Structural Insights into Substrate Recognition and Product Expulsion in CTX-M Enzymes

Delmas, Julien; Leyssene, D.; Dubois, Damien; Birck, Catherine; Vazeille, Emilie; Robin, Frédéric; Bonnet, Richard

doi:10.1016/j.jmb.2010.04.062

Cited by 43 publications

(88 citation statements)

References 77 publications

(87 reference statements)

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“…This adds an additional level of complexity to the understanding of ␤-lactamases and their variants, particularly ESBLs. We note that a ligand-dependent shift was also observed previously for an ESBL phenotype CTX-M ␤-lactamase as the distance between residues 170 and 240 increased 0.8 Å upon ligand binding (7). These R164 changes in SHV-1 do not disrupt the overall fold of the enzyme or of the ⍀ loop in the absence of an inhibitor, thus allowing the variant apo enzymes to retain the basic wt active-site configuration.…”

Section: Discussionsupporting

confidence: 78%

Ligand-Dependent Disorder of the Ω Loop Observed in Extended-Spectrum SHV-Type β-Lactamase

Sampson

Wei

Bethel

et al. 2011

Antimicrob Agents Chemother

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Among Gram-negative bacteria, resistance to ␤-lactams is mediated primarily by ␤-lactamases (EC 3.2.6.5), periplasmic enzymes that inactivate ␤-lactam antibiotics. Substitutions at critical amino acid positions in the class A ␤-lactamase families result in enzymes that can hydrolyze extended-spectrum cephalosporins, thus demonstrating an "extended-spectrum" ␤-lactamase (ESBL) phenotype. Using SHV ESBLs with substitutions in the ⍀ loop (R164H and R164S) as target enzymes to understand this enhanced biochemical capability and to serve as a basis for novel ␤-lactamase inhibitor development, we determined the spectra of activity and crystal structures of these variants. We also studied the inactivation of the R164H and R164S mutants with tazobactam and SA2-13, a unique ␤-lactamase inhibitor that undergoes a distinctive reaction chemistry in the active site. We noted that the reduced K i values for the R164H and R164S mutants with SA2-13 are comparable to those with tazobactam (submicromolar). The apo enzyme crystal structures of the R164H and R164S SHV variants revealed an ordered ⍀ loop architecture that became disordered when SA2-13 was bound. Important structural alterations that result from the binding of SA2-13 explain the enhanced susceptibility of these ESBL enzymes to this inhibitor and highlight ligand-dependent ⍀ loop flexibility as a mechanism for accommodating and hydrolyzing ␤-lactam substrates.The most common ␤-lactamases found in Escherichia coli and Klebsiella pneumoniae are the class A enzymes TEM, SHV, and CTX-M (4, 23). Point mutations in the bla TEM and bla SHV genes result in single-amino-acid substitutions that broaden the substrate spectrum of the common TEM and SHV family enzymes to include extended-spectrum cephalosporin antibiotics like ceftazidime or cefotaxime (9). These altered ␤-lactamases are referred to as "extended-spectrum" ␤-lactamases (ESBLs), and this property is referred to as the "ESBL phenotype" (23). The amino acid substitutions responsible for this phenotype in both TEM and SHV enzymes are located in three areas of the class A ␤-lactamase, the ⍀ loop (Ambler residues 164 to 179), the B3 ␤-strand, and residue 104 (2).The ⍀ loop borders the active site on one side and includes the catalytic residue Glu166, which, in addition to aiding in acylation, primes a water molecule needed in the regeneration of the enzyme via the deacylation of the enzyme-substrate complex at Ser70 (2, 16). In wild-type (wt) TEM and SHV, two salt bridges, between residues Arg164 and Asp179 and between residues Arg164 and Glu171, stabilize the loop in place, thereby ensuring the presence of the catalytic Glu166 in the active site. Naturally occurring variants with single-amino-acid changes disrupting the salt bridge at residues 164 to 179, including D179N (SHV-8) in the SHV family and R164H (TEM-29) and R164S (TEM-12) in the closely related TEM ␤-lactamases, have been isolated in the clinic and were shown to have ESBL phenotypes (18,24). Such ESBL activity is hypothesized to result in the increased flexi...

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Section: Discussionsupporting

confidence: 78%

Ligand-Dependent Disorder of the Ω Loop Observed in Extended-Spectrum SHV-Type β-Lactamase

Sampson

Wei

Bethel

et al. 2011

Antimicrob Agents Chemother

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“…Crystals of CTX-M-9-S70G were obtained by vapor diffusion in hanging drops, using microseeding techniques with CTX-M-9 crystals. To a solution of 10 mg/ml protein in 5 mM Tris-HCl (pH 7.0) and 50 mM NaCl was added an equal volume of 1.2 M potassium phosphate buffer (pH 8.2) as previously described (10). Crystals appeared within 24 to 48 h after equilibration at 20°C and were soaked overnight at 20°C with hydrolyzed and native ␤-lactams at a concentration of 50 mM in 1.2 M potassium phosphate buffer (pH 8.2).…”

Section: Methodsmentioning

confidence: 99%

“…Several studies have looked at the crystal structure of CTX-M enzymes in complexes with ␤-lactams or transitionstate analogs (8,10,11,26). Surprisingly, CTX-M-9 efficiently binds and hydrolyzes the large oximino cephalosporin cefotaxime despite its small active site.…”

mentioning

confidence: 99%

“…Surprisingly, CTX-M-9 efficiently binds and hydrolyzes the large oximino cephalosporin cefotaxime despite its small active site. Recently, the structures of benzylpenicillin and cefotaxime CTX-M-9 Michaelis complexes revealed that the accommodation of the bulky cephalosporin cefotaxime can be accommodated by a widening of the catalytic pocket, which is not observed for the small molecule benzylpenicillin (10). In this work, we determined the crystal structure of an inactive Ser70Gly mutant of CTX-M-9 in complex with the hydrolyzed and nonhydrolyzed forms of piperacillin to investigate the recognition of this bulky penicillin by CTX-M-type ESBLs.…”

mentioning

confidence: 99%

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Noncovalent Complexes of an Inactive Mutant of CTX-M-9 with the Substrate Piperacillin and the Corresponding Product

Leyssene

Delmas

Robin

et al. 2011

Antimicrob Agents Chemother

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We determined the crystal structure of an inactive Ser70Gly mutant of CTX-M-9 in complex with the bulky penicillin piperacillin at precovalent and posthydrolytic stages in the catalytic process. The structures obtained at high resolution were compared with the corresponding structures for the small penicillin benzylpenicillin and the bulky cephalosporin cefotaxime. The findings highlight the key role of the configuration of the carbon adjacent to the acylamino group of the side chain of ␤-lactams in the precovalent recognition of substrates.The production of ␤-lactamases is the predominant cause of resistance to ␤-lactam antibiotics in Gram-negative bacteria. These enzymes are divided on the basis of amino acid sequences into four classes designated A to D, with class A ␤-lactamases being the most prevalent in Enterobacteriaceae (1).The oximino cephalosporins such as cefotaxime have been used since the 1980s to overcome these resistance mechanisms because of their relative stability to serine ␤-lactamases and their broad antibacterial spectrum. These ␤-lactams typically contain a bulky group such as a 2-(2-aminothiazole-4-yl)-2-oximino substituent at position C-7 of the cephalosporin nucleus, which prevents hydrolysis by serine ␤-lactamases. However, the intensive use of these molecules was quickly followed by the emergence of extended-spectrum ␤-lactamases (ESBLs), which can accommodate and hydrolyze such large ␤-lactams. The CTX-M enzymes of the class A family, first described in 1989, have spread worldwide since the late 1990s and are now the most prevalent extended-spectrum ␤-lactamases (2,3,5,6,25,29).The hydrolysis process catalyzed by these enzymes is based on an acid-base catalytic mechanism, as for the other class A enzymes. It comprises three major steps (8,9,14,19): the formation of a precovalent encounter complex with the substrate; the nucleophilic attack on the ␤-lactam ring by the active serine residue resulting in an acyl-enzyme; and the nucleophilic attack on this covalent complex by the catalytic water molecule, which produces a transient noncovalent productenzyme complex and subsequently results in the release of both the product and the active enzyme.Several studies have looked at the crystal structure of CTX-M enzymes in complexes with ␤-lactams or transitionstate analogs (8,10,11,26). Surprisingly, CTX-M-9 efficiently binds and hydrolyzes the large oximino cephalosporin cefotaxime despite its small active site. Recently, the structures of benzylpenicillin and cefotaxime CTX-M-9 Michaelis complexes revealed that the accommodation of the bulky cephalosporin cefotaxime can be accommodated by a widening of the catalytic pocket, which is not observed for the small molecule benzylpenicillin (10). In this work, we determined the crystal structure of an inactive Ser70Gly mutant of CTX-M-9 in complex with the hydrolyzed and nonhydrolyzed forms of piperacillin to investigate the recognition of this bulky penicillin by CTX-M-type ESBLs. The structure shows that the size of the substrate is not the exc...

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“…The CTX-M enzymes usually have higher activity against cefotaxime than against ceftazidime and aztreonam (31). The cefotaxime-hydrolyzing activity of CTX-M enzymes is related to the flexibility of the ␤3 strand and omega loop and asparagine, serine, aspartate, and arginine at Ambler positions 104, 237, 240, and 276, respectively (1,13,14,31,33). Several CTX-Ms exhibiting an increased enzymatic activity against ceftazidime have recently been reported: the P167S mutant of CTX-M-18 (also called CTX-M-14), designated CTX-M-19 (29); the P167Q mutant of CTX-M-3, designated CTX-M-54 (2); the P167T mutant of CTX-M-1, designated CTX-M-23 (34); and D240G mutants of CTX-M-3, CTX-M-9, and CTX-M-14, designated CTX-M-15, CTX-M-16, and CTX-M-27, respectively (3,4,27).…”

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confidence: 99%

CTX-M-93, a CTX-M Variant Lacking Penicillin Hydrolytic Activity

Djamdjian¹,

Naas²,

Tandé³

et al. 2011

Antimicrob Agents Chemother

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Structural Insights into Substrate Recognition and Product Expulsion in CTX-M Enzymes

Cited by 43 publications

References 77 publications

Ligand-Dependent Disorder of the Ω Loop Observed in Extended-Spectrum SHV-Type β-Lactamase

Ligand-Dependent Disorder of the Ω Loop Observed in Extended-Spectrum SHV-Type β-Lactamase

Noncovalent Complexes of an Inactive Mutant of CTX-M-9 with the Substrate Piperacillin and the Corresponding Product

CTX-M-93, a CTX-M Variant Lacking Penicillin Hydrolytic Activity

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