scholarly journals Sequence analysis and enzyme kinetics of the L2 serine beta-lactamase from Stenotrophomonas maltophilia.

1997 ◽  
Vol 41 (7) ◽  
pp. 1460-1464 ◽  
Author(s):  
T R Walsh ◽  
A P MacGowan ◽  
P M Bennett
Keyword(s):  
Amino Acids ◽  
Sequence Analysis ◽  
Clavulanic Acid ◽  
Active Site ◽  
Stenotrophomonas Maltophilia ◽  
Leader Peptide ◽  
Beta Lactamase ◽  
Class A ◽  
Cloned Gene ◽  
Kinetics Of

The L2 serine active-site beta-lactamase from Stenotrophomonas maltophilia has been classified as a clavulanic acid-sensitive cephalosporinase. The gene encoding this enzyme from S. maltophilia 1275 IID has been cloned on a 3.3-kb fragment into pK18 under the control of a Ptac promoter to generate recombinant plasmid pUB5840; when expressed in Escherichia coli, this gene confers resistance to cephalosporins and penicillins. Sequence analysis has revealed an open reading frame (ORF) of 909 bp with a GC content of 71.6%, comparable to that of the L1 metallo-beta-lactamase gene (68.4%) from the same bacterium. The ORF encodes an unmodified protein of 303 amino acids with a predicted molecular mass of 31.5 kDa, accommodating a putative leader peptide of 27 amino acids. Comparison of the amino acid sequence with those of other beta-lactamases showed it to be most closely related (54% identity) to the BLA-A beta-lactamase from Yersinia enterocolitica. Sequence identity is most obvious near the STXK active-site motif and the SDN loop motif common to all serine active-site penicillinases. Sequences outside the conserved regions display low homology with comparable regions of other class A penicillinases. Kinetics of the enzyme from the cloned gene demonstrated an increase in activity with cefotaxime but markedly less activity with imipenem than previously reported. Hence, the S. maltophilia L2 beta-lactamase is an inducible Ambler class A beta-lactamase which would account for the sensitivity to clavulanic acid.

scholarly journals Burkholderia pseudomallei Class A β-Lactamase Mutations That Confer Selective Resistance against Ceftazidime or Clavulanic Acid Inhibition

2003 ◽  
Vol 47 (7) ◽  
pp. 2082-2087 ◽  
Author(s):  
Chanwit Tribuddharat ◽  
Richard A. Moore ◽  
Patricia Baker ◽  
Donald E. Woods
Keyword(s):  
Sequence Analysis ◽  
Enzyme Kinetics ◽  
Clavulanic Acid ◽  
Burkholderia Pseudomallei ◽  
Acid Inhibition ◽  
Regulator Gene ◽  
Class A ◽  
Resistance Patterns ◽  
Kinetics Of ◽  
Lactamase Inhibitor

ABSTRACT Burkholderia pseudomallei, the causative agent of melioidosis, is inherently resistant to a variety of antibiotics including aminoglycosides, macrolides, polymyxins, and β-lactam antibiotics. Despite resistance to many β-lactams, ceftazidime and β-lactamase inhibitor-β-lactam combinations are commonly used for treatment of melioidosis. Here, we examine the enzyme kinetics of β-lactamase isolated from mutants resistant to ceftazidime and clavulanic acid inhibition and describe specific mutations within conserved motifs of the β-lactamase enzyme which account for these resistance patterns. Sequence analysis of regions flanking the B. pseudomallei penA gene revealed a putative regulator gene located downstream of penA. We have cloned and sequenced the penA gene from B. mallei and found it to be identical to penA from B. pseudomallei.

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 IMI-1 beta-lactamase, a class A carbapenem-hydrolyzing enzyme from Enterobacter cloacae.

1996 ◽  
Vol 40 (9) ◽  
pp. 2080-2086 ◽  
Author(s):  
B A Rasmussen ◽  
K Bush ◽  
D Keeney ◽  
Y Yang ◽  
R Hare ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Clavulanic Acid ◽  
Regulatory Protein ◽  
Enterobacter Cloacae ◽  
Amino Acid Sequences ◽  
Beta Lactamase ◽  
Gene Encoding ◽  
Class A ◽  
Lactamase Gene

In 1984, a year prior to the U.S. approval of imipenem for clinical use, a wound isolate and a bile isolate of Enterobacter cloacae were obtained from two patients in a California hospital. These isolates were resistant to imipenem, penicillins, and inhibitor combinations; early cephalosporins such as cephalothin, cefamandole, and cefoxitin; and cefoperazone. However, they were susceptible (MICs, < 4 micrograms/ml) to cefotaxime, ceftriaxone, ceftazidime, and moxalactam. Both strains produced an apparent TEM-1 beta-lactamase; an inducible NmcA-type imipenem-hydrolyzing beta-lactamase, IMI-1, with a pl of 7.05; and an inducible beta-lactamase with a pI of 8.1, typical of an E. cloacae AmpC beta-lactamase. Purified IMI-1 hydrolyzed imipenem and benzylpenicillin at modest rates, but more slowly than cephaloridine. The enzyme was inhibited by clavulanic acid and tazobactam. EDTA did not inhibit the cephaloridine-hydrolyzing activity. The beta-lactamase gene encoding IMI-1, imiA1, was cloned from E. cloacae 1413B. Sequence analysis identified the imiA1 gene as encoding a class A serine beta-lactamase. Both the imiA1 DNA and encoded amino acid sequences shared greater than 95% identity with the NmcA gene and its encoded protein. DNA sequence analysis also identified a gene upstream of imiA1 that shares > 95% identity with nmcR and that may encode a regulatory protein. In conclusion, IMI-1, a carbapenem-hydrolyzing beta-lactamase inhibited by clavulanic acid, was identified as a group 2f, class A, carbapenem-hydrolyzing cephalosporinase.

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 Imipenem as substrate and inhibitor of β-lactamases

1988 ◽  
Vol 253 (2) ◽  
pp. 323-328 ◽  
Author(s):  
J Monks ◽  
S G Waley
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacillus Cereus ◽  
Reversible Reaction ◽  
Clinical Use ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Class A ◽  
Class C ◽  
Kinetics Of ◽  
Carbapenem Antibiotic

The interaction between imipenem, a carbapenem antibiotic, and two representative beta-lactamases has been studied. The first enzyme was beta-lactamase I, a class-A beta-lactamase from Bacillus cereus; imipenem behaved as a slow substrate (kcat. 6.7 min-1, Km 0.4 mM at 30 degrees C and at pH 7) that reacted by a branched pathway. There was transient formation of an altered species formed in a reversible reaction; this species was probably an acyl-enzyme in a slightly altered, but considerably more labile, conformation. The kinetics of the reaction were investigated by measuring both the concentration of the substrate and the activity of the enzyme, which fell and then rose again more slowly. The second enzyme was the chromosomal class-C beta-lactamase from Pseudomonas aeruginosa; imipenem was a substrate with a low kcat. (0.8 min-1) and a low Km (0.7 microM). Possible implications for the clinical use of imipenem are considered.

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 In VitroandIn VivoProperties of BAL30376, a β-Lactam and Dual β-Lactamase Inhibitor Combination with Enhanced Activity against Gram-Negative Bacilli That Express Multiple β-Lactamases

2011 ◽  
Vol 55 (4) ◽  
pp. 1510-1519 ◽  
Author(s):  
Malcolm G. P. Page ◽  
Clothilde Dantier ◽  
Eric Desarbre ◽  
Bérangère Gaucher ◽  
Klaus Gebhardt ◽  
...  
Keyword(s):  
Clavulanic Acid ◽  
Stenotrophomonas Maltophilia ◽  
Multidrug Resistant ◽  
Triple Combination ◽  
Class A ◽  
Link Type ◽  
Class D ◽  
Class C ◽  
Inhibitor Combination ◽  
Lactamase Inhibitor

ABSTRACTBAL30376 is a triple combination comprising a siderophore monobactam,BAL19764; a novel bridged monobactam,BAL29880, which specifically inhibits class C β-lactamases; and clavulanic acid, which inhibits many class A and some class D β-lactamases. The MIC90was ≤4 μg/ml (expressed as the concentration ofBAL19764) for most species of theEnterobacteriaceaefamily, including strains that produced metallo-β-lactamases and were resistant to all of the other β-lactams tested. The MIC90forStenotrophomonas maltophiliawas 2 μg/ml, for multidrug-resistant (MDR)Pseudomonas aeruginosait was 8 μg/ml, and for MDRAcinetobacterandBurkholderiaspp. it was 16 μg/ml. The presence of the class C β-lactamase inhibitorBAL29880contributed significantly to the activity ofBAL30376against strains ofCitrobacter freundii,Enterobacterspecies,Serratia marcescens, andP. aeruginosa. The presence of clavulanic acid contributed significantly to the activity against many strains ofEscherichia coliandKlebsiella pneumoniaethat produced class A extended-spectrum β-lactamases. The activity ofBAL30376against strains with metallo-β-lactamases was largely attributable to the intrinsic stability of the monobactamBAL19764toward these enzymes. Considering its three components,BAL30376was unexpectedly refractory toward the development of stable resistance.

scholarly journals Cloning, Expression, and Sequence Analysis of the Gene Encoding the Alkali-Stable, Thermostable Endoxylanase from Alkalophilic, Mesophilic Bacillus sp. Strain NG-27

2000 ◽  
Vol 66 (6) ◽  
pp. 2631-2635 ◽  
Author(s):  
Naveen Gupta ◽  
Vanga Shiva Reddy ◽  
Sankar Maiti ◽  
Amit Ghosh
Keyword(s):  
Amino Acids ◽  
Sequence Analysis ◽  
Active Site ◽  
Cysteine Residue ◽  
Open Reading Frame ◽  
Bacillus Sp ◽  
Gene Encoding ◽  
Reading Frame ◽  
The Family ◽  
Site Characteristic

ABSTRACT Alkalophilic Bacillus sp. strain NG-27 produces a 42-kDa endoxylanase active at 70°C and at a pH of 8.4. The gene for this endoxylanase was cloned and sequenced. The gene contained one open reading frame of 1,215 bases. An active site characteristic of the family 10 β-glycanases was recognized between amino acids 303 and 313, with the active glutamate at position 310. Though highly thermostable, the enzyme contains no cysteine residue.

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 Structure of CARB-4 and AER-1 CarbenicillinHydrolyzing β-Lactamases

1998 ◽  
Vol 42 (8) ◽  
pp. 1966-1972 ◽  
Author(s):  
François Sanschagrin ◽  
Noureddine Bejaoui ◽  
Roger C. Levesque
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Dna Sequences ◽  
Active Site ◽  
Multiple Alignment ◽  
Dna Homology ◽  
Class A ◽  
Canonical Amino Acid ◽  
Multiple Alignment Analysis ◽  
Alignment Analysis

ABSTRACT We determined the nucleotide sequences ofblaCARB-4 encoding CARB-4 and deduced a polypeptide of 288 amino acids. The gene was characterized as a variant of group 2c carbenicillin-hydrolyzing β-lactamases such as PSE-4, PSE-1, and CARB-3. The level of DNA homology between thebla genes for these β-lactamases varied from 98.7 to 99.9%, while that between these genes andblaCARB-4 encoding CARB-4 was 86.3%. TheblaCARB-4 gene was acquired from some other source because it has a G+C content of 39.1%, compared to a G+C content of 67% for typical Pseudomonas aeruginosa genes. DNA sequencing revealed that blaAER-1 shared 60.8% DNA identity with blaPSE-3 encoding PSE-3. The deduced AER-1 β-lactamase peptide was compared to class A, B, C, and D enzymes and had 57.6% identity with PSE-3, including an STHK tetrad at the active site. For CARB-4 and AER-1, conserved canonical amino acid boxes typical of class A β-lactamases were identified in a multiple alignment. Analysis of the DNA sequences flankingblaCARB-4 and blaAER-1 confirmed the importance of gene cassettes acquired via integrons inbla gene distribution.

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