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 Site-directed mutagenesis of the Actinomadura R39 DD-peptidase

1997 ◽  
Vol 327 (2) ◽  
pp. 377-381 ◽  
Author(s):  
Guo-Hua ZHAO ◽  
Colette DUEZ ◽  
Sophie LEPAGE ◽  
Christine FORCEILLE ◽  
Noureddine RHAZI ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Catalytic Properties ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Structural Elements ◽  
Penicillin Binding Protein ◽  
Peptide Substrate ◽  
Binding Properties ◽  
Km Value

The role of various residues in the conserved structural elements of the Actinomadura R39 penicillin-sensitive DD-peptidase has been studied by site-directed mutagenesis. Replacement of Ser-298 of the ‘SDN loop’ by Ala or Gly significantly decreased the kcat/Km value for the peptide substrate, but only by a factor of 15 and had little effect on the other catalytic properties. Mutations of Asn-300 of the same loop and of Lys-410 of the KTG triad yielded very unstable proteins. However, the N300S mutant could be purified as a fusion protein with thioredoxin that exhibited decreased rates of acylation by the peptide substrate and various cephalosporins. Similar fusion proteins obtained with the N300A, K410H and K410N mutants were unstable and their catalytic and penicillin-binding properties were very strongly affected. In transpeptidation reactions, the presence of the acceptor influenced the kcat/Km values, which suggested a catalytic pathway more complex than a simple partition of the acyl-enzyme between hydrolysis and aminolysis. These results are compared with those obtained with two other penicillin-sensitive enzymes, the Streptomyces R61 DD-peptidase and Escherichia coli penicillin-binding protein (PBP) 5.

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.

Access of the substrate to the active site of squalene and oxidosqualene cyclases: comparative inhibition, site-directed mutagenesis and homology-modelling studies

2005 ◽  
Vol 33 (5) ◽  
pp. 1202-1205 ◽  
Author(s):  
S. Oliaro-Bosso ◽  
T. Schulz-Gasch ◽  
S. Taramino ◽  
M. Scaldaferri ◽  
F. Viola ◽  
...  
Keyword(s):  
Active Site ◽  
Homology Modelling ◽  
Critical Role ◽  
Homology Model ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Specific Substrate ◽  
Oxidosqualene Cyclase ◽  
Active Site Cavity

Substrate access to the active-site cavity of squalene-hopene cyclase from Alicyclobacillus acidocaldarious and lanosterol synthase [OSC (oxidosqualene cyclase)] from Saccharomyces cerevisiae was studied by an inhibition, mutagenesis and homology-modelling approach. Crystal structure and homology modelling indicate that both enzymes possess a narrow constriction that separates an entrance lipophilic channel from the active-site cavity. The role of the constriction as a mobile gate that permits substrate passage was investigated by experiments in which critically located Cys residues, either present in native protein or inserted by site-directed mutagenesis, were labelled with specifically designed thiol-reacting molecules. Some amino acid residues of the yeast enzyme, selected on the basis of sequence alignment and a homology model, were individually replaced by residues bearing side chains of different lengths, charges or hydrophobicities. In some of these mutants, substitution severely reduced enzymatic activity and thermal stability. Homology modelling revealed that in these mutants some critical stabilizing interactions could no longer occur. The possible critical role of entrance channel and constriction in specific substrate recognition by eukaryotic OSC is discussed.

Role of the amino acid invariants in the active site of MurG as evaluated by site-directed mutagenesis

Biochimie ◽  
2007 ◽  
Vol 89 (12) ◽  
pp. 1498-1508 ◽  
Author(s):  
Muriel Crouvoisier ◽  
Geneviève Auger ◽  
Didier Blanot ◽  
Dominique Mengin-Lecreulx
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Role of Acidic and Aromatic Amino Acids inRhodobacter CapsulatusCytochromec1. A Site-Directed Mutagenesis Study†

Biochemistry ◽  
2003 ◽  
Vol 42 (29) ◽  
pp. 8818-8830 ◽  
Author(s):  
Jun Li ◽  
Artur Osyczka ◽  
Richard C. Conover ◽  
Michael K. Johnson ◽  
Hong Qin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Aromatic Amino Acids ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
A Site ◽  
Mutagenesis Study

Site-Directed Mutagenesis of Firefly Luciferase Active Site Amino Acids:  A Proposed Model for Bioluminescence Color†

Biochemistry ◽  
1999 ◽  
Vol 38 (40) ◽  
pp. 13223-13230 ◽  
Author(s):  
Bruce R. Branchini ◽  
Rachelle A. Magyar ◽  
Martha H. Murtiashaw ◽  
Shannon M. Anderson ◽  
Lisa C. Helgerson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Firefly Luciferase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Proposed Model
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