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 A dramatic change in the rate-limiting step of β-lactam hydrolysis results from the substitution of the active-site serine residue by a cysteine in the class-C β-lactamase of Enterobacter cloacae 908R

1993 ◽  
Vol 292 (2) ◽  
pp. 537-543 ◽  
Author(s):  
A Dubus ◽  
D Monnaie ◽  
C Jacobs ◽  
S Normark ◽  
J M FrÉre
Keyword(s):  
Active Site ◽  
Enterobacter Cloacae ◽  
Order Rate Constant ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Serine Residue ◽  
Rate Limiting Step ◽  
Class C ◽  
Rate Limiting ◽  
Selective Influence

A cysteine residue has been substituted for the active-site serine of the class-C beta-lactamase produced by Enterobacter cloacae 908R by site-directed mutagenesis. The modified protein exhibited drastically reduced kcat./Km values on all tested substrates. However, this decrease was due to increased Km values with some substrates and to decreased kcat. values with others. These apparently contradictory results could be explained by a selective influence of the mutation on the first-order rate constant characteristic of the acylation step, a hypothesis which was confirmed by the absence of detectable acylenzyme accumulation with all the tested substrates, with the sole exception of cefoxitin.

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 β-lactamase of Enterobacter cloacae P99. Chemical properties, N-terminal sequence and interaction with 6β-halogenopenicillanates

1985 ◽  
Vol 228 (1) ◽  
pp. 241-248 ◽  
Author(s):  
B Joris ◽  
F De Meester ◽  
M Galleni ◽  
G Reckinger ◽  
J Coyette ◽  
...  
Keyword(s):  
Active Site ◽  
Chemical Properties ◽  
Enterobacter Cloacae ◽  
Order Rate Constant ◽  
Beta Lactamase ◽  
Serine Residue ◽  
Terminal Sequence ◽  
Beta Lactamases ◽  
A Value ◽  
K 12

The beta-lactamase of Enterobacter cloacae P99 consists of one polypeptide chain of Mr 39000 devoid of disulphide bridges and free thiol groups. It contains an unusually high proportion of tyrosine and tryptophan. The N-terminal sequence exhibits overlaps with the tryptic peptide obtained after labelling the active site with 6 beta-iodopenicillanate. The active-site serine residue is at position 64. The homology with the chromosomal beta-lactamase of Escherichia coli K 12 (ampC gene) is lower within the 25 residues of the N-terminal portion than around the active-site serine residue. The P99 beta-lactamase is inactivated by 6 beta-bromo- and 6 beta-iodo-penicillanate, with a second-order rate constant of 110-140M-1 X s-1 at 30 degrees C and pH 7.0, a value that is much lower than that observed with class-A beta-lactamases.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

scholarly journals A comparative study of class-D β-lactamases

1993 ◽  
Vol 292 (2) ◽  
pp. 555-562 ◽  
Author(s):  
P Ledent ◽  
X Raquet ◽  
B Joris ◽  
J Van Beeumen ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Gene Sequence ◽  
Catalytic Properties ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Beta Lactamase ◽  
Serine Residue ◽  
Beta Lactamases ◽  
Class D ◽  
Burst Kinetics

Three class-D beta-lactamases (OXA2, OXA1 and PSE2) were produced and purified to protein homogeneity. 6 beta-Iodopenicillanate inactivated the OXA2 enzyme without detectable turnover. Labelling of the same beta-lactamase with 6 beta-iodo[3H]penicillanate allowed the identification of Ser-70 as the active-site serine residue. In agreement with previous reports, the apparent M(r) of the OXA2 enzyme as determined by molecular-sieve filtration, was significantly higher than that deduced from the gene sequence, but this was not due to an equilibrium between a monomer and a dimer. The heterogeneity of the OXA2 beta-lactamase on ion-exchange chromatography contrasted with the similarity of the catalytic properties of the various forms. A first overview of the enzymic properties of the three ‘oxacillinases’ is presented. With the OXA2 enzyme, ‘burst’ kinetics, implying branched pathways, seemed to prevail with many substrates.

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 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.

Role of lysine-67 in the active site of class C beta-lactamase from Citrobacter freundii GN346

1990 ◽  
Vol 188 (1) ◽  
pp. 15-22 ◽  
Author(s):  
Kikuo TSUKAMOTO ◽  
Katsumi TACHIBANA ◽  
Noriko YAMAZAKI ◽  
Yuko ISHII ◽  
Kumiko UJHE ◽  
...  
Keyword(s):  
Active Site ◽  
Citrobacter Freundii ◽  
Beta Lactamase ◽  
Class C

scholarly journals Identification of the site of covalent attachment of nafcillin, a reversible suicide inhibitor of β-lactamase

1992 ◽  
Vol 281 (1) ◽  
pp. 191-196 ◽  
Author(s):  
A K Tan ◽  
A L Fink
Keyword(s):  
Staphylococcus Aureus ◽  
Rate Constant ◽  
Active Site ◽  
Covalent Attachment ◽  
Beta Lactamase ◽  
Peptide Fragment ◽  
Serine Residue ◽  
Complete Inactivation ◽  
Enzyme Intermediate ◽  
Inhibited Enzyme

Nafcillin was shown to reversibly inhibit beta-lactamase from Staphylococcus aureus PC1 with characteristics indicative of a type A inhibitor [Citri, Samuni & Zyk (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1048-1052]. At nafcillin concentrations above 80 mM, complete inactivation occurred within 200 s. Upon removal of the excess nafcillin the inhibited enzyme was re-activated completely, with a rate constant of 2.0 x 10(-3) s-1 (25 degrees C). The inhibited enzyme was shown to be in the form of a covalent acyl-enzyme intermediate. Digestion by pepsin and trypsin yielded a single nafcillin-labelled peptide fragment which was isolated, sequenced and shown to be: Ala-Tyr-Ala-Ser-Thr-Ser-Lys. This sequence corresponds to the region surrounding the active-site serine residue, Ser-70, indicating that the inhibitor is covalently attached to the same residue as productive substrates.

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