Acyl-intermediate Structures of the Extended-spectrum Class A β-Lactamase, Toho-1, in Complex with Cefotaxime, Cephalothin, and Benzylpenicillin
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m207884200
ISSN1083-351X
AutoresTatsuro Shimamura, Akiko Shimizu‐Ibuka, Shinya Fushinobu, Takayoshi Wakagi, Masaji Ishiguro, Yoshikazu Ishii, Hiroshi Matsuzawa,
Tópico(s)Pneumonia and Respiratory Infections
ResumoBacterial resistance to β-lactam antibiotics is a serious problem limiting current clinical therapy. The most common form of resistance is the production of β-lactamases that inactivate β-lactam antibiotics. Toho-1 is an extended-spectrum β-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 β-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 Ω-loop of Toho-1 is displaced to avoid the steric contacts with the bulky side chain of cefotaxime. Second, the conserved residues Asn104 and Asp240 form unique interactions with the bulky side chain of cefotaxime to fix it tightly. Finally, the unique interaction between the conserved Ser237 and cephalosporins probably helps to bring the β-lactam carbonyl group to the suitable position in the oxyanion hole, thus increasing the cephalosporinase activity. Bacterial resistance to β-lactam antibiotics is a serious problem limiting current clinical therapy. The most common form of resistance is the production of β-lactamases that inactivate β-lactam antibiotics. Toho-1 is an extended-spectrum β-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 β-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 Ω-loop of Toho-1 is displaced to avoid the steric contacts with the bulky side chain of cefotaxime. Second, the conserved residues Asn104 and Asp240 form unique interactions with the bulky side chain of cefotaxime to fix it tightly. Finally, the unique interaction between the conserved Ser237 and cephalosporins probably helps to bring the β-lactam carbonyl group to the suitable position in the oxyanion hole, thus increasing the cephalosporinase activity. β-Lactam antibiotics are effectively used against a wide range of bacterial infectious diseases (1Matagne A. Lamotte-Brasseur J. Frère J.M. Biochem. J. 1998; 330: 581-598Crossref PubMed Scopus (321) Google Scholar). The antibiotics form stable acyl-enzymes with penicillin-binding proteins (PBPs) 1The abbreviations used for: PBP, penicillin-binding protein; ESBL, extended spectrum β-lactamase. 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 β-lactam antibiotics mainly by producing β-lactamases that hydrolyze the antibiotics. β-Lactamases are acylated by β-lactam antibiotics in a similar manner as PBPs; however, they are rapidly deacylated. β-Lactamases are classified into four groups (classes A, B, C, and D) according to their amino acid sequences and substrate profiles (1Matagne A. Lamotte-Brasseur J. Frère J.M. Biochem. J. 1998; 330: 581-598Crossref PubMed Scopus (321) Google Scholar). Classes A, C, and D β-lactamases are serine β-lactamases, whereas class B β-lactamases are zinc-containing β-lactamases. Among these four classes of β-lactamases, class A β-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 β-lactamases such as TEM-1 and SHV-1 (1Matagne A. Lamotte-Brasseur J. Frère J.M. Biochem. J. 1998; 330: 581-598Crossref PubMed Scopus (321) Google Scholar, 2Knox J.R. Antimicrob. Agents Chemother. 1995; 39: 2593-2601Crossref PubMed Scopus (301) Google Scholar). 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 β-lactamases capable of hydrolyzing oxyimino-cephalosporins called extended- spectrum β-lactamases (ESBLs) (1Matagne A. Lamotte-Brasseur J. Frère J.M. Biochem. J. 1998; 330: 581-598Crossref PubMed Scopus (321) Google Scholar, 2Knox J.R. Antimicrob. Agents Chemother. 1995; 39: 2593-2601Crossref PubMed Scopus (301) Google Scholar). 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 (2Knox J.R. Antimicrob. Agents Chemother. 1995; 39: 2593-2601Crossref PubMed Scopus (301) Google Scholar). 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 β-lactamases, CTX-M-type ESBLs have an efficient hydrolytic activity toward oxyimino-cephalosporins but exhibit lower activity toward penicillins than non-ESBLs (3Tzouvelekis L.S. Tzelepi E. Tassios P.T. Legakis N.J. Int. J. Antimicrob. Agents. 2000; 14: 137-142Crossref PubMed Scopus (246) Google Scholar). Toho-1 belongs to the CTX-M-type ESBLs (4Ishii Y. Ohno A. Taguchi H. Imajo S. Ishiguro M. Matsuzawa H. Antimicrob. Agents Chemother. 1995; 39: 2269-2275Crossref PubMed Scopus (185) Google Scholar). We have previously reported the structure of the Toho-1 mutant E166A whose overall fold was shown to be similar to non-ESBLs (5Ibuka A. Taguchi A. Ishiguro M. Fushinobu S. Ishii Y. Kamitori S. Okuyama K. Yamaguchi K. Konno M. Matsuzawa H. J. Mol. Biol. 1999; 285: 2079-2087Crossref PubMed Scopus (86) Google Scholar). Toho-1 has some variations in hydrogen-bonding patterns and an increase in flexibility of the β-strand B3 as well as the Ω-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 β-lactamases solved (TEM-52 (6Orencia M.C. Yoon J.S. Ness J.E. Stemmer W.P.C. Stevens R.C. Nat. Struct. Biol. 2001; 8: 238-242Crossref PubMed Scopus (194) Google Scholar), TEM-64 (7Wang X. Minasov G. Shoichet B.K. J. Mol. Biol. 2002; 320: 85-95Crossref PubMed Scopus (370) Google Scholar), TEM-1 G238A mutant (7Wang X. Minasov G. Shoichet B.K. J. Mol. Biol. 2002; 320: 85-95Crossref PubMed Scopus (370) Google Scholar), PER-1 (8Tranier S. Bouthors A.T. Maveyraud L. Guillet V. Sougakoff W. Samama J.P. J. Biol. Chem. 2000; 275: 28075-28082Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), a β-lactamase from Proteus vulgaris K1 (9Nukaga M. Mayama K. Crichlow G.V. Knox J.R. J. Mol. Biol. 2002; 317: 109-117Crossref PubMed Scopus (18) Google Scholar), NMC-A (10Swaren P. Maveyraud L. Raquet X. Cabantous S. Duez C. Pedelacq J.D. Mariotte-Boyer S. Mourey L. Labia R. Nicolas-Chanoine M.H. Nordmann P. Frère J.M. Samama J.P. J. Biol. Chem. 1998; 273: 26714-26721Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), and a class C β-lactamase from Enterobacter cloacae GC1 (11Crichlow G.V. Kuzin A.P. Nukaga M. Mayama K. Sawai T. Knox J.R. Biochemistry. 1999; 38: 10256-10261Crossref PubMed Scopus (93) Google Scholar)), but none of these β-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. Toho-1 E166A mutant was expressed and purified as described previously (5Ibuka A. Taguchi A. Ishiguro M. Fushinobu S. Ishii Y. Kamitori S. Okuyama K. Yamaguchi K. Konno M. Matsuzawa H. J. Mol. Biol. 1999; 285: 2079-2087Crossref PubMed Scopus (86) Google Scholar). 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). Images were processed with Denzo and Scalepack (12Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). The soaked crystals were isomorphous with the unbound crystal. Phases were calculated by molecular replacement with the program CNS (13Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) using the structure of Toho-1 E166A (5Ibuka A. Taguchi A. Ishiguro M. Fushinobu S. Ishii Y. Kamitori S. Okuyama K. Yamaguchi K. Konno M. Matsuzawa H. J. Mol. Biol. 1999; 285: 2079-2087Crossref PubMed Scopus (86) Google Scholar) as a search model. The refinement and model building were performed with programs CNS and O (14Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). The chemical topologies of the acylated form of substrates were estimated from the structures covalently bound to a PBP from Streptomyces sp. R61 (15Kuzin A.P. Liu H. Kelly J.A. Knox J.R. Biochemistry. 1995; 34: 9532-9540Crossref PubMed Scopus (106) Google Scholar),Staphylococcus aureus β-lactamase (16Chen C.C.H. Herzberg O. Biochemistry. 2001; 40: 2351-2358Crossref PubMed Scopus (36) Google Scholar), and TEM-1 (17Strynadka N.C.J. Adachi H. Jensen S.E. Johns K. Sielecki A. Betzel C. Sutoh K. James M.N.G. Nature. 1992; 359: 700-705Crossref PubMed Scopus (533) Google Scholar). 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.Table IData collection and refinement statisticsSubstrateCefotaximeCephalothinBenzylpenicillinData collectionWavelength (Å)1.01.01.0Resolution (Å)1.81.81.8Measured reflections96,844117,11070,280Unique reflections26,98528,50726,625Completeness (%)95.296.992.3R mergeaRmerge = ΣΣi‖I(h)i − 〈I(h)〉‖/ΣΣi‖I(h)i‖, where 〈I(h)〉 is the mean intensity of equivalent reflections.(%)7.35.012.8Space groupP3221P3221P3221Unit cell (Å)a = b= 72.6a = b = 73.2a =b = 72.8c = 98.2c = 100.2c = 99.5α = β = 90°α = β = 90°α = β = 90°γ = 120°γ = 120°γ = 120°RefinementResolution (Å)40–1.840–2.040–2.1No. of protein residues260260261No. of water molecules216138155No. of sulfate ions333R bR = Σ‖Fo−Fc‖/Σ‖Fo‖, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. (%)17.819.919.2R freecRfree = Σ‖Fo−Fc‖/Σ‖Fo‖, calculated using a test data set, 5% of total data randomly selected from the observed reflections. (%)20.624.522.7Average B factor (Å2)Protein12.833.633.4Substrate17.236.327.6Solvent23.539.839.1r.m.s. deviationsBond lengths (Å)0.0050.0070.007Bond angles (°)1.331.371.40a Rmerge = ΣΣi‖I(h)i − 〈I(h)〉‖/ΣΣi‖I(h)i‖, where 〈I(h)〉 is the mean intensity of equivalent reflections.b R = Σ‖Fo−Fc‖/Σ‖Fo‖, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively.c Rfree = Σ‖Fo−Fc‖/Σ‖Fo‖, calculated using a test data set, 5% of total data randomly selected from the observed reflections. Open table in a new tab β-Lactamases hydrolyze β-lactam antibiotics so rapidly that it is difficult to trap the acyl-intermediate. In class A β-lactamases, the conserved residue Glu166 positions and activates the hydrolytic water for the deacylation (1Matagne A. Lamotte-Brasseur J. Frère J.M. Biochem. J. 1998; 330: 581-598Crossref PubMed Scopus (321) Google Scholar, 18Adachi H. Ohta T. Matsuzawa H. J. Biol. Chem. 1991; 266: 3186-3191Abstract Full Text PDF PubMed Google Scholar). Therefore, we used a Glu166mutant of Toho-1 to trap the acyl-intermediate in a similar manner as was done for the acyl-intermediate structures of non-ESBLs (16Chen C.C.H. Herzberg O. Biochemistry. 2001; 40: 2351-2358Crossref PubMed Scopus (36) Google Scholar, 17Strynadka N.C.J. Adachi H. Jensen S.E. Johns K. Sielecki A. Betzel C. Sutoh K. James M.N.G. Nature. 1992; 359: 700-705Crossref PubMed Scopus (533) Google Scholar). In a class A β-lactamase from Bacillus licheniformis, the structure of E166A is known to display few differences with the wild type enzyme (19Knox J.R. Moews P.C. Escobar W.A. Fink A.L. Protein Eng. 1993; 6: 11-18Crossref PubMed Scopus (59) Google Scholar). 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 (TableI). The overall fold of these acyl-intermediate structures is the same as the previously reported unbound Toho-1 E166A (5Ibuka A. Taguchi A. Ishiguro M. Fushinobu S. Ishii Y. Kamitori S. Okuyama K. Yamaguchi K. Konno M. Matsuzawa H. J. Mol. Biol. 1999; 285: 2079-2087Crossref PubMed Scopus (86) Google Scholar) with root mean square deviations among the Cα positions of 0.25, 0.21, and 0.25 Å for the cefotaxime-, cephalothin- and benzylpenicillin-intermediate structures, respectively. The active sites of these acyl-intermediate structures are shown in Fig. 1, A–C, with 2F o − F c electron density maps around the bound substrates. In the cephalosporin-intermediate structures (Fig. 1, A andB), the C3′-leaving group has been removed as observed in other acyl-enzyme structures in complex with cephalosporins (15Kuzin A.P. Liu H. Kelly J.A. Knox J.R. Biochemistry. 1995; 34: 9532-9540Crossref PubMed Scopus (106) Google Scholar, 16Chen C.C.H. Herzberg O. Biochemistry. 2001; 40: 2351-2358Crossref PubMed Scopus (36) Google Scholar,20Fonze E. Vanhove M. Dive G. Sauvage E. Frère J.M. Charlier P. Biochemistry. 2002; 41: 1877-1885Crossref PubMed Scopus (51) Google Scholar, 21Powers R.A. Caselli E. Focia P.J. Prati F. Shoichet B.K. Biochemistry. 2001; 40: 9207-9214Crossref PubMed Scopus (104) Google Scholar, 22Beadle B.M. Trehan I. Focia P.J. Shoichet B.K. Structure. 2002; 10: 413-424Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In the cefotaxime-intermediate structure, residues Pro167, Asn170, Ser237, Asp240, and Arg274 surround the bulky side chain of cefotaxime. In addition, both side chain oxygens of Asp240 interact with the amino group in the aminothiazole ring, which may be involved in the binding of cefotaxime (Fig.1 A). 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). Substrate binding does not induce conformational changes in the Ω-loop and the β-strand B3 but rather causes a structural rearrangement of residues Arg274, Ser237, and Asn104 (Fig.2, A and B). Arg274 is a unique residue in Toho-1 not found in any other CTX-M-type ESBLs (Fig. 3). In the unbound Toho-1 structure, Arg274 is obstructing the substrate binding pocket, but upon substrate binding, Arg274 is forced out of the active site by the side chain of the substrate (Fig.2 A).Figure 3Sequence alignments of class A β-lactamases. The eight β-lactamases above the black line are ESBLs, and the others are non-ESBLs. The CTX-M-type and the chromosomal ESBLs are colored blue andgreen, respectively. The three conserved regions and Glu166 are highlighted in red. The residues mentioned in the text are highlighted in green. Arrows above the sequences indicate the Ω-loop and the β-strand B3. KLEOX, the β-lactamase from K. oxytoca (4Ishii Y. Ohno A. Taguchi H. Imajo S. Ishiguro M. Matsuzawa H. Antimicrob. Agents Chemother. 1995; 39: 2269-2275Crossref PubMed Scopus (185) Google Scholar); CITDI, β-lactamase from C. diversus (4Ishii Y. Ohno A. Taguchi H. Imajo S. Ishiguro M. Matsuzawa H. Antimicrob. Agents Chemother. 1995; 39: 2269-2275Crossref PubMed Scopus (185) Google Scholar); PROVU, β-lactamase from P. vulgaris (4Ishii Y. Ohno A. Taguchi H. Imajo S. Ishiguro M. Matsuzawa H. Antimicrob. Agents Chemother. 1995; 39: 2269-2275Crossref PubMed Scopus (185) Google Scholar); PC-1, β-lactamase from S. aureus PC-1 (4Ishii Y. Ohno A. Taguchi H. Imajo S. Ishiguro M. Matsuzawa H. Antimicrob. Agents Chemother. 1995; 39: 2269-2275Crossref PubMed Scopus (185) Google Scholar); STRAL, β-lactamase fromStreptomyces albus G (4Ishii Y. Ohno A. Taguchi H. Imajo S. Ishiguro M. Matsuzawa H. Antimicrob. Agents Chemother. 1995; 39: 2269-2275Crossref PubMed Scopus (185) Google Scholar); BLICH, β-lactamase from B. licheniformis (4Ishii Y. Ohno A. Taguchi H. Imajo S. Ishiguro M. Matsuzawa H. Antimicrob. Agents Chemother. 1995; 39: 2269-2275Crossref PubMed Scopus (185) Google Scholar); YEREN, β-lactamase from Y. enterocolitica (36Seoane A. Garcia Lobo J.M. J. Gen. Microbiol. 1991; 137: 141-146Crossref PubMed Scopus (20) Google Scholar).View Large Image Figure ViewerDownload (PPT) A comparison of the acyl-intermediate structures with the unbound Toho-1 structure shows the side chain of Ser237 in two different conformations (Fig. 2 A). In the cephalothin-intermediate structure, Ser237 has rotated ∼30°, whereas in the cefotaxime-intermediate and benzylpenicillin-intermediate, there is an ∼150° rotation of the side chain. This rotation prevents steric clashes with the methoxyimino (cefotaxime) and the methyl group (benzylpenicillin) of the substrate. Asn104 is positioned at a bend in the binding site formed by Val103-Asn104-Tyr105-Asn106(Fig. 2 B). 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 Asn106 makes hydrogen bonds with the backbone groups of Val103 to maintain this bent conformation. However, in the acyl-intermediate structures, the Nδ of Asn106 changes its hydrogen bond acceptor from backbone oxygen of Val103 to that of Asn106 by causing a slight rotation of the peptide bond between Asn104 and Tyr105, which forces Val103 and Asn106 to be held in unfavorable conformations. This exchange is induced by the side chain movement of Asn104 to interact with the substrate. The movement of Asn104 also induces the movement of Asn132 whose side chain interacts with Asn104 and the substrate (Fig. 2 B). The main differences between the benzylpenicillin-intermediate and the cephalosporin-intermediates are observed around Ser70 and the oxyanion hole. In the benzylpenicillin-intermediate, the carbonyl group of the β-lactam ring does not position suitably for the hydrolysis in the oxyanion hole (Fig. 2 C). This is caused by van der Waals contact between the methyl group of the thiazolidine ring, and the Cβ and the Oγ of Ser237. An oxyanion hole formed by the backbone amides of Ser70 and residue 237 interacts with the carbonyl oxygen of the β-lactam ring in the substrate (1Matagne A. Lamotte-Brasseur J. Frère J.M. Biochem. J. 1998; 330: 581-598Crossref PubMed Scopus (321) Google Scholar). These interactions play essential roles for the hydrolysis by stabilizing the negative charge appeared on the β-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 β-lactam ring, thus favoring the nucleophilic attack to the carbonyl carbon by Ser70 in acylation and by the hydrolytic water in deacylation. In the cephalosporin-intermediate structures of Toho-1, the carbonyl oxygens of the β-lactam ring are located at similar distances from both backbone nitrogens (2.6–2.9 Å) to those in other class A β-lactamase acyl-intermediate structures with substrate (2.7–3.0 Å) (16Chen C.C.H. Herzberg O. Biochemistry. 2001; 40: 2351-2358Crossref PubMed Scopus (36) Google Scholar, 17Strynadka N.C.J. Adachi H. Jensen S.E. Johns K. Sielecki A. Betzel C. Sutoh K. James M.N.G. Nature. 1992; 359: 700-705Crossref PubMed Scopus (533) Google Scholar, 20Fonze E. Vanhove M. Dive G. Sauvage E. Frère J.M. Charlier P. Biochemistry. 2002; 41: 1877-1885Crossref PubMed Scopus (51) Google Scholar). In the benzylpenicillin-intermediate structure, however, the carbonyl oxygen exists at a distance of 2.6 Å from the backbone nitrogen of Ser70, but 3.2 Å from the backbone nitrogen of Ser237, 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 Ser237 (Fig. 3). Actually, in CTX-M-4 and a chromosomal β-lactamase from P. vulgaris K1 sharing high sequence homologies with CTX-M-type ESBLs, the S237A mutants exhibited increasedk cat/K m− value toward benzylpenicillin (23Gazouli M. Tzelepi E. Sidorenko S.V. Tzouvelekis L.S. Antimicrob. Agents Chemother. 1998; 42: 1259-1262Crossref PubMed Google Scholar, 24Tamaki M. Nukaga M. Sawai T. Biochemistry. 1994; 33: 10200-10206Crossref PubMed Scopus (30) Google Scholar). This information may be useful for the development of new type inhibitors against CTX-M-type ESBLs. The acyl-intermediate structures of Toho-1 and non-ESBLs show similar interactions with the substrates (16Chen C.C.H. Herzberg O. Biochemistry. 2001; 40: 2351-2358Crossref PubMed Scopus (36) Google Scholar, 17Strynadka N.C.J. Adachi H. Jensen S.E. Johns K. Sielecki A. Betzel C. Sutoh K. James M.N.G. Nature. 1992; 359: 700-705Crossref PubMed Scopus (533) Google Scholar) (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 Arg244 in non-ESBLs, because CTX-M-type ESBLs do not contain Arg244present in most non-ESBLs (Fig. 3). In the acyl-intermediate structures of non-ESBLs, Arg244 interacts with the carboxylate oxygen of the substrate and the backbone oxygen of Gly236 (Fig.4). Site-directed mutagenesis analyses indicate that Arg244 is critical for the catalysis of TEM-1 (25Delaire M. Labia R. Samama J.P. Masson J.M. J. Biol. Chem. 1992; 267: 20600-20606Abstract Full Text PDF PubMed Google Scholar, 26Zafaralla G. Manvathu E.K. Lerner S.A. Mobashery S. Biochemistry. 1992; 31: 3847-3852Crossref PubMed Scopus (111) Google Scholar). In Toho-1, Arg276 was predicted to be a substitute for Arg244 (4Ishii Y. Ohno A. Taguchi H. Imajo S. Ishiguro M. Matsuzawa H. Antimicrob. Agents Chemother. 1995; 39: 2269-2275Crossref PubMed Scopus (185) Google Scholar); however, this residue has no interactions with the substrates (Figs. 1, A–C, and2 A). Instead, in Toho-1, Ser237 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 acyl- intermediate structures of Toho-1 compared with those of non-ESBLs. The residue Ser237 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 Streptomycessp. R61, Thr301, the corresponding residue to Ser237 of Toho-1, interacts with the amide group of acylamide side chain in cefotaxime to stabilize substrate binding, whereas in the cephalothin-intermediate structure (15Kuzin A.P. Liu H. Kelly J.A. Knox J.R. Biochemistry. 1995; 34: 9532-9540Crossref PubMed Scopus (106) Google Scholar), Thr301 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 acyl- intermediate structures of class A β-lactamases, the acylamide side chain of the substrate interacts with the Nδ of Asn132 and the backbone oxygen of residue 237 (Fig. 4). The residue Asn132 is the third residue of the conserved Ser130-Asp131-Asn132 motif in class A β-lactamases, and the hydrogen bond between the Nδ of Asn132 and the carbonyl oxygen of the substrate is proposed to be important for the recognition of cephalosporins (27Jacob F. Joris B. Dideberg O. Dusart J. Ghuysen J.M. Frère J.M. Protein Eng. 1990; 4: 79-86Crossref PubMed Scopus (30) Google Scholar). The residue 237 exists just after the conserved Lys234-Thr235-Gly236 motif on the β-strand B3 in class A β-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 (28Jelsch C. Mourey L. Masson J.M. Samama J.P. Proteins. 1993; 16: 364-383Crossref PubMed Scopus (361) Google Scholar). Considering that the oxyimino group and the aminothiazole ring are introduced in the acylamide side chain of oxyimino-cephalosporins, the different positions of Asn132 and Ser237 would contribute to the oxyimino-cephalosporinase activity of Toho-1. In non-ESBLs, the position of Asn132 is fixed by the interactions between Asp131 and Thr/Ser109 as well as the interaction between the Nδ of Asn132 and the backbone oxygen of residue 104 (Fig. 4) (28Jelsch C. Mourey L. Masson J.M. Samama J.P. Proteins. 1993; 16: 364-383Crossref PubMed Scopus (361) Google Scholar,29Petit A. Maveyraud L. Lenfant F. Samama J.P. Labia R. Masson J.M. Biochem. J. 1995; 305: 33-40Crossref PubMed Scopus (50) Google Scholar). 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 Oδ of Asn104 interacts with the Nδ of Asn132 in Toho-1. This unique peptide bond conformation in the vicinity of Asn104 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 β-strand B3 and the side chain of residue 69 in class A β-lactamases (28Jelsch C. Mourey L. Masson J.M. Samama J.P. Proteins. 1993; 16: 364-383Crossref PubMed Scopus (361) Google Scholar, 30Knox J.R. Moews P.C. Frère J.M. Chem. Biol. 1996; 3: 937-947Abstract Full Text PDF PubMed Scopus (148) Google Scholar). Although residue 69 is variable in class A β-lactamases, a cysteine is conserved at this position in CTX-M-type ESBLs (Fig. 3), which may place Ser237 at a unique position. The lack of an interaction between Gly236and Arg244 present in non-ESBLs and the flexibility of the β-strand B3 as mentioned previously (5Ibuka A. Taguchi A. Ishiguro M. Fushinobu S. Ishii Y. Kamitori S. Okuyama K. Yamaguchi K. Konno M. Matsuzawa H. J. Mol. Biol. 1999; 285: 2079-2087Crossref PubMed Scopus (86) Google Scholar) also affect the position of Ser237 in Toho-1. In fact, in Toho-1, the distance between the backbone amide groups of Ser70 and Ser237constructing an oxyanion hole (4.3 Å) is shorter than that in TEM-1 (4.9 Å) (28Jelsch C. Mourey L. Masson J.M. Samama J.P. Proteins. 1993; 16: 364-383Crossref PubMed Scopus (361) Google Scholar) and the average in class A β-lactamases (4.7 Å) (30Knox J.R. Moews P.C. Frère J.M. Chem. Biol. 1996; 3: 937-947Abstract Full Text PDF PubMed Scopus (148) Google Scholar). Moreover, in Toho-1, a unique hydrogen bond is formed between the Nδ of Asn104 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, Asp240 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. Asn104 and Asp240 are conserved in CTX-M-type ESBLs (Fig. 3), indicating that these interactions promote oxyimino-cephalosporinase activity. Non-ESBLs have a low oxyimino-cephalosporinase activity (1Matagne A. Lamotte-Brasseur J. Frère J.M. Biochem. J. 1998; 330: 581-598Crossref PubMed Scopus (321) Google Scholar). It is thought that steric hindrance between the bulky side chains of oxyimino-cephalosporins and the Ω-loop of non-ESBLs reduces substrate affi
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