Mechanism of inhibition of the class C .beta.-lactamase of Enterobacter cloacae P99 by phosphonate monoesters

Biochemistry ◽  
1992 ◽  
Vol 31 (25) ◽  
pp. 5869-5878 ◽  
Author(s):  
Jubrail Rahil ◽  
R. F. Pratt
Keyword(s):  
Enterobacter Cloacae ◽  
Beta Lactamase ◽  
Mechanism Of Inhibition ◽  
Class C

scholarly journals The active site of the P99 β-lactamase from Enterobacter cloacae

1984 ◽  
Vol 223 (1) ◽  
pp. 271-274 ◽  
Author(s):  
B Joris ◽  
J Dusart ◽  
J M Frere ◽  
J van Beeumen ◽  
E L Emanuel ◽  
...  
Keyword(s):  
Active Site ◽  
Enterobacter Cloacae ◽  
Beta Lactamase ◽  
Serine Residue ◽  
Class C

Labelling the beta-lactamase of Enterobacter cloacae P99 with a poor substrate or a mechanism-based inactivator points to an active-site serine residue in a sequence closely resembling that of the ampC beta-lactamase. These results establish the P99 enzyme as a class-C beta-lactamase, and the concurrence of the two approaches helps to confirm the reliability of determining active-site sequences with the aid of mechanism-based inactivators.

scholarly journals Breakdown of the stereospecificity of dd-peptidases and β-lactamases with thiolester substrates

1995 ◽  
Vol 309 (2) ◽  
pp. 431-436 ◽  
Author(s):  
C Damblon ◽  
G H Zhao ◽  
M Jamin ◽  
P Ledent ◽  
A Dubus ◽  
...  
Keyword(s):  
Enterobacter Cloacae ◽  
Beta Lactamase ◽  
Strict Preference ◽  
Beta Lactamases ◽  
Class D ◽  
Leaving Group ◽  
Class C ◽  
Strict Specificity ◽  
Peptide Analogues ◽  
Penultimate Position

With peptide analogues of their natural substrates (the glycopeptide units of nascent peptidoglycan), the DD-peptidases exhibit a strict preference for D-Ala-D-Xaa C-termini. Gly is tolerated as the C-terminal residue, but with a significantly decreased activity. These enzymes were also known to hydrolyse various ester and thiolester analogues of their natural substrates. Some thiolesters with a C-terminal leaving group that exhibited L stereochemistry were significantly hydrolysed by some of the enzymes, particularly the Actinomadura R39 DD-peptidase, but the strict specificity for a D residue in the penultimate position was fully retained. These esters and thiolesters also behave as substrates for beta-lactamases. In this case, thiolesters exhibiting L stereochemistry in the ultimate position could also be hydrolysed, mainly by the class-C and class-D enzymes. However, more surprisingly, the class-C Enterobacter cloacae P99 beta-lactamase also hydrolysed thiolesters containing an L residue in the penultimate position, sometimes with a higher efficiency than the D isomer.

scholarly journals AmpC beta-lactamase enzymes are ubiquitous in catfish (Clarias gariepinus) cultured in the Nigerian catfish grow-out pond systems

2019 ◽  
Vol 30 (1) ◽  
pp. 25-28
Author(s):  
Faith I. Akinnibosun ◽  
Odaro S. Imade
Keyword(s):  
Bacterial Pathogens ◽  
Clarias Gariepinus ◽  
Enterobacter Cloacae ◽  
Regulatory Agencies ◽  
Beta Lactamase ◽  
Bacterial Strains ◽  
Class C ◽  
Catfish Ponds ◽  
Almost All ◽  
Family Class

Abstract This research was performed to ascertain the ubiquity of bacterial pathogens which hyper-produced AmpC β-lactamase enzymes in adult catfish cultured in the Nigerian catfish grow-out pond systems. Phenotypic and molecular methods were used to isolate and identify bacterial pathogens that hyper-produced AmpC β-lactamase enzymes. The AmpC β-lactamase enzymes produced by the bacterial pathogens were subsequently characterized by BLASTX and RPS-BLAST bioinformatics software as well as with the Cn3D molecular modelling software. Findings from the present study indicated that pathogenic bacterial strains which hyper-produced the AmpC β-lactamase enzymes were isolated from 49 catfish samples out of the 54 catfish samples which were examined. The pathogenic bacterial strains were mainly identified as Citrobacter freundii MGH 150, Enterobacter cloacae NG 14, and Enterobacter cloacae subspecies dissolvens HKE 15. The AmpC β-lactamase enzymes produced by the bacterial pathogens were also respectively identified as cephalosporinase hydrolyzing class C CMY-LAT-MOX-ACT-MIR-FOX, CMY2/MIR/ACT/EC family class C beta-lactamase, and CMY2/MIR/ACT/EC family class C beta-lactamase. The presence of AmpC enzymes that are hyper-produced by bacterial pathogens which were isolated from almost all the catfish examined calls for urgent monitoring/surveillance of the Nigerian catfish ponds by the relevant regulatory agencies.

scholarly journals Purification of a class C A-type β-lactamase from a derepressed strain of Enterobacter cloacae. Comparison of the wild-type and mutant enzyme with those from strains P99, 208 and GN7471

1989 ◽  
Vol 260 (3) ◽  
pp. 705-710 ◽  
Author(s):  
M N Graham ◽  
T J Mantle
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Gene ◽  
Enterobacter Cloacae ◽  
Cross Reactivity ◽  
Permeability Barrier ◽  
Beta Lactamase ◽  
Wild Type ◽  
Beta Lactams ◽  
Class C

Enterobacter cloacae strain 5822 expresses low levels of a class C beta-lactamase which can be induced 100-fold by imipenem. Mutants that constitutively express high levels of beta-lactamase can be selected on aztreonam or cefotaxime. The beta-lactamase from one such mutant (5822M2) has been purified to homogeneity and compared on the basis of subunit Mr, pI, substrate specificity, inhibitor sensitivity and immunological cross-reactivity with the enzyme from strains P99, GN7471 and 208, which have been studied previously. The enzyme from strain 5822M2 is clearly related to these other forms and is of the A-type according to the criteria of Seeberg, Tolxdorff-Neutzling & Wiedemann [Antimicrob. Agents Chemother. (1983) 23, 918-925]. The enzyme from the wild-type strain (5822) is shown to be identical to that found in the depressed strain (5822M2), indicating that the mutation is in a regulatory gene. A detailed analysis of the kinetics of the enzyme from strain 5822M2 shows that all of the beta-lactams studied are substrates and that a mechanism involving the formation of an acyl-enzyme is probably applicable in every case. The substrates however can clearly be grouped into two classes, i.e. ‘good’ substrates with kcat. values of 80-1200 s-1 and ‘poor’ substrates/good inhibitors with kcat. values of 0.009-0.00007 s-1. The permeability barrier to aztreonam is 4-fold less in the derepressed strain when compared with the wild-type strain. This is associated with significant changes in the expression of outer membrane porins. The observed resistance in the derepressed mutant appears to be linked to the elevated levels of beta-lactamase (3000-fold) rather than to the modest changes in the permeability barrier.

scholarly journals Evolution of an enzyme activity: crystallographic structure at 2-A resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase

1993 ◽  
Vol 90 (23) ◽  
pp. 11257-11261 ◽  
Author(s):  
E Lobkovsky ◽  
P C Moews ◽  
H Liu ◽  
H Zhao ◽  
J M Frere ◽  
...  
Keyword(s):  
Binding Site ◽  
Enterobacter Cloacae ◽  
Crystallographic Structure ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
X Ray Crystallography ◽  
Class A ◽  
Beta Lactam Antibiotics ◽  
Class C

The structure of the class C ampC beta-lactamase (cephalosporinase) from Enterobacter cloacae strain P99 has been established by x-ray crystallography to 2-A resolution and compared to a class A beta-lactamase (penicillinase) structure. The binding site for beta-lactam (penicillinase) structure. The binding site for beta-lactam antibiotics is generally more open than that in penicillinases, in agreement with the ability of the class C beta-lactamases to better bind third-generation cephalosporins. Four corresponding catalytic residues (Ser-64/70, Lys-67/73, Lys-315/234, and Tyr-150/Ser-130 in class C/A) lie in equivalent positions within 0.4 A. Significant differences in positions and accessibilities of Arg-349/244 may explain the inability of clavulanate-type inhibitors to effectively inactivate the class C beta-lactamases. Glu-166, required for deacylation of the beta-lactamoyl intermediate in class A penicillinases, has no counterpart in this cephalosporinase; the nearest candidate, Asp-217, is 10 A from the reactive Ser-64. A comparison of overall tertiary folding shows that the cephalosporinase, more than the penicillinase, is broadly similar to the ancestral beta-lactam-inhibited enzymes of bacterial cell wall synthesis. On this basis, it is proposed that the cephalosporinase is the older of the two beta-lactamases, and, therefore, that a local refolding in the active site, rather than a simple point mutation, was required for the primordial class C beta-lactamase to evolve to the class A beta-lactamase having an improved ability to catalyze the deacylation step of beta-lactam hydrolysis.

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 Peptidase activity of β-lactamases

1999 ◽  
Vol 341 (2) ◽  
pp. 409-413 ◽  
Author(s):  
Noureddine RHAZI ◽  
Moreno GALLENI ◽  
Michael I. PAGE ◽  
Jean-Marie FRÈRE
Keyword(s):  
Molecular Mass ◽  
Enterobacter Cloacae ◽  
Peptidase Activity ◽  
Rate Enhancement ◽  
Enhancement Factors ◽  
Lactam Ring ◽  
Class C ◽  
Hydrolysis Of

Although β-lactamases have generally been considered as being devoid of peptidase activity, a low but significant hydrolysis of various N-acylated dipeptides was observed with representatives of each class of β-lactamases. The kcat/Km values were below 0.1 M-1˙s-1, but the enzyme rate enhancement factors were in the range 5000-20000 for the best substrates. Not unexpectedly, the best ‘peptidase’ was the class C β-lactamase of Enterobacter cloacae P99, but, more surprisingly, the activity was always higher with the phenylacetyl- and benzoyl-D-Ala-D-Ala dipeptides than with the diacetyl- and α-acetyl-L-Lys-D-Ala-D-Ala tripeptides, which are the preferred substrates of the low-molecular-mass, soluble DD-peptidases. A comparison between the β-lactamases and DD-peptidases showed that it might be as difficult for a DD-peptidase to open the β-lactam ring as it is for the β-lactamases to hydrolyse the peptides, an observation which can be explained by geometric and stereoelectronic considerations.

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