scholarly journals Structural Insights into the TLA-3 Extended-Spectrum β-Lactamase and Its Inhibition by Avibactam and OP0595

2017 ◽  
Vol 61 (10) ◽  
Author(s):  
Wanchun Jin ◽  
Jun-ichi Wachino ◽  
Yoshihiro Yamaguchi ◽  
Kouji Kimura ◽  
Anupriya Kumar ◽  
...  
Keyword(s):  
Rate Constant ◽  
Catalytic Efficiency ◽  
Amino Acid Residues ◽  
Inhibitory Effects ◽  
Content Type ◽  
Class A ◽  
Extended Spectrum ◽  
Structural Template ◽  
Structural Insights ◽  
Positively Charged

ABSTRACT The development of effective inhibitors that block extended-spectrum β-lactamases (ESBLs) and restore the action of β-lactams represents an effective strategy against ESBL-producing Enterobacteriaceae. We evaluated the inhibitory effects of the diazabicyclooctanes avibactam and OP0595 against TLA-3, an ESBL that we identified previously. Avibactam and OP0595 inhibited TLA-3 with apparent inhibitor constants (Ki app) of 1.71 ± 0.10 and 1.49 ± 0.05 μM, respectively, and could restore susceptibility to cephalosporins in the TLA-3-producing Escherichia coli strain. The value of the second-order acylation rate constant (k 2/K, where k 2 is the acylation rate constant and K is the equilibrium constant) of avibactam [(3.25 ± 0.03) × 103 M−1 · s−1] was closer to that of class C and D β-lactamases (k 2/K, <104 M−1 · s−1) than that of class A β-lactamases (k 2/K, >104 M−1 · s−1). In addition, we determined the structure of TLA-3 and that of TLA-3 complexed with avibactam or OP0595 at resolutions of 1.6, 1.6, and 2.0 Å, respectively. TLA-3 contains an inverted Ω loop and an extended loop between the β5 and β6 strands (insertion after Ser237), which appear only in PER-type class A β-lactamases. These structures might favor the accommodation of cephalosporins harboring bulky R1 side chains. TLA-3 presented a high catalytic efficiency (k cat/Km ) against cephalosporins, including cephalothin, cefuroxime, and cefotaxime. Avibactam and OP0595 bound covalently to TLA-3 via the Ser70 residue and made contacts with residues Ser130, Thr235, and Ser237, which are conserved in ESBLs. Additionally, the sulfate group of the inhibitors formed polar contacts with amino acid residues in a positively charged pocket of TLA-3. Our findings provide a structural template for designing improved diazabicyclooctane-based inhibitors that are effective against ESBL-producing Enterobacteriaceae.

scholarly journals Structural Insights into the Inhibition of the Extended-Spectrum β-Lactamase PER-2 by Avibactam

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Melina Ruggiero ◽  
Krisztina M. Papp-Wallace ◽  
Florencia Brunetti ◽  
Melissa D. Barnes ◽  
Robert A. Bonomo ◽  
...  
Keyword(s):  
Side Chain ◽  
Structural Studies ◽  
Content Type ◽  
Class A ◽  
Extended Spectrum ◽  
Inhibition Constants ◽  
The Rich ◽  
Structural Insights ◽  
Insight Into ◽  
Made In

ABSTRACT The diazabicyclooctane (DBO) avibactam (AVI) reversibly inactivates most serine-β-lactamases. Previous investigations showed that inhibition constants of AVI toward class A PER-2 are reminiscent of values observed for class C and D β-lactamases (i.e., k2/K of ≈103 M−1 s−1) but lower than other class A β-lactamases (i.e., k2/K = 104 to 105 M−1 s−1). Herein, biochemical and structural studies were conducted with PER-2 and AVI to explore these differences. Furthermore, biochemical studies on Arg220 and Thr237 variants with AVI were conducted to gain deeper insight into the mechanism of PER-2 inactivation. The main biochemical and structural observations revealed the following: (i) both amino-acid substitutions in Arg220 and the rich hydrophobic content in the active site hinder the binding of catalytic waters and acylation, impairing AVI inhibition; (ii) movement of Ser130 upon binding of AVI favors the formation of a hydrogen bond with the sulfate group of AVI; and (iii) the Thr237Ala substitution alters the AVI inhibition constants. The acylation constant (k2/K) of PER-2 by AVI is primarily influenced by stabilizing hydrogen bonds involving AVI and important residues such as Thr237 and Arg220. (Variants in Arg220 demonstrate a dramatic reduction in k2/K.) We also observed that displacement of Ser130 side chain impairs AVI acylation, an observation not made in other extended-spectrum β-lactamases (ESBLs). Comparatively, relebactam combined with a β-lactam is more potent against Escherichia coli producing PER-2 variants than β-lactam–AVI combinations. Our findings provide a rationale for evaluating the utility of the currently available DBO inhibitors against unique ESBLs like PER-2 and anticipate the effectiveness of these inhibitors toward variants that may eventually be selected upon AVI usage.

scholarly journals Hydrolysis Spectrum Extension of CMY-2-Like β-Lactamases Resulting from Structural Alteration in the Y-X-N Loop

2012 ◽  
Vol 56 (3) ◽  
pp. 1151-1156 ◽  
Author(s):  
Sandrine Dahyot ◽  
Hedi Mammeri
Keyword(s):  
Hot Spot ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Hydrolysis Rate ◽  
Directed Mutagenesis ◽  
Content Type ◽  
E Coli ◽  
Class A ◽  
Extended Spectrum ◽  
Cloning Experiment

ABSTRACTTheCitrobacter freundiiisolate CHA, which was responsible for postoperative peritonitis after 10 days of cefepime therapy, displayed a phenotype of resistance consistent with extended-spectrum AmpC (ESAC) β-lactamase. The chromosome-borneblaAmpC-CHAgene was amplified and sequenced, revealing five amino acid substitutions, I125V, R148H, Q196H, V305A, and V348A, in the product compared to the sequence of native AmpC. A cloning experiment yielded theEscherichia coliTOP10(pAmpC-CHA) strain, which was resistant to all extended-spectrum cephalosporins (ESCs), including cefepime. To ascertain whether the R148H substitution accounted for the hydrolysis spectrum extension, it was reverted by site-directed mutagenesis. The resultingE. coliTOP10(pAmpC-CHA-H148R) strain was fully susceptible to cefepime, thus confirming that the Arg-148 replacement was mandatory for substrate profile enlargement. To further characterize the phenotypical and biochemical effects induced by the R148H change, it was introduced by site-directed mutagenesis into the CMY-2 β-lactamase, which is structurally related to the chromosome-borne cephalosporinase ofC. freundii. The CMY-2-R148H variant conferred increased MICs of ESCs, whereas those of carbapenems were unchanged even in a porin-deficientE. colistrain. Moreover, it exhibited increased catalytic efficiency (kcat/Km) toward ceftazidime (100-fold) due to an enhanced hydrolysis rate (kcat), whereas the enzymatic parameters toward imipenem were unchanged. The structural analysis of the AmpC variant showed that the R148H replacement occurred in the loop containing the Y-X-N motif, which is the counterpart of the SDN loop in class A β-lactamases. This study shows that the Y-X-N loop is a novel hot spot for mutations accounting for hydrolysis spectrum extension in CMY-2-type enzymes.

scholarly journals Effects of external pH on substrate binding and on the inward chloride translocation rate constant of band 3.

1996 ◽  
Vol 107 (2) ◽  
pp. 271-291 ◽  
Author(s):  
S Q Liu ◽  
F Y Law ◽  
P A Knauf
Keyword(s):  
Rate Constant ◽  
Band 3 ◽  
Complex Model ◽  
Amino Acid Residues ◽  
Ping Pong ◽  
Extracellular Transport ◽  
Charged Form ◽  
Transport Site ◽  
External Ph ◽  
Positively Charged

To test the hypothesis that amino acid residues in band 3 with titratable positive charges play a role in the binding of anions to the outside-facing transport site, we measured the effects of changing external pH (pH(O)) on the dissociation constant for binding of external iodide to the transport site, K(O)(I). K(O)(I) increased with increasing pH(O), and a significant increase was seen even at pH(O) values as low as 9.9. The dependence of K(O)(I) on pH(O) can be explained by a model with one titratable site with pK 9.5 +/- 0.2 (probably lysine), which increases anion affinity for the external transport site when it is in the positively charged form. A more complex model, analogous to one recently proposed by Bjerrum (1992), with two titratable sites, one with pK 9.3 +/- 0.3 (probably lysine) and another with pK &gt; 11 (probably arginine), gives a slightly better fit to the data. Thus, titratable positively charged residues seem to be functionally important for the binding of substrate anions to the outward-facing anion transport site. In addition, analysis of Dixon plot slopes for L inhibition of Cl- exchange at different pH 0 values, coupled with the assumption that pH(O) has parallel effects on external I- and Cl- binding, indicates that k', the rate-constant for inward translocation of the complex of Cl- with the extracellular transport site, decreases with increasing pH(O). The data are compatible with a model in which titration of the pK 9.3 residue decreases k to 14 +/- 10% of its value at neutral pH(O). This result, however, together with Bjerrum's (1992) observation that the maximum flux J(M)) increases 1.6-fold when this residue is deprotonated, makes quantitative predictions that raise significant questions about the adequacy of the two titratable site ping-pong model or the assumptions used in analyzing the data.

scholarly journals Substitutions at Position 105 in SHV Family β-Lactamases Decrease Catalytic Efficiency and Cause Inhibitor Resistance

2012 ◽  
Vol 56 (11) ◽  
pp. 5678-5686 ◽  
Author(s):  
Mei Li ◽  
Benjamin C. Conklin ◽  
Magdalena A. Taracila ◽  
Rebecca A. Hutton ◽  
Marion J. Skalweit
Keyword(s):  
Clavulanic Acid ◽  
Susceptibility Testing ◽  
Catalytic Efficiency ◽  
Wild Type ◽  
Oxyanion Hole ◽  
Content Type ◽  
Class A ◽  
Inhibitor Resistance ◽  
Structural Probes ◽  
Variant Residue

ABSTRACTAmbler position 105 in class A β-lactamases is implicated in resistance to clavulanic acid, although no clinical isolates with mutations at this site have been reported. We hypothesized that Y105 is important in resistance to clavulanic acid because changes in positioning of the inhibitor for ring oxygen protonation could occur. In addition, resistance to bicyclic 6-methylidene penems, which are interesting structural probes that inhibit all classes of serine β-lactamases with nanomolar affinity, might emerge with substitutions at position 105, especially with nonaromatic substitutions. All 19 variants of SHV-1 with variations at position 105 were prepared. Antimicrobial susceptibility testing showed thatEscherichia coliDH10B expressing Y105 variants retained activity against ampicillin, except for the Y105L variant, which was susceptible to all β-lactams, similar to the case for the host control strain. Several variants had elevated MICs to ampicillin-clavulanate. However, all the variants remained susceptible to piperacillin in combination with a penem inhibitor (MIC, ≤2/4 mg/liter). The Y105E, -F, -M, and -R variants demonstrated reduced catalytic efficiency toward ampicillin compared to the wild-type (WT) enzyme, which was caused by increasedKm. Clavulanic acid and penemKivalues were also increased for some of the variants, especially Y105E. Mutagenesis at position 105 in SHV yields mutants resistant to clavulanate with reduced catalytic efficiency for ampicillin and nitrocefin, similar to the case for the class A carbapenemase KPC-2. Our modeling analyses suggest that resistance is due to oxyanion hole distortion. Susceptibility to a penem inhibitor is retained although affinity is decreased, especially for the Y105E variant. Residue 105 is important to consider when designing new inhibitors.

scholarly journals Characterization of the Inhibitor-Resistant SHV β-Lactamase SHV-107 in a Clinical Klebsiella pneumoniae Strain Coproducing GES-7 Enzyme

2011 ◽  
Vol 56 (2) ◽  
pp. 1042-1046 ◽  
Author(s):  
Vera Manageiro ◽  
Eugénia Ferreira ◽  
Antony Cougnoux ◽  
Luís Albuquerque ◽  
Manuela Caniça ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Inhibitory Concentration ◽  
Catalytic Efficiency ◽  
Dynamics Simulation ◽  
Inhibitory Effects ◽  
Content Type ◽  
High Affinity ◽  
Amoxicillin Clavulanate ◽  
Lactamase Gene

ABSTRACTThe clinicalKlebsiella pneumoniaeINSRA6884 strain exhibited nonsusceptibility to all penicillins tested (MICs of 64 to >2,048 μg/ml). The MICs of penicillins were weakly reduced by clavulanate (from 2,048 to 512 μg/ml), and tazobactam restored piperacillin susceptibility. Molecular characterization identified the genesblaGES-7and a new β-lactamase gene,blaSHV-107, which encoded an enzyme that differed from SHV-1 by the amino acid substitutions Leu35Gln and Thr235Ala. The SHV-107-producingEscherichia colistrain exhibited only a β-lactam resistance phenotype with respect to amoxicillin, ticarcillin, and amoxicillin-clavulanate combination. The kinetic parameters of the purified SHV-107 enzyme revealed a high affinity for penicillins. However, catalytic efficiency for these antibiotics was lower for SHV-107 than for SHV-1. No hydrolysis was detected against oxyimino-β-lactams. The 50% inhibitory concentration (IC50) for clavulanic acid was 9-fold higher for SHV-107 than for SHV-1, but the inhibitory effects of tazobactam were unchanged. Molecular dynamics simulation suggested that the Thr235Ala substitution affects the accommodation of clavulanate in the binding site and therefore its inhibitory activity.

scholarly journals Identification of Novel VEB β-Lactamase Enzymes and Their Impact on Avibactam Inhibition

2016 ◽  
Vol 60 (5) ◽  
pp. 3183-3186 ◽  
Author(s):  
Sushmita D. Lahiri ◽  
Richard A. Alm
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Pocket ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Content Type ◽  
Class A ◽  
Extended Spectrum ◽  
Class C

ABSTRACTCeftazidime-avibactam has activity againstPseudomonas aeruginosaandEnterobacteriaceaeexpressing numerous class A and class C β-lactamases, although the ability to inhibit many minor enzyme variants has not been established. Novel VEB class A β-lactamases were identified during characterization of surveillance isolates. The cloned novel VEB β-lactamases possessed an extended-spectrum β-lactamase phenotype and were inhibited by avibactam in a concentration-dependent manner. The residues that comprised the avibactam binding pocket were either identical or functionally conserved. These data demonstrate that avibactam can inhibit VEB β-lactamases.

scholarly journals PME-1, an Extended-Spectrum β-Lactamase Identified in Pseudomonas aeruginosa

2011 ◽  
Vol 55 (6) ◽  
pp. 2710-2713 ◽  
Author(s):  
Guo-Bao Tian ◽  
Jennifer M. Adams-Haduch ◽  
Tatiana Bogdanovich ◽  
Hong-Ning Wang ◽  
Yohei Doi
Keyword(s):  
Pseudomonas Aeruginosa ◽  
United Arab Emirates ◽  
Stenotrophomonas Maltophilia ◽  
Hydrolytic Activity ◽  
The United States ◽  
Amino Acid Identity ◽  
The Novel ◽  
Content Type ◽  
Class A ◽  
Extended Spectrum

ABSTRACTA novel extended-spectrum β-lactamase (ESBL) was identified in aPseudomonas aeruginosaclinical isolate obtained from a patient admitted to a hospital in Pennsylvania in 2008. The patient had a prolonged hospitalization in a hospital in Dubai, United Arab Emirates, before being transferred to the United States. The novel ESBL, designated PME-1 (Pseudomonas aeruginosaESBL 1), is a molecular class A, Bush-Jacoby-Medeiros group 2be enzyme and shared 50, 43, and 41% amino acid identity with the L2 β-lactamase ofStenotrophomonas maltophilia, CTX-M-9, and KPC-2, respectively. PME-1 conferred clinically relevant resistance to ceftazidime, cefotaxime, cefepime, and aztreonam inP. aeruginosaPAO1 but not to carbapenems. Purified PME-1 showed good hydrolytic activity against ceftazidime, cefotaxime, and aztreonam, while activity against carbapenems and cefepime could not be measured. PME-1 was inhibited well by β-lactamase inhibitors, including clavulanic acid, sulbactam, and tazobactam. TheblaPME-1gene was carried by an approximately 9-kb plasmid and flanked by tandem ISCR24elements.

scholarly journals Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Susann Skagseth ◽  
Tony Christopeit ◽  
Sundus Akhter ◽  
Annette Bayer ◽  
Ørjan Samuelsen ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Global Public Health ◽  
Equilibrium Dissociation Constant ◽  
Inhibitor Binding ◽  
Class A ◽  
Class D ◽  
Equilibrium Dissociation ◽  
Structural Insights

ABSTRACT Metallo-β-lactamases (MBLs) threaten the effectiveness of β-lactam antibiotics, including carbapenems, and are a concern for global public health. β-Lactam/β-lactamase inhibitor combinations active against class A and class D carbapenemases are used, but no clinically useful MBL inhibitor is currently available. Tripoli metallo-β-lactamase-1 (TMB-1) and TMB-2 are members of MBL subclass B1a, where TMB-2 is an S228P variant of TMB-1. The role of S228P was studied by comparisons of TMB-1 and TMB-2, and E119 was investigated through the construction of site-directed mutants of TMB-1, E119Q, E119S, and E119A (E119Q/S/A). All TMB variants were characterized through enzyme kinetic studies. Thermostability and crystallization analyses of TMB-1 were performed. Thiol-based inhibitors were investigated by determining the 50% inhibitory concentrations (IC50) and binding using surface plasmon resonance (SPR) for analysis of TMB-1. Thermostability measurements found TMB-1 to be stabilized by high NaCl concentrations. Steady-state enzyme kinetics analyses found substitutions of E119, in particular, substitutions associated with the penicillins, to affect hydrolysis to some extent. TMB-2 with S228P showed slightly reduced catalytic efficiency compared to TMB-1. The IC50 levels of the new thiol-based inhibitors were 0.66 μM (inhibitor 2a) and 0.62 μM (inhibitor 2b), and the equilibrium dissociation constant (KD ) of inhibitor 2a was 1.6 μM; thus, both were more potent inhibitors than l-captopril (IC50 = 47 μM; KD = 25 μM). The crystal structure of TMB-1 was resolved to 1.75 Å. Modeling of inhibitor 2b in the TMB-1 active site suggested that the presence of the W64 residue results in T-shaped π-π stacking and R224 cation-π interactions with the phenyl ring of the inhibitor. In sum, the results suggest that residues 119 and 228 affect the catalytic efficiency of TMB-1 and that inhibitors 2a and 2b are more potent inhibitors for TMB-1 than l-captopril.

scholarly journals Characterization of CIA-1, an Ambler Class A Extended-Spectrum β-Lactamase from Chryseobacterium indologenes

2011 ◽  
Vol 56 (1) ◽  
pp. 588-590 ◽  
Author(s):  
Takehisa Matsumoto ◽  
Mika Nagata ◽  
Nau Ishimine ◽  
Kenji Kawasaki ◽  
Kazuyoshi Yamauchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Reference Strain ◽  
Content Type ◽  
Chryseobacterium Indologenes ◽  
Esbl Gene ◽  
Class A ◽  
Extended Spectrum ◽  
Class B ◽  
Lactamase Gene

ABSTRACTAn Ambler class A β-lactamase gene,blaCIA-1, was cloned from the reference strainChryseobacterium indologenesATCC 29897 and expressed inEscherichia coliBL21. TheblaCIA-1gene encodes a novel extended-spectrum β-lactamase (ESBL) that shared 68% and 60% identities with the CGA-1 and CME-1 β-lactamases, respectively.blaCIA-1-like genes were detected from clinical isolates. In addition to the metallo-β-lactamase IND of Ambler class B,C. indologeneshas a class A ESBL gene,blaCIA-1, located on the chromosome.

scholarly journals Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
Venu Gopal Vandavasi ◽  
Patricia S. Langan ◽  
Kevin L. Weiss ◽  
Jerry M. Parks ◽  
Jonathan B. Cooper ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Active Site Residues ◽  
Hydrogen Atoms ◽  
Content Type ◽  
X Ray Crystallography ◽  
General Base ◽  
Class A ◽  
Extended Spectrum ◽  
Enzyme Intermediate

ABSTRACT The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed.

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