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Literature summary for 3.5.2.6 extracted from

  • Avci, F.G.; Altinisik, F.E.; Vardar Ulu, D.; Ozkirimli Olmez, E.; Sariyar Akbulut, B.
    An evolutionarily conserved allosteric site modulates beta-lactamase activity (2016), J. Enzyme Inhib. Med. Chem., 31, 33-40 .
    View publication on PubMed

Application

Application Comment Organism
drug development the PWP triad is an evolutionarily conserved motif unique to class A beta-lactamases aligning its allosteric site and hence is an effective potential target for enzyme regulation and selective drug design Escherichia coli

Cloned(Commentary)

Cloned (Comment) Organism
gene blaTEM-1, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Escherichia coli

Protein Variants

Protein Variants Comment Organism
additional information no significant loss in thermal stability for the point mutants compared to the wild-type enzyme Escherichia coli
P226A site-directed mutagenesis, the mutant has 92% reduced activity compared to wild-type Escherichia coli
P252A site-directed mutagenesis, the mutant has 85% reduced activity compared to wild-type Escherichia coli
W229A site-directed mutagenesis, the mutant has 98% reduced activity compared to wild-type Escherichia coli
W229F site-directed mutagenesis, the mutant has 84% reduced activity compared to wild-type Escherichia coli
W229Y site-directed mutagenesis, the mutant has 82% reduced activity compared to wild-type Escherichia coli

Inhibitors

Inhibitors Comment Organism Structure
additional information the PWP triad is an evolutionarily conserved motif unique to class A beta-lactamases aligning its allosteric site and hence is an effective potential target for enzyme regulation and selective drug design Escherichia coli

Organism

Organism UniProt Comment Textmining
Escherichia coli Q6SJ61
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Escherichia coli

Renatured (Commentary)

Renatured (Comment) Organism
refolding of recombinant enzyme from periplasmic protein pellets by resuspension in 5 mL 50 mM Tris buffer, pH 7.8, with addition pf 10 ml 8 M urea Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
CENTA + H2O commercial chromogenic substrate Escherichia coli ?
-
?

Synonyms

Synonyms Comment Organism
blaTEM-1
-
Escherichia coli
TEM-1 beta-lactamase
-
Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Escherichia coli

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
48
-
the wild-type Tm is around 48°C occurring over a temperature range of about 23°C (35-58°C) for both the wild-type and the three W229 mutants Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
assay at Escherichia coli

General Information

General Information Comment Organism
evolution The PWP triad is sequentially and structurally conserved in class a beta-lactamase family, the evolutionarily conserved allosteric site modulates beta-lactamase activity Escherichia coli
malfunction while point mutations in the PWP triad preserve the overall secondary structures around the allosteric site, they result in a more open and dynamic global structure with decreased chemical stability and increased aggregation propensity. These mutant enzymes with a less compact hydrophobic core around the allosteric site display significant activity loss Escherichia coli
additional information the evolutionarily conserved PWP triad located at the N-terminus of the H10 helix directly interacts with the allosteric site in TEM-1 beta-lactamase and regulates its activity. The PWP triad is an evolutionarily conserved motif unique to class A beta-lactamases aligning its allosteric site. The H10 helix C-terminus forms part of the active site, specifically K243 participating in catalysis and substrate binding, while P226-W229-P252 (PWP triad) residues at the N-terminus participate in aromatic ring stacking that stabilize the helix. Together with the T-shaped aromatic interaction between W229 and W290, this extensive interaction network modulates the flexibility of H10 and its interactions with other secondary structural elements in the hydrophobic inhibitor binding pocket, suggesting the contribution of these residues to allostery. Structure comparisons, overview Escherichia coli