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

Chen, Yu; Delmas, Julien; Sirot, J.; Shoichet, Brian K.; Bonnet, Richard

doi:10.1016/j.jmb.2005.02.010

Cited by 135 publications

(207 citation statements)

References 48 publications

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“…The bla CTX-M-9 gene was cloned into the pET9a (ϩ) plasmid vector (Novagen) as described previously and was maintained in E. coli BL21(DE3) cells (11,16).…”

Section: Methodsmentioning

confidence: 99%

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Exploring the Inhibition of CTX-M-9 by β-Lactamase Inhibitors and Carbapenems

Bethel

Taracila

Shyr

et al. 2011

Antimicrob Agents Chemother

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Currently, CTX-M ␤-lactamases are among the most prevalent and most heterogeneous extended-spectrum ␤-lactamases (ESBLs). In general, CTX-M enzymes are susceptible to inhibition by ␤-lactamase inhibitors. However, it is unknown if the pathway to inhibition by ␤-lactamase inhibitors for CTX-M ESBLs is similar to TEM and SHV ␤-lactamases and why bacteria possessing only CTX-M ESBLs are so susceptible to carbapenems. Here, we have performed a kinetic analysis and timed electrospray ionization mass spectrometry (ESI-MS) studies to reveal the intermediates of inhibition of CTX-M-9, an ESBL representative of this family of enzymes. CTX-M-9 ␤-lactamase was inactivated by sulbactam, tazobactam, clavulanate, meropenem, doripenem, ertapenem, and a 6-methylidene penem, penem 1. CTX-M enzymes are becoming one of the most prevalent extended-spectrum ␤-lactamases (ESBLs) (3,8,9,(30)(31)(32) in the world. The widespread dissemination of CTX-M ␤-lactamases, especially Escherichia coli ST131 possessing CTX-M-15, has had a significant impact on the treatment of hospital-and community-acquired infections caused by E. coli and other enteric bacilli (6, 7, 13, 23, 36, 41, 44-49, 55, 59).As class A family ␤-lactamases, CTX-Ms are the most genetically heterogeneous (5 major divisions, CTX-M-1, -2, -8, -25, and -9-like groups) (1, 24, 35, 44-46, 58, 60). Most CTX-M enzymes expressed in E. coli provide a high level of resistance to the oxyimino-cephalosporins, cefotaxime and ceftriaxone, and variable levels of resistance to cefepime and cefpirome (3,43). Depending on the type of CTX-M expressed by the isolates, the MICs of ceftazidime are also increased (43). In addition, the MICs of combinations of clavulanate with amoxicillin or ticarcillin vary, and in some cases, low-level resistance has been observed (3).As a consequence of their clinical importance, the reaction mechanism of CTX-M ESBLs has been the subject of intense study (10-12, 14, 16, 42). However, the molecular properties and characteristics of CTX-M that determine the level of susceptibility and resistance to ␤-lactam-␤-lactamase inhibitor combinations and carbapenems are still unknown. Of the currently available inhibitors, tazobactam is the most active (50% inhibitory concentrations [IC 50 s] are 2 to 10 nM for tazobactam and 9 to 90 nM for clavulanate), and sulbactam is the least active (IC 50 s are 0.1 to 4.5 M) (3).In order to develop more effective and broader-spectrum ␤-lactamase inhibitors (18), detailed kinetic and biochemical measurements are needed to reveal the important intermediates in the inactivation of the CTX-M family. In TEM-1 and SHV-1, Arg244 is important in the mechanism of inactivation of carbapenems (imipenem and meropenem), clavulanic acid, sulbactam, and tazobactam (27,28,51,53,63). CTX-M-9 does not contain Arg244, and mutagenesis of a potential corresponding position, Arg276, does not firmly establish this amino acid as an "Arg244 equivalent" (42). Given the differences among class A enzymes, we wondered what the intermediates of inactivation by ...

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“…The bla CTX-M-9 gene was cloned into the pET9a (ϩ) plasmid vector (Novagen) as described previously and was maintained in E. coli BL21(DE3) cells (11,16).…”

Section: Methodsmentioning

confidence: 99%

“…coli BL21(DE3) cells (Novagen) containing the pET 9a (ϩ) expression vector were grown in superoptimal broth (SOB) containing 50 g/ml kanamycin (11,16). These cells were grown with agitation at 37°C until an optical density at 600 nm of 0.8 was attained.…”

Section: Methodsmentioning

confidence: 99%

Exploring the Inhibition of CTX-M-9 by β-Lactamase Inhibitors and Carbapenems

Bethel

Taracila

Shyr

et al. 2011

Antimicrob Agents Chemother

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“…Phases were calculated by molecular replacement with the program EPMR 22 using the apo-enzyme structure of CTX-M-9, with water molecules and ions removed. 13 The complex structures of CTX-M-9 and CTX-M-14 with compound 6 at 1.35 Å resolution and CTX-M-9 with compound 5 at 1.16 Å resolution were refined in SHELX97. 23 The refinement was carried out using ADPs (anisotropic displacement parameters) with standard DELU (rigid-bond), SIMU (spatially adjacent atoms), and ISOR (isolated atoms to be approximately isotropic) restraints.…”

Section: Crystallizationmentioning

confidence: 99%

“…As previously observed, all melts were over 95% reversible and appeared to be two-state. 13 The cefoxitin adduct with CTX-M-9 was formed by mixing an enzyme solution in a buffer with a 350-fold molar excess of the compound at room temperature. The enzyme or enzyme adduct was denatured by raising the temperature in 0.1 °C increments at a ramp rate of 2 °C/min.…”

Section: Thermal Denaturation Of Ctx-m-9 In Complex With Cefoxitinmentioning

confidence: 99%

“…This is based on comparing the occupancies of the two conformations in this 1.16 Å structure and those in previously determined ultrahigh-resolution apo structures. 13 Corresponding to the conformational changes of Lys73 along the reaction coordinate, the side chain of Glu166 also changes conformation. From the apo-enzyme to acylation transition state and acyl-enzyme intermediate, Glu166, together with the catalytic water, moves by 0.4 to 0.6 Å away from the ligand.…”

Section: Movements Of Catalytic Residues Lys73 and Glu166mentioning

confidence: 99%

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Structure, Function, and Inhibition along the Reaction Coordinate of CTX-M β-Lactamases

2005

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CTX-M enzymes are an emerging group of extended spectrum β-lactamases (ESBLs) that hydrolyze not only the penicillins but also the first-, second-, and third-generation cephalosporins. Although they have become the most frequently observed ESBLs in certain areas, there are few effective inhibitors and relatively little is known about their detailed mechanism. Here we describe the X-ray crystal structures of CTX-M enzymes in complex with different transition-state analogues and β-lactam inhibitors, representing the enzyme as it progresses from its acylation transition state to its acyl enzyme complex to the deacylation transition state. As the enzyme moves along this reaction coordinate, two key catalytic residues, Lys73 and Glu166, change conformations, tracking the state of the reaction. Unexpectedly, the acyl enzyme complex with the β-lactam inhibitor cefoxitin still has the catalytic water bound; this water had been predicted to be displaced by the unusual 7α-methoxy of the inhibitor. Instead, the 7α-group appears to inhibit by preventing the formation of the deacylation transition state through steric hindrance. From an inhibitor design standpoint, we note that the best of the reversible inhibitors, a ceftazidime-like boronic acid compound, binds to CTX-M-16 with a K i value of 4 nM. When used together in cell culture, this inhibitor reversed cefotaxime resistance in CTX-M-producing bacteria. The structure of its complex with CTX-M enzyme and the structural view of the reaction coordinate described here provide templates for inhibitor design and intervention to combat this family of antibiotic resistance enzymes.

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Mutation of the conserved Asp‐Asp pair impairs the structure, function, and inhibition of CTX‐M Class A β‐lactamase

et al. 2021

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The Asp233‐Asp246 pair is highly conserved in Class A β‐lactamases, which hydrolyze β‐lactam antibiotics. Here, we characterize its function using CTX‐M‐14 β‐lactamase. The D233N mutant displayed decreased activity that is substrate‐dependent, with reductions in kcat/Km ranging from 20% for nitrocefin to 6‐fold for cefotaxime. In comparison, the mutation reduced the binding of a known reversible inhibitor by 10‐fold. The mutant structures showed movement of the 213‐219 loop and the loss of the Thr216‐Thr235 hydrogen bond, which was restored by inhibitor binding. Mutagenesis of Thr216 further highlighted its contribution to CTX‐M activity. These results demonstrate the importance of the aspartate pair to CTX‐M hydrolysis of substrates with bulky side chains, while suggesting increased protein flexibility as a means to evolve drug resistance.

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

Cited by 135 publications

References 48 publications

Exploring the Inhibition of CTX-M-9 by β-Lactamase Inhibitors and Carbapenems

Exploring the Inhibition of CTX-M-9 by β-Lactamase Inhibitors and Carbapenems

Structure, Function, and Inhibition along the Reaction Coordinate of CTX-M β-Lactamases

Mutation of the conserved Asp‐Asp pair impairs the structure, function, and inhibition of CTX‐M Class A β‐lactamase

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