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Literature summary extracted from

  • Golden, E.; Paterson, R.; Tie, W.; Anandan, A.; Flematti, G.; Molla, G.; Rosini, E.; Pollegioni, L.; Vrielink, A.
    Structure of a class III engineered cephalosporin acylase: Comparisons with class I acylase and implications for differences in substrate specificity and catalytic activity (2013), Biochem. J., 451, 217-226.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.1.93 recombinant expression of wild-type enzyme and of unaltered mutant and selenomethionine-labeled mutant H57betaS/H70betaS in Escherichia coli strain BL21(DE3)pLysS Pseudomonas sp.

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.5.1.93 recombinant wild-type enzyme, and unaltered mutant and selenomethionine-labeled mutant H57betaS/H70betaS enzyme, hanging drop vapour diffusion method, reservoir solutions containing 30% PEG, 20% glycerol, and 100 mM Tris, pH 8.0, for 2-4 h, the crystals are cryoprotected using paratone, X-ray diffraction structure determination and analysis at 1.57-2.48 A resolution, modeling Pseudomonas sp.

Protein Variants

EC Number Protein Variants Comment Organism
3.5.1.93 H57betaS/H70betaS site-directed mutagenesis, interest in designing a single step enzymatic conversion of cephalosporin C to (7R)-7-aminocephalosporanate catalyzed by a true cephalosporin C acylase, the engineered mutant displays enhanced catalytic efficiency on cephalosporin C over glutaryl-7-aminocephalosporanic acid compared to the wild-type enzyme. The nucleophilic catalytic serine residue, Ser1beta, is situated at the base of the active site cavity Pseudomonas sp.
3.5.1.93 M165alphaS/H57betaS/H70betaS site-directed mutagenesis, the engineered mutant shows higher activity on both cephalosporin C and (7R)-7-(4-carboxybutanamido)cephalosporanate compared to the double H57betaS/H70betaS mutant Pseudomonas sp.

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.5.1.93 (7R)-7-(4-carboxybutanamido)cephalosporanate inhibition of mutant M165alphaS/H57betaS/H70betaS, no inhibition of wild-type enzyme and mutant H57betaS/H70betaS Pseudomonas sp.

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.1.93 1.5
-
 (7R)-7-(4-carboxybutanamido)cephalosporanate recombinant wild-type enzyme, pH 8.0, 25°C Pseudomonas sp.
3.5.1.93 6.9
-
 (7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS, pH 8.0, 25°C Pseudomonas sp.
3.5.1.93 9.5
-
 cephalosporin C recombinant wild-type enzyme, pH 8.0, 25°C Pseudomonas sp.
3.5.1.93 12.2
-
 cephalosporin C recombinant mutant H57betaS/H70betaS, pH 8.0, 25°C Pseudomonas sp.
3.5.1.93 21.9
-
 (7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant M165alphaS/H57betaS/H70betaS, pH 8.0, 25°C Pseudomonas sp.
3.5.1.93 27.7
-
 cephalosporin C recombinant mutant M165alphaS/H57betaS/H70betaS, pH 8.0, 25°C Pseudomonas sp.

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.5.1.93 (7R)-7-(4-carboxybutanamido)cephalosporanate + H2O Pseudomonas sp.
-
 (7R)-7-aminocephalosporanate + glutarate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.5.1.93 Pseudomonas sp.
-
-
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
3.5.1.93 proteolytic modification the enzyme is synthesized as a single folded precursor protein made of 782 amino acid residues that, once folded, undergoes an autocatalytic processing event to produce the mature alpha/beta heterodimer, an internal linker segment of ten residues (GDASDAAGGG) is removed from the pre-enzyme between the final alpha- and beta-chains, maturation process, overview Pseudomonas sp.

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.1.93 recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)pLysS Pseudomonas sp.

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.1.93 (7R)-7-(4-carboxybutanamido)cephalosporanate + H2O
-
 Pseudomonas sp. (7R)-7-aminocephalosporanate + glutarate
-
 ?
3.5.1.93 cephalosporin C + H2O the wild-type enzyme shows weak activity with cephalosporin C, while the activity of double mutant H57betaS/H70betaS with this substrate is enhanced Pseudomonas sp. (7R)-7-aminocephalosporanate + 2-aminoadipate
-
 ?

Subunits

EC Number Subunits Comment Organism
3.5.1.93 heterodimer alphabeta Pseudomonas sp.
3.5.1.93 More a deep cavity constitutes the active site. The nucleophilic catalytic serine residue, Ser1beta, is situated at the base of the active site cavity Pseudomonas sp.

Synonyms

EC Number Synonyms Comment Organism
3.5.1.93 cephalosporin acylase
-
 Pseudomonas sp.
3.5.1.93 class III acylase
-
 Pseudomonas sp.
3.5.1.93 class III GA
-
 Pseudomonas sp.
3.5.1.93 class III glutaryl acylase
-
 Pseudomonas sp.
3.5.1.93 glutaryl acylase
-
 Pseudomonas sp.
3.5.1.93 glutaryl-7-(7-aminocephalosporanic acid) acylase
-
 Pseudomonas sp.
3.5.1.93 glutaryl-7-ACA acylase
-
 Pseudomonas sp.
3.5.1.93 VAC
-
 Pseudomonas sp.
3.5.1.93 VAC acylase
-
 Pseudomonas sp.

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.1.93 25
-
 assay at Pseudomonas sp.

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.5.1.93 8
-
 assay at Pseudomonas sp.

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
3.5.1.93 1
-
 (7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant M165alphaS/H57betaS/H70betaS, pH 8.0, 25°C Pseudomonas sp.

General Information

EC Number General Information Comment Organism
3.5.1.93 additional information a deep cavity constitutes the active site, structure overview. The nucleophilic catalytic serine residue, Ser1beta, is situated at the base of the active site cavity, ligand covalently binds to the catalytic serine residue forming a tetrahedral adduct and mimickingc the transition state of the enzyme for both the maturation step and the catalysis of the substrates Pseudomonas sp.