Application | Comment | Organism |
---|---|---|
synthesis | biocatalytic synthesis by the cephalosporin acylase from Pseudomonas sp. strain N176 is a promising alternative to chemical semisynthesis of cephalosporins antibiotics | Pseudomonas sp. N176 |
Protein Variants | Comment | Organism |
---|---|---|
F58betaN/H70betaS/I176betaT | site-directed mutagenesis, mutation of wild-type variant M31betaF, comparison of substrate binding structures and abilities with the wild-type, molecular dynamics simulations and modeling, overview | Pseudomonas sp. N176 |
additional information | optimization of enzyme protein engineering for biocatalytic production of cephalosporins, molecular dynamics simulations with wild-type enzyme and mutant M6, analysis of access of the substrate cephalosporin C from the bulk to the active site and stability of the enzyme-substrate complex. In both variants, cephalosporin C is binding to a non-productive substrate binding site (E86alpha, S369beta, S460beta) at the entrance to the binding pocket, preventing substrate access. A second non-productive binding site (G372beta, W376beta, L457beta) is identified within the binding pocket, which competes with the active site for substrate binding | Pseudomonas sp. N176 |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | substrate binding to the protein surface follows a Langmuir model resulting in binding constants K = 7.4 and 9.2 mM for wild-type and mutant M6 enzymes, respectively, which are similar to the experimentally determined Michaelis constants, thermodynamics, overview | Pseudomonas sp. N176 | |
8.1 | - |
cephalosporin C | recombinant enzyme mutant M6, pH and temperature not specified in the publication | Pseudomonas sp. N176 | |
11 | - |
cephalosporin C | recombinant wild-type enzyme, pH and temperature not specified in the publication | Pseudomonas sp. N176 |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
(7R)-7-(4-carboxybutanamido)cephalosporanate + H2O | Pseudomonas sp. N176 | - |
(7R)-7-aminocephalosporanate + glutarate | - |
? | |
cephalosporin C + H2O | Pseudomonas sp. N176 | low activity | 7-aminocephalosporanic acid + 2-amino-5-hydroxypentanoate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas sp. N176 | A0A1D8GRD5 | wild-type M31betaF variant | - |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(7R)-7-(4-carboxybutanamido)cephalosporanate + H2O | - |
Pseudomonas sp. N176 | (7R)-7-aminocephalosporanate + glutarate | - |
? | |
cephalosporin C + H2O | low activity | Pseudomonas sp. N176 | 7-aminocephalosporanic acid + 2-amino-5-hydroxypentanoate | - |
? |
Synonyms | Comment | Organism |
---|---|---|
cephalosporin acylase | - |
Pseudomonas sp. N176 |
General Information | Comment | Organism |
---|---|---|
additional information | based on structural information, a catalytic mechanism of the class III enzyme from Pseudomonas sp. strain N176 is proposed: like other N-terminal hydrolases, the N-terminal amine group acts as a base to deprotonate the hydroxyl group of the same residue. Subsequently, the N-terminal Ser1beta performs a nucleophilic attack to the carbonyl group of the substrate, resulting in the formation of a tetrahedral intermediate, which is stabilized by the oxyanion hole formed by the side chain of Asn242beta and the backbone amino group of His70beta. The reaction proceeds to cephalosporin C hydrolysis via the release of 7-aminocephalosporanic acid and the subsequent nucleophilic attack of a water molecule. Cephalosporin C (CPC) substrate binding structure and kinetics, simulation of the enzyme-substrate complex, detailed overview. The barrier results from the binding of a CPC molecule to three gatekeeper residues (S369beta, S460beta, E86alpha) at the entrance to the binding pocket, modeling using the enzyme's crystal structure, PDB ID 4HSR | Pseudomonas sp. N176 |