scholarly journals Structural and kinetic studies on β-lactamase K1 from Klebsiella aerogenes

1986 ◽  
Vol 234 (2) ◽  
pp. 343-347 ◽  
Author(s):  
E L Emanuel ◽  
J Gagnon ◽  
S G Waley
Keyword(s):  
Active Site ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Thiol Group ◽  
Klebsiella Aerogenes ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Class A ◽  
Rate Determining Step

beta-Lactamase K1 from Klebsiella aerogenes 1082E hydrolyses both penicillins and cephalosporins comparably and is inhibited by mercurials but not by cloxacillin. These properties distinguish it from those other beta-lactamases that have been allotted to classes on the basis of their amino sequences. beta-Lactamase K1 has been isolated by affinity chromatography; its composition shows resemblances to class A beta-lactamases. Moreover, the N-terminal sequence is similar to those of class A beta-lactamases: there is about 30% identity over the first 32 residues. Furthermore, a putative active-site octapeptide has been isolated and its sequence is similar to the region around the active-site serine residue in class A beta-lactamases. There is one thiol group in beta-lactamase K1; it is not essential for activity. The pH-dependence of kcat. and kcat./Km for the hydrolysis of benzylpenicillin by beta-lactamase K1 were closely similar, suggesting that the rate-determining step is cleavage of the beta-lactam ring.

Interaction of β-lactamases I and II from Bacillus cereus with semisynthetic cephamycins. Kinetic studies

1991 ◽  
Vol 279 (1) ◽  
pp. 111-114 ◽  
Author(s):  
J Martin Villacorta ◽  
P Arriaga ◽  
J Laynez ◽  
M Menendez
Keyword(s):  
Bacillus Cereus ◽  
Kinetic Studies ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Class A ◽  
Rate Determining Step ◽  
Class B ◽  
Lactam Ring ◽  
Beta Lactamase Ii

The influence of C-6 alpha- or C-7 alpha-methoxylation of the beta-lactam ring in the catalytic action of class A and B beta-lactamases has been investigated. For this purpose the kinetic behaviour of beta-lactamases I (class A) and II (class B) from Bacillus cereus was analysed by using several cephamycins, moxalactam, temocillin and related antibiotics. These compounds behaved as poor substrates for beta-lactamase II, with high Km values and very low catalytic efficiencies. In the case of beta-lactamase I, the substitution of a methoxy group for a H atom at C-7 alpha or C-6 alpha decreased the affinity of the substrates for the enzyme. Furthermore, the acylation of cephamycins was completely blocked, whereas that of penicillins was slowed down by a factor of 10(4)-10(5), acylation being the rate-determining step of the process.

scholarly journals The mutation Lys234His yields a class A β-lactamase with a novel pH-dependence

1991 ◽  
Vol 278 (3) ◽  
pp. 673-678 ◽  
Author(s):  
J Brannigan ◽  
A Matagne ◽  
F Jacob ◽  
C Damblon ◽  
B Joris ◽  
...  
Keyword(s):  
Active Site ◽  
Positive Charge ◽  
Ph Dependence ◽  
Side Chain ◽  
Beta Lactamase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Beta Lactamases ◽  
Class A ◽  
Transition State Stabilization

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A beta-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the kcat, value for benzylpenicillin was as high as 50% of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both kcat. and kcat/Km dramatically decreased above pH 6 but the decrease in kcat./Km could not be attributed to larger Km values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.

scholarly journals The K1 β-lactamase of Klebsiella pneumoniae

1987 ◽  
Vol 243 (2) ◽  
pp. 561-567 ◽  
Author(s):  
B Joris ◽  
F De Meester ◽  
M Galleni ◽  
J M Frère ◽  
J Van Beeumen
Keyword(s):  
Klebsiella Pneumoniae ◽  
Active Site ◽  
Cysteine Residue ◽  
Klebsiella Aerogenes ◽  
Beta Lactamase ◽  
Serine Residue ◽  
Class A

beta-Lactamase K1 was purified from Klebsiella pneumoniae SC10436. It is very similar to the enzyme produced by Klebsiella aerogenes 1082E and described by Emanuel, Gagnon & Waley [Biochem. J. (1986) 234, 343-347]. An active-site peptide was isolated after labelling of the enzyme with tritiated beta-iodopenicillanate. A cysteine residue was found just before the active-site serine residue. This result could explain the properties of the enzyme after modification by thiol-blocking reagents. The sequence of the active-site peptide clearly established the enzyme as a class A beta-lactamase.

scholarly journals Phosphonate monoester inhibitors of class A β-lactamases

1991 ◽  
Vol 275 (3) ◽  
pp. 793-795 ◽  
Author(s):  
J Rahil ◽  
R F Pratt
Keyword(s):  
Enzyme Inhibition ◽  
Mechanism Of Action ◽  
Active Site ◽  
General Structure ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Inhibitory Ability ◽  
Class A ◽  
Structure Formula ◽  
Slow Turnover

Phosphonate monoesters with the general structure: [formula: see text] are inhibitors of representative class A and class C beta-lactamases. This result extends the range of this type of inhibitor to the class A enzymes. Compounds where X is an electron-withdrawing substituent are better inhibitors than the unsubstituted analogue (X = H), and enzyme inhibition is concerted with stoichiometric release of the substituted phenol. Slow turnover of the phosphonates also occurs. These observations support the proposition that the mechanism of action of these inhibitors involves phosphorylation of the beta-lactamase active site. The inhibitory ability of these phosphonates suggests that the beta-lactamase active site is very effective at stabilizing negatively charged transition states. One of the compounds described also inactivated the Streptomyces R61 D-alanyl-D-alanine carboxypeptidase/transpeptidase.

scholarly journals Streptomyces albus G serine β-lactamase. Probing of the catalytic mechanism via molecular modelling of mutant enzymes

1992 ◽  
Vol 282 (1) ◽  
pp. 189-195 ◽  
Author(s):  
J Lamotte-Brasseur ◽  
F Jacob-Dubuisson ◽  
G Dive ◽  
J M Frère ◽  
J M Ghuysen
Keyword(s):  
Hydrogen Bonding ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Network Configuration ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Class A ◽  
Streptomyces Albus ◽  
Hydrogen Bonding Network

In previous studies, several amino acids of the active site of class A beta-lactamases have been modified by site-directed mutagenesis. On the basis of the catalytic mechanism proposed for the Streptomyces albus G beta-lactamase [Lamotte-Brasseur, Dive, Dideberg, Charlier, Frère & Ghuysen (1991) Biochem. J. 279, 213-221], the influence that these mutations exert on the hydrogen-bonding network of the active site has been analysed by molecular mechanics. The results satisfactorily explain the effects of the mutations on the kinetic parameters of the enzyme's activity towards a set of substrates. The present study also shows that, upon binding a properly structured beta-lactam compound, the impaired cavity of a mutant enzyme can readopt a functional hydrogen-bonding-network configuration.

scholarly journals Vaborbactam: Spectrum of Beta-Lactamase Inhibition and Impact of Resistance Mechanisms on Activity in Enterobacteriaceae

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Olga Lomovskaya ◽  
Dongxu Sun ◽  
Debora Rubio-Aparicio ◽  
Kirk Nelson ◽  
Ruslan Tsivkovski ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Content Type ◽  
Beta Lactamases ◽  
Class A ◽  
Class C ◽  
The Impact ◽  
Isogenic Strains ◽  
Lactamase Inhibitor

ABSTRACT Vaborbactam (formerly RPX7009) is a new beta-lactamase inhibitor based on a cyclic boronic acid pharmacophore. The spectrum of beta-lactamase inhibition by vaborbactam and the impact of bacterial efflux and permeability on its activity were determined using a panel of strains with beta-lactamases cloned from various classes and a panel of Klebsiella pneumoniae carbapenemase 3 (KPC-3)-producing isogenic strains with various combinations of efflux and porin mutations. Vaborbactam is a potent inhibitor of class A carbapenemases, such as KPC, as well as an inhibitor of other class A (CTX-M, SHV, TEM) and class C (P99, MIR, FOX) beta-lactamases. Vaborbactam does not inhibit class D or class B carbapenemases. When combined with meropenem, vaborbactam had the highest potency compared to the potencies of vaborbactam in combination with other antibiotics against strains producing the KPC beta-lactamase. Consistent with broad-spectrum beta-lactamase inhibition, vaborbactam reduced the meropenem MICs for engineered isogenic strains of K. pneumoniae with increased meropenem MICs due to a combination of extended-spectrum beta-lactamase production, class C beta-lactamase production, and reduced permeability due to porin mutations. Vaborbactam crosses the outer membrane of K. pneumoniae using both OmpK35 and OmpK36, but OmpK36 is the preferred porin. Efflux by the multidrug resistance efflux pump AcrAB-TolC had a minimal impact on vaborbactam activity. Investigation of the vaborbactam concentration necessary for restoration of meropenem potency showed that vaborbactam at 8 μg/ml results in meropenem MICs of ≤2 μg/ml in the most resistant engineered strains containing multiple mutations. Vaborbactam is a highly active beta-lactamase inhibitor that restores the activity of meropenem and other beta-lactam antibiotics in beta-lactamase-producing bacteria, particularly KPC-producing carbapenem-resistant Enterobacteriaceae.

scholarly journals The pH-dependence of class B and class C β-lactamases

1983 ◽  
Vol 213 (1) ◽  
pp. 61-66 ◽  
Author(s):  
R Bicknell ◽  
V Knott-Hunziker ◽  
S G Waley
Keyword(s):  
Ph Dependence ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Class A ◽  
Class B ◽  
Class C ◽  
Beta Lactamase Ii ◽  
Hydrolysis Of ◽  
Ionic Forms ◽  
Do So

The classification by structure allots beta-lactamases to (at present) three classes, A, B and C. The pH-dependence of the kinetic parameters for class B and class C have been determined. They differ from each other and from class A beta-lactamases. The class B enzyme was beta-lactamase II from Bacillus cereus 569/H/9. The plots of kcat against pH for the hydrolysis of benzylpenicillin by Zn(II)-requiring beta-lactamase II and Co(II)-requiring beta-lactamase II were not symmetrical, but those of kcat/Km were. A similar feature was observed for the hydrolysis of both benzylpenicillin and cephalosporin C by a class C beta-lactamase from Pseudomonas aeruginosa. The results have been interpreted by a scheme in which two ionic forms of an intermediate can give product, but do so at differing rates.

scholarly journals Interactions between active-site-serine β-lactamases and mechanism-based inactivators: a kinetic study and an overview

1993 ◽  
Vol 295 (3) ◽  
pp. 705-711 ◽  
Author(s):  
A Matagne ◽  
M F Ghuysen ◽  
J M Frère
Keyword(s):  
Kinetic Study ◽  
Clavulanic Acid ◽  
Active Site ◽  
Rate Constants ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Class A

The interactions between three class A beta-lactamases and three beta-lactamase inactivators (clavulanic acid, sulbactam and olivanic acid MM13902) were studied. Interestingly, the interaction between the Streptomyces cacaoi beta-lactamase and clavulanate indicated little irreversible inactivation. With sulbactam, irreversible inactivation was found to occur with the three studied enzymes, but no evidence for transiently inactivated adducts was found. Irreversible inactivation of the S. albus G and S. cacaoi enzymes was particularly slow. With olivanate, irreversible inactivation was also observed with the three enzymes, but with the S. cacaoi enzyme, no hydrolysis could be detected. A tentative summary of the results found in the literature is also presented (including 6 beta-halogenopenicillanates), and the general conclusions underline the diversity of the mechanisms and the wide variations of the rate constants observed when class A beta-lactamases interact with beta-lactamase inactivators, in agreement with the behaviours of the same enzymes towards their good and poor substrates.

scholarly journals Characterization of the penA and penR genes of Burkholderia cepacia 249 which encode the chromosomal class A penicillinase and its LysR-type transcriptional regulator.

1997 ◽  
Vol 41 (11) ◽  
pp. 2399-2405 ◽  
Author(s):  
S Trépanier ◽  
A Prince ◽  
A Huletsky
Keyword(s):  
Burkholderia Cepacia ◽  
Klebsiella Oxytoca ◽  
Molecular Size ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Reading Frame ◽  
Beta Lactamases ◽  
Class A ◽  
High Degree ◽  
Degree Of Similarity

Burkholderia cepacia is recognized as an important pathogen in the lung infections of patients with cystic fibrosis. An inducible beta-lactamase activity has been associated with increased resistance to beta-lactam antibiotics in clinical isolates of B. cepacia. In this study, we report the revised sequence of the penA gene, which encodes the inducible penicillinase of B. cepacia, and show that it belongs to the molecular class A beta-lactamases and exhibits a high degree of similarity to the chromosomal beta-lactamase of Klebsiella oxytoca. Analysis of the nucleotide sequence of the DNA region directly upstream of the penA coding sequence revealed an open reading frame (penR), the transcription of which was oriented opposite to that of penA and whose initiation was 130 bp away from that of penA. Two potential ribosome-binding sites and two overlapping -10 and -35 promoter sequences were identified in the intercistronic region. The predicted translation product of penR was a polypeptide of 301 amino acids with an estimated molecular size of 33.2 kDa. The deduced polypeptide of penR showed a high degree of similarity with AmpR-like transcriptional activators of class A and C beta-lactamases, with identities of 59 and 58.7% with Pseudomonas aeruginosa PAO1 AmpR and Proteus vulgaris B317 CumR, respectively. The N-terminal portion of B. cepacia PenR was predicted to include a helix-turn-helix motif, which may bind the LysR motif identified in the intercistronic region. Induction of PenA by imipenem was shown to be dependent upon the presence of PenR. Expression of the cloned B. cepacia penA and penR genes in Escherichia coli SNO302 (ampD) resulted in a high basal and hyperinducible PenA activity. These results suggest that the regulation of the PenA penicillinase of B. cepacia 249 is similar to that observed in other class A and class C beta-lactamases that are under the control of a divergently transcribed AmpR-like regulator.

scholarly journals Kinetics of inactivation of β-lactamase I by 6 β-bromopenicillanic acid

1980 ◽  
Vol 187 (3) ◽  
pp. 797-802 ◽  
Author(s):  
V Knott-Hunziker ◽  
B S Orlek ◽  
P G Sammes ◽  
S G Waley
Keyword(s):  
Bacillus Cereus ◽  
Rate Constant ◽  
Active Site ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Rate Determining Step ◽  
Competitive Model ◽  
Lactam Ring ◽  
Acid Inactivation ◽  
Kinetics Of

The kinetics of the inactivation of beta-lactamase I from Bacillus cereus 569 by preparations of 6 alpha-bromopenicillanic acid showed unexpected features. These can be quantitatively accounted for on the basis of the inactivator being the epimer, 6 beta-bromopenicillanic acid. At pH 9.2, the rate-determining step in the inactivation is the formation of the inactivator. When pure 6 beta-bromopenicillanic acid is used to inactivate beta-lactamase I, simple second-order kinetics are observed. The inactivated enzyme has a new absorption peak at 326 nm. The rate constant for inactivation has the same value as the rate constant for appearance of absorption at 326 nm; the rate-determining step may thus be fission of the beta-lactam ring of 6 beta-bromopenicillanic acid. Inactivation is slower in the presence of substrate, and the observed kinetics can be quantitatively accounted for on a simple competitive model. The results strongly suggest that inactivation is a consequence of reaction at the active site.

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