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Literature summary for 3.5.1.93 extracted from

  • Ferrario, V.; Fischer, M.; Zhu, Y.; Pleiss, J.
    Modelling of substrate access and substrate binding to cephalosporin acylases (2019), Sci. Rep., 9, 12402 .
    View publication on PubMedView publication on EuropePMC

Application

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

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 [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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Pseudomonas sp. N176 A0A1D8GRD5 wild-type M31betaF variant
-

Substrates and Products (Substrate)

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

Synonyms Comment Organism
cephalosporin acylase
-
Pseudomonas sp. N176

General Information

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