scholarly journals Multiple substitutions at position 104 of β-lactamase TEM-1: assessing the role of this residue in substrate specificity

1995 ◽  
Vol 305 (1) ◽  
pp. 33-40 ◽  
Author(s):  
A Petit ◽  
L Maveyraud ◽  
F Lenfant ◽  
J P Samama ◽  
R Labia ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Kinetic Properties ◽  
Third Generation ◽  
Wild Type Enzyme ◽  
Beta Lactamases ◽  
Class A ◽  
Extended Spectrum ◽  
Third Generation Cephalosporins

Residue 104 is frequently mutated from a glutamic acid to a lysine in the extended-spectrum TEM beta-lactamases responsible for the resistance to third-generation cephalosporins in clinical Gram negative strains. Among class A beta-lactamases, it is the most variable residue within a highly conserved loop which delineates one side of the active site of the enzymes. To investigate the role of this residue in the extended-spectrum phenotype, it has been replaced by serine, threonine, lysine, arginine, tyrosine and proline. All these substitutions yield active enzymes, with no drastic changes in kinetic properties compared with the wild-type enzyme, except with cefaclor, but an overall improved affinity for second- and third-generation cephalosporins. Only mutant E104K exhibits a significant ability to hydrolyse cefotaxime. Molecular modelling shows that the substitutions have generally no impact on the conformation of the 101-111 loop as the side chains of residues at position 104 are all turned towards the solvent. Unexpectedly, the E104P mutant turns out to be the most efficient enzyme. All our results argue in favour of an indirect role for this residue 104 in the substrate specificity of the class A beta-lactamases. This residue contributes to the precise positioning of residues 130-132 which are involved in substrate binding and catalysis. Changing residue 104 could also modify slightly the local electrostatic potential in this part of the active site. The limited kinetic impact of the mutations at this position have to be analysed in the context of the microbiological problem of resistance to third-generation cephalosporins. Although mutation E104K improves the ability of the enzyme to hydrolyse these compounds, it is not sufficient to confer true resistance, and is always found in clinical isolates associated with at least one mutation at another part of the active site. It is the combined effect of the two mutations that synergistically enhances the hydrolytic capability of the enzyme towards third-generation cephalosporins.

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 Roles of Residues Cys69, Asn104, Phe160, Gly232, Ser237, and Asp240 in Extended-Spectrum β-Lactamase Toho-1

2010 ◽  
Vol 55 (1) ◽  
pp. 284-290 ◽  
Author(s):  
Akiko Shimizu-Ibuka ◽  
Mika Oishi ◽  
Shoko Yamada ◽  
Yoshikazu Ishii ◽  
Kiyoshi Mura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Binding Site ◽  
Kinetic Properties ◽  
Amino Acid Residues ◽  
Class A ◽  
Extended Spectrum ◽  
Hydrolysis Of

ABSTRACTToho-1, which is also designated CTX-M-44, is an extended-spectrum class A β-lactamase that has high activity toward cefotaxime. In this study, we investigated the roles of residues suggested to be critical for the substrate specificity expansion of Toho-1 in previous structural analyses. Six amino acid residues were replaced one by one with amino acids that are often observed in the corresponding position of non-extended-spectrum β-lactamases. The mutants produced inEscherichia colistrains were analyzed both for their kinetic properties and their effect on drug susceptibilities. The results indicate that the substitutions of Asn104 and Ser237 have certain effects on expansion of substrate specificity, while those of Cys69 and Phe160 have less effect, and that of Asp240 has no effect on the hydrolysis of any substrates tested. Gly232, which had been assumed to increase the flexibility of the substrate binding site, was revealed not to be critical for the expansion of substrate specificity of this enzyme, although this substitution resulted in deleterious effects on expression and stability of the enzyme.

scholarly journals Site-directed mutagenesis of β-lactamase I: role of Glu-166

1994 ◽  
Vol 299 (3) ◽  
pp. 671-678 ◽  
Author(s):  
Y C Leung ◽  
C V Robinson ◽  
R T Aplin ◽  
S G Waley
Keyword(s):  
Active Site ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Kinetic Properties ◽  
Ionization Mass ◽  
Beta Lactamase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Important Residue

Two Glu-166 mutants of beta-lactamase I from Bacillus cereus 569/H were constructed: one with a lengthened side chain (E166Cmc, the S-carboxymethylcysteine mutant) and the other with the side chain shortened and made non-polar (E166A). Their kinetic properties were studied and compared with those of the wild-type and the E166D mutant (with a shortened side chain) previously made by Gibson, Christensen and Waley (1990) (Biochem. J. 272, 613-619). Surprisingly, with good penicillin substrates, Km, kcat. and kcat./Km of the two conservative mutants (E166Cmc and E166D) are similar to those of the non-conservative mutant E166A. Their kcat. values are 3000-fold lower than that of the wild-type enzyme, showing that Glu-166 is a very important residue. The acylenzyme intermediate of E166A and a good substrate, penicillin V, was trapped by acid-quench and observed by electrospray ionization mass spectrometry, suggesting that Glu-166 is more important in catalysing the deacylation step than the acylation step. The beta-lactamase I E166A mutant is about 200-fold more active than the Bacillus licheniformis E166A mutant with nitrocefin or 6 beta-furylacryloyl-amidopenicillanic acid as substrate. This suggested that other groups in the active site of the beta-lactamase I mutant may activate the catalytic water molecule for deacylation.

scholarly journals Crystal Structure of the Extended-Spectrum β-Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β-Lactams and β-Lactamase Inhibitors

2014 ◽  
Vol 58 (10) ◽  
pp. 5994-6002 ◽  
Author(s):  
Melina Ruggiero ◽  
Frédéric Kerff ◽  
Raphaël Herman ◽  
Frédéric Sapunaric ◽  
Moreno Galleni ◽  
...  
Keyword(s):  
Active Site ◽  
Amino Acid Identity ◽  
Conserved Residues ◽  
Class A ◽  
Extended Spectrum ◽  
Active Site Cavity ◽  
Bond Network ◽  
Hydrolysis Of ◽  
Structure And Activity

ABSTRACTPER-2 belongs to a small (7 members to date) group of extended-spectrum β-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most β-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward β-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singulartransbond between residues 166 to 167, which generates an inverted Ω loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A β-lactamases. PER β-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A (“A” indicates an insertion according to Ambler's scheme for residue numbering in PER β-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different β-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior.

scholarly journals Survey and molecular genetics of SHV beta-lactamases in Enterobacteriaceae in Switzerland: two novel enzymes, SHV-11 and SHV-12.

1997 ◽  
Vol 41 (5) ◽  
pp. 943-949 ◽  
Author(s):  
M T Nüesch-Inderbinen ◽  
F H Kayser ◽  
H Hächler
Keyword(s):  
Molecular Genetics ◽  
Third Generation ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Low Resistance ◽  
Beta Lactam Antibiotics ◽  
Extended Spectrum ◽  
Extended Spectrum Beta Lactamases ◽  
Third Generation Cephalosporins

Sixty isolates of Enterobacteriaceae resistant to beta-lactam antibiotics were collected over a period of 2 years in Switzerland and screened by hybridization for the carriage of SHV genes. Thirty-four positive strains were found, and their SHV genes were amplified and sequenced. SHV extended-spectrum beta-lactamases (ESBLs) were found: 13 strains contained SHV-2a, 12 harbored SHV-2, and SHV-5 was found twice. Four strains were shown to contain SHV-1. In addition, we report two new SHV variants, termed SHV-11 (non-ESBL) and SHV-12 (ESBL). In spite of the carriage of SHV ESBLs, many strains showed only low resistance to one or more third-generation cephalosporins. In addition, 26 did not transfer the blaSHV gene in mating experiments.

scholarly journals Effect of the 3′-leaving group on turnover of cephem antibiotics by a class C β-lactamase

1989 ◽  
Vol 259 (1) ◽  
pp. 255-260 ◽  
Author(s):  
L J Mazzella ◽  
R F Pratt
Keyword(s):  
Active Site ◽  
First Generation ◽  
Enterobacter Cloacae ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Class A ◽  
Leaving Group ◽  
Enzyme Intermediates ◽  
Class C ◽  
Third Generation Cephalosporins

It has been previously demonstrated for class A beta-lactamases and the DD-peptidase of Streptomyces R61 that the presence of a leaving group at the 3′-position of a cephalosporin can lead to the generation of more-inert acyl-enzyme intermediates than from cephalosporins lacking such a leaving group, and thus to beta-lactamase inhibitors and potentially better antibiotics. In the present work we extend this result to a class C beta-lactamase, that of Enterobacter cloacae P99. The effect is not seen with first-generation cephalosporins, since here deacylation generally seems faster than elimination of the leaving group, but it does clearly appear with cephamycins and third-generation cephalosporins. The structural and/or mechanistic features of the active site giving rise to this phenomenon may thus be common to all serine beta-lactamases and transpeptidases.

scholarly journals The role of prophylactic antibiotics in hepatitis B virus-related acute-on-chronic liver failure patients at risk of bacterial infection: a retrospective study

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Xiao-Qin Liu ◽  
Xue-Yun Zhang ◽  
Yue Ying ◽  
Jian-Ming Zheng ◽  
Jian Sun ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Clinical Course ◽  
Prophylactic Antibiotics ◽  
Third Generation ◽  
Chronic Liver Failure ◽  
Free Survival ◽  
B Virus ◽  
Third Generation Cephalosporins

Abstract Background Acute-on-chronic liver failure (ACLF) is characterized by an excessive systemic inflammatory response and organ failure and has high mortality. Bacterial infections (BIs) worsen the clinical course of ACLF and carry a poor prognosis in ACLF patients. The efficacy of third-generation cephalosporins has been challenged in recent years. The aim of this study was to characterize the difference between ACLF patients with and without BIs and to provide a reference for medical intervention. Methods A total of 140 patients with hepatitis B virus-related ACLF (HBV-ACLF) hospitalized at the Department of Infectious Diseases, Huashan Hospital, Fudan University (Shanghai, China) between May 2013 and January 2020 were enrolled. Mann-Whitney U test was used to compare the baseline characteristics of HBV-ACLF patients with and without BIs. Univariate and multivariate analyses were performed to find predictors of BIs. The characteristics of BIs and the role of prophylactic antibiotics were profiled. Results A total of 97 episodes of BIs occurred in patients during the course of HBV-ACLF. Patients with and without BIs differed in clinical characteristics. The incidence of BIs showed a positive correlation with the ACLF grade (P = 0.003) and the clinical course (P = 0.003). The 90-day transplant-free survival of patients with BIs was lower than those without BIs (P < 0.0001). Patients administered prophylactic antibiotics showed a lower incidence of BIs and had a higher transplant-free survival probability than those who did not (P = 0.046). No statistical differences in antibiotic efficacy between third-generation and other antibiotics were observed (P = 0.108). Conclusions BIs affected the clinical course and prognosis of patients with HBV-ACLF. Prophylactic antibiotics were of potential clinical importance in the prevention of BIs and improving the clinical course and prognosis in HBV-ACLF patients. Third-generation cephalosporins were qualified for use in antibiotic prophylaxis.

scholarly journals Characterization of beta-lactamase gene blaPER-2, which encodes an extended-spectrum class A beta-lactamase.

1996 ◽  
Vol 40 (3) ◽  
pp. 616-620 ◽  
Author(s):  
A Bauernfeind ◽  
I Stemplinger ◽  
R Jungwirth ◽  
P Mangold ◽  
S Amann ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Beta Lactamase ◽  
Reading Frame ◽  
Beta Lactamases ◽  
Class A ◽  
Extended Spectrum ◽  
The Family ◽  
Extended Spectrum Beta Lactamases ◽  
Lactamase Gene

Plasmidic extended-spectrum beta-lactamases of Ambler class A are mostly inactive against ceftibuten. Salmonella typhimurium JMC isolated in Argentina harbors a bla gene located on a plasmid (pMVP-5) which confers transferable resistance to oxyiminocephalosporins, aztreonam, and ceftibuten. The beta-lactamase PER-2 (formerly ceftibutenase-1; CTI-1) is highly susceptible to inhibition by clavulanate and is located at a pI of 5.4 after isoelectric focusing. The blaPER-2 gene was cloned and sequenced. The nucleotide sequence of a 2.2-kb insert in vector pBluescript includes an open reading frame of 927 bp. Comparison of the deduced amino acid sequence of PER-2 with those of other beta-lactamases indicates that PER-2 is not closely related to TEM or SHV enzymes (25 to 26% homology). PER-2 is most closely related to PER-1 (86.4% homology), an Ambler class A enzyme first detected in Pseudomonas aeruginosa. An enzyme with an amino acid sequence identical to that of PER-1, meanwhile, was found in various members of the family Enterobacteriaceae isolated from patients in Turkey. Our data indicate that PER-2 and PER-1 represent a new group of Ambler class A extended-spectrum beta-lactamases. PER-2 so far has been detected only in pathogens (S. typhimurium, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis) isolated from patients in South America, while the incidence of PER-1-producing strains so far has been restricted to Turkey, where it occurs both in members of the family Enterobacteriaceae and in P. aeruginosa.

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