scholarly journals Site-directed mutagenesis of the Streptomyces R61 DD-peptidase. Catalytic function of the conserved residues around the active site and a comparison with class-A and class-C beta-lactamases

1992 ◽  
Vol 207 (1) ◽  
pp. 97-102 ◽  
Author(s):  
Ayaovi M. HADONOU ◽  
Jean-Marc WILKIN ◽  
Louis VARETTO ◽  
Bernard JORIS ◽  
Josette LAMOTTE-BRASSEUR ◽  
...  
Keyword(s):  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Conserved Residues ◽  
Beta Lactamases ◽  
Class A ◽  
Catalytic Function ◽  
Class C

scholarly journals The mechanism of action of dd-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 dd-peptidase

1994 ◽  
Vol 301 (2) ◽  
pp. 477-483 ◽  
Author(s):  
J M Wilkin ◽  
A Dubus ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Peptide Substrate ◽  
Binding Properties ◽  
Beta Lactamases ◽  
Penicillin Binding Proteins ◽  
Active Site Cavity

The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the active-site cavity. Thr299 corresponds to the second residue of the Lys-Thr(Ser)-Gly triad, highly conserved in active-site beta-lactamases and penicillin-binding proteins (PBPs). Modification of Thr301 resulted only in minor alterations of the catalytic and penicillin-binding properties of the enzyme. No selective decrease of the rate of acylation was observed for any particular class of compounds. By contrast, the loss of the hydroxy group of the residue in position 299 yielded a seriously impaired enzyme. The rates of inactivation by penicillins were decreased 30-50-fold, whereas the reactions with cephalosporins were even more affected. The efficiency of hydrolysis against the peptide substrate was also seriously decreased. More surprisingly, the mutant was completely unable to catalyse transpeptidation reactions. The conservation of an hydroxylated residue in this position in PBPs is thus easily explained by these results.

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 Amino Acid Residues That Contribute to Substrate Specificity of Class A β-Lactamase SME-1

2005 ◽  
Vol 49 (8) ◽  
pp. 3421-3427 ◽  
Author(s):  
Fahd K. Majiduddin ◽  
Timothy Palzkill
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Class A ◽  
Study Support ◽  
Functional Mutants ◽  
Hydrolysis Of ◽  
Traditional Class

ABSTRACT Carbapenem antibiotics are used as antibiotics of last resort because they possess a broad spectrum of antimicrobial activity and are not easily hydrolyzed by β-lactamases. Recently, class A enzymes, such as the SME-1, NMC-A, and IMI-1 β-lactamases, have been identified with the capacity to hydrolyze carbapenem antibiotics. Traditional class A β-lactamases, such as TEM-1 and SHV-1, are unable to hydrolyze carbapenem antibiotics and exhibit some differences in sequence from those that are able to hydrolyze carbapenem antibiotics. The positions that differ may contribute to the unique substrate specificity of the class A carbapenemase SME-1. Codons in the SME-1 gene representing residues 104, 105, 132, 167, 237, and 241 were randomized by site-directed mutagenesis, and functional mutants were selected for the ability to hydrolyze imipenem, ampicillin, or cefotaxime. Although several positions are important for hydrolysis of β-lactam antibiotics, no single position was found to uniquely contribute to carbapenem hydrolysis. The results of this study support a model whereby the carbapenemase activity of SME-1 is due to a highly distributed set of interactions that subtly alter the structure of the active-site pocket.

scholarly journals Catalytic mechanism of active-site serine β-lactamases: role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly triad

1994 ◽  
Vol 301 (2) ◽  
pp. 485-494 ◽  
Author(s):  
A Dubus ◽  
J M Wilkin ◽  
X Raquet ◽  
S Normark ◽  
J M Frère
Keyword(s):  
Hydroxy Group ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Peptidase Activity ◽  
Beta Lactams ◽  
Beta Lactamases ◽  
Penicillin Binding Proteins ◽  
Class A ◽  
Class C

The role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly [KT(S)G] triad has been studied for a class A and a class C beta-lactamase by site-directed mutagenesis. Surprisingly, the disappearance of this functional group had little impact on the penicillinase activity of both enzymes. The cephalosporinase activity was much more affected for the class A S235A (Ser235-->Ala) and the class C T316V (Thr315-->Val) mutants, but the class C T316A mutant was less impaired. Studies were extended to beta-lactams, where the carboxy group on C-3 of penicillins or C-4 of cephalosporins had been modified. The effects of the mutations were the same on these compounds as on the unmodified regular penicillins and cephalosporins. The results are compared with those obtained with a similar mutant (T299V) of the Streptomyces R61 DD-peptidase. With this enzyme the mutation also affected the interactions with penicillins and severely decreased the peptidase activity. The strict conservation of the hydroxy group on the second residue of the KT(S)G triad is thus much more easy to understand for the DD-peptidase and the penicillin-binding proteins than for beta-lactamases, especially those of class C.

scholarly journals The active sites of the β-lactamases of Streptomyces cacaoi and Streptomyces albus G.

1987 ◽  
Vol 244 (2) ◽  
pp. 427-432 ◽  
Author(s):  
F De Meester ◽  
B Joris ◽  
M V Lenzini ◽  
P Dehottay ◽  
T Erpicium ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Trypsin Digestion ◽  
Conserved Residues ◽  
Beta Lactamases ◽  
Class A ◽  
Denatured Proteins ◽  
Streptomyces Albus ◽  
High Degree

The active-site serine of the extracellular beta-lactamases of Streptomyces cacaoi and Streptomyces albus G has been labelled with beta-iodopenicillanate. The determination of the sequence of the labelled peptides obtained after trypsin digestion of the denatured proteins indicate both enzymes to be class A beta-lactamases. Surprisingly the two Streptomyces enzymes do not appear to be especially homologous, and none of them exhibited a high degree of homology with the Streptomyces R61 DD-peptidase. Our data confirm that, as a family of homologous enzymes, class A is rather heterogeneous, with only a small number of conserved residues in all members of the class.

scholarly journals Role of the conserved amino acids of the ‘SDN’ loop (Ser130, Asp131 and Asn132) in a class A β-lactamase studied by site-directed mutagenesis

1990 ◽  
Vol 271 (2) ◽  
pp. 399-406 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
S Lepage ◽  
J Dusart ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Enzyme Stability ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Mutant Proteins ◽  
Bond Forming ◽  
Class A ◽  
Active Site Cavity

Ser130, Asp131 and Asn132 (‘SDN’) are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.

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.

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