A and B). However, the fact that a W165S mutant of TEM-1 does not hydrolyze cefotaxime (35) indicates the likelihood of multiple factors influencing the unfavorable disposition of the ⍀-loop in non-ESBLs.
In TEM-1, Leu 162 and Leu 169 on the ⍀-loop form van der Waals contacts with Phe 72 on the ␣-helix H2 in a hydrophobic core (Fig. 5B). However, Toho-1 has a serine at position 72 (Fig.  3) and lacks these van der Waals contacts. This residue substitution permits the ⍀-loop of Toho-1 to approach the ␣-helix H2. A decrease in hydrophobicity in this core region along with the replacement of a large hydrophobic side chain at position 72 is compensated for by Met 135 and Phe 160 in Toho-1 (Fig. 5B). These interpretations are supported by the fact that Trp 165 and Phe 72 are highly conserved in non-ESBLs, whereas Thr 165 , Ser 72 , Met 135 , and Phe 160 are conserved in CTX-M-type ESBLs (Fig. 3). Further support is obtained by comparing a ␤-lacta- mase from Yersinia enterocolitica. This ␤-lactamase has a high sequence homology with Toho-1 (36) but still does not hydrolyze oxyimino-cephalosporins because of the existence of the residues Phe 72 and Trp 165 as in non-ESBLs (Fig. 3).
CTX-M-type ESBLs share high sequence similarity with the chromosomal ␤-lactamases from Klebsiella oxytoca, Citrobacter diversus, and P. vulgaris (Fig. 3). However, CTX-M-type ESBLs exhibit more efficient oxyimino-cephalosporinase activity than these chromosomal ESBLs (1). Recently, the structure of a chromosomal ESBL from P. vulgaris K1 was determined (9). Interestingly, the position of the ⍀-loop in this ESBL shows higher similarity to those of non-ESBLs than that of Toho-1 (Fig. 5A), indicating that the affinities for oxyimino-cephalosporins are lower than Toho-1 in this ESBL. Actually, the K m value for cefuroxime, an oxyimino-cephalosporin, was ϳ8ϫ larger than that for cephalothin in a ␤-lactamase from P. vulgaris K1 (24), whereas the K m values for cefotaxime and cephalothin are almost identical in Toho-1 (4). Thus, together with the lack of Asn 104 (Fig. 3), the unfavorable position of the ⍀-loop for the binding of oxyimino-cephalosporins could be one of the reasons for the lower activity in a ␤-lactamase from P. vulgaris K1 than CTX-M-type ESBLs. More specifically, this unfavorable position of the ⍀-loop may be attributed to the different conformations of the ␤-strand B3 between these enzymes. In class A ␤-lactamases, the ␤-strand B3 has contacts with residue 69 and the ⍀-loop (30). In a ␤-lactamase from P. vulgaris K1, the shorter side chain of Ala 69 compared with that of Cys 69 in Toho-1 (Fig. 3) allows the ␤-strand B3 to extend The unbound Toho-1 (5) and a ␤-lactamase from P. vulgaris K1 (9) are colored yellow and cyan, respectively, whereas the other non-ESBLs are colored gray (TEM-1 (28), SHV-1 (43), a ␤-lactamase from S. aureus (44), a ␤-lactamase from S. albus G (Protein Data Bank code 1BSG), and that from B. licheniformis (45)). The acylated cefotaxime is colored pink. B, the overlay of Toho-1 (5) (orange) and TEM-1 (28) (gray) around the ⍀-loop and the hydrophobic core. Van der Waals interactions in TEM-1 are shown as green dotted lines. S/F72 denotes that Toho-1 has a serine and TEM-1 has a phenylalanine at position 72, respectively. inward toward the binding site (Fig. 5A), which may hinder the shift of the ⍀-loop as observed in Toho-1. In addition, the interaction between Arg 274 and Gly 238 in Toho-1 may also contribute to the difference of the ␤-strand conformation, although this is lost in the cephalothin-intermediate structure ( Fig. 2A). This interpretation is not applicable to chromosomal ESBLs from K. oxytoca and C. diversus, because they possess Cys 69 (Fig. 3). The lack of Asn 104 and/or Ser 237 may be the main cause of lower oxyimino-cephalosporinase activity in these enzymes.
Mechanism for the Extended-spectrum of Toho-1-In addition to Toho-1, there have been several oxyimino-cephalosporin-hydrolyzing ␤-lactamase structures solved (TEM-52 (6), TEM-64 (7), TEM-1 G238A mutant (7), PER-1 (8), a ␤-lactamase from P. vulgaris K1 (9), NMC-A (10), and a class C ␤-lactamase from E. cloacae GC1 (11)). Compared with the former ␤-lactamases, these enzymes have sufficient space to accommodate the bulky side chain of oxyimino-cephalosporins. TEM-64 and PER-1 have each changed the conformation of the ⍀-loop to generate broad binding cavities as mentioned above (7,8). TEM-52 is an E104K/M182T/G238S mutant of TEM-1 (6). In TEM-52, Ser 238 forms two new hydrogen bonds with the main and side chains of Ser 243 that alter the conformation of loop 238 -243 and widen the active site cavity. In TEM-1 G238A mutant, the steric contact between the side chain of Ala 238 and the backbone oxygen of Asn 170 enlarges the active site (7). In NMC-A, the unique disulfide bond between Cys 69 and Cys 238 induces distortion in the ␤-strand B3, which increases the space between residue 170 and this strand (10). GC1 ␤-lactamase has a three-residue insertion in the ⍀-loop, which produces the wider binding cavity (11). The complex structure of class C ␤-lactamase AmpC with ceftazidime (21) proposes the importance of this insertion for the improved activity of GC1. In the case of a ␤-lactamase from P. vulgaris K1, such conformational changes are not reported, but replacements of glutamates at positions 104 and 240 with shorter alanine and aspartate, respectively, are thought to be more accommodating to aztreonam and ceftazidime with negatively charged oxyimino groups (9).
In Toho-1, the shift of the ⍀-loop avoids the steric interactions with the bulky side chain of oxyimino-cephalosporins. Interestingly, this shift narrows the binding site but creates space for the bulky side chain to bind without bad contacts. This shift is attained by a structural rearrangement in the hydrophobic core in the vicinity of the ⍀-loop. In addition, Asn 104 and Asp 240 interact with the bulky acylamide side chain of oxyimino-cephalosporins, which assist in the binding of the substrate. The characteristic arrangements of Asn 132 and Ser 237 in Toho-1 may also aid the binding of a bulky acylamide side chain. Thus, Toho-1 has improved affinities for oxyiminocephalosporins. Many ESBLs have improved affinities for oxyimino-cephalosporins by changing the conformation of the ⍀-loop as mentioned above (2,7,8,31). However, their k cat values against oxyimino-cephalosporins remain low (7,31,37), because the hydrolytic water is positioned and activated by Glu 166 and Asn 170 on the ⍀-loop in class A ␤-lactamases. In contrast, Toho-1 retains high k cat values (4), because the shift of the ⍀-loop does not induce the conformational change observed in other ESBLs. Moreover, Ser 237 probably contributes to the high k cat values against oxyimino-cephalosporins. Ser 237 forms a unique hydrogen bond with the carboxylate oxygen of the substrate. Ser 237 is conserved in all CTX-M-type ESBLs and in some of the chromosomal ESBLs. The S237A mutant of CTX-M-4 decreased the relative hydrolytic efficiencies against oxyimino-cephalosporins (23). The S237A mutant of a chromosomal ESBL from P. vulgaris K1 decreased k cat and k cat /K m Ϫ values not only toward cephalothin but also toward cefuroxime (24). These results indicate that the interaction formed by Ser 237 is important for the acylation of cephalosporins in CTX-M-type ESBLs. Penicillins and cephalosporins differ in the relative positions of the carboxylate group against the ␤-lactam ring, and the active site residues in class A ␤-lactamases are thought to be ideally located for hydrolyzing penicillins but not cephalosporins (38). Considering the acyl-intermediate structures of Toho-1 and the kinetic analyses of the mutants of the related enzymes, the unique interactions formed by Ser 237 in Toho-1 would assist in bringing the carbonyl group of the ␤-lactam ring of cephalosporins to the optimal position in the oxyanion hole for the acylation. However, in the case of the hydrolysis of penicillins, this interaction seems to produce undesirable contacts with the substrate, which prevent the proper positioning of the carbonyl group of the ␤-lactam ring in the oxyanion hole. Similarly, the introduction of A237T mutation into TEM-1 or TEM-derived ESBLs increased cephalosporinase activities (2,39,40). In contrast, the cefotaxime-resistant PBP2x from Streptococcus pneumoniae has a threonine to an alanine replacement at the corresponding position to 237 in Toho-1, which caused the 90% reduction of the acylation for cefotaxime, whereas the acylation by benzylpenicillin was not affected (41).
The acyl-intermediate structures of Toho-1 reveal that the mechanism of the extended-spectrum of CTX-M-type ESBLs is characterized by the increased affinity for oxyimino-cephalosporins and the efficient acylation of cephalosporins. These features are accomplished through interactions with the conserved residues Cys 69 , Ser 72 , Asn 104 , Asn 106 , Met 135 , Thr 165 , Phe 160 , Ser 237 , and Asp 240 , and it is these residues that discriminate CTX-M-type ESBLs from other class A ␤-lactamases.