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Literature summary for 3.5.1.93 extracted from

  • Conti, G.; Pollegioni, L.; Molla, G.; Rosini, E.
    Strategic manipulation of an industrial biocatalyst - evolution of a cephalosporin C acylase (2014), FEBS J., 281, 2443-2455.
    View publication on PubMed

Application

Application Comment Organism
synthesis the enzyme mutant may be suitable for industrial application of the mono-step process for cephalosporin C conversion to (7R)-7-aminocephalosporanate, which is the precursor for production of semisynthetic cephalosporins Pseudomonas sp.

Cloned(Commentary)

Cloned (Comment) Organism
phylogenetic analysis, recombinant exxpression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)pLysS Pseudomonas sp.

Protein Variants

Protein Variants Comment Organism
H57betaS/H70betaS site-directed mutagenesis, the engineered mutant shows increased Vmax on cephalosporin C compared to the wild-type enzyme Pseudomonas sp.
H57betaS/H70betaS/A215alphaY site-directed mutagenesis, the engineered mutant shows increased Vmax on cephalosporin C compared to the wild-type enzyme Pseudomonas sp.
H57betaS/H70betaS/F58betaD random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme Pseudomonas sp.
H57betaS/H70betaS/F58betaI random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme Pseudomonas sp.
H57betaS/H70betaS/F72betaR random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme Pseudomonas sp.
H57betaS/H70betaS/I176betaK random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme Pseudomonas sp.
H57betaS/H70betaS/I176betaQ random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme Pseudomonas sp.
H57betaS/H70betaS/I176betaT random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme Pseudomonas sp.
H57betaS/H70betaS/L154betaY random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme Pseudomonas sp.
H57betaS/H70betaS/L154betaY 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 highly enhanced catalytic efficiency, with a 11000fold increase in specificity constant for CephC versus glutaryl-7-amino cephalosporanic acid, on cephalosporin C over glutaryl-7-aminocephalosporanic acid compared to the wild-type enzyme under conditions resembling those used at industrial level because of its high kinetic efficiency and the absence of substrate or product inhibition effects Pseudomonas sp.
H57betaS/H70betaS/L154betaY/M165alphaS site-directed mutagenesis, the engineered mutant shows increased Vmax on cephalosporin C compared to the wild-type enzyme Pseudomonas sp.
H57betaS/H70betaS/L175betaT random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme Pseudomonas sp.
H57betaS/H70betaS/M165alphaS site-directed mutagenesis, the engineered mutant shows increased Vmax on cephalosporin C compared to the wild-type enzyme Pseudomonas sp.
H57betaS/H70betaS/M165alphaS/I176betaS site-directed mutagenesis, the engineered mutant shows increased Vmax on cephalosporin C compared to the wild-type enzyme Pseudomonas sp.
H57betaS/H70betaS/R24betaP random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme Pseudomonas sp.
H57betaS/H70betaS/V25betaR random mutagenesis of mutant H57betaS/H70betaS, the mutant shows altered substrate specificity compared to the wild-type enzyme Pseudomonas sp.
additional information mutant screening of H57betaS/H70betaS triple mutants generated by site-saturation mutagenesis, introduction of multiple mutations, overview Pseudomonas sp.

Inhibitors

Inhibitors Comment Organism Structure
(7R)-7-(4-carboxybutanamido)cephalosporanate substrate inhibition Pseudomonas sp.
(7R)-7-aminocephalosporanate product inhibition Pseudomonas sp.
cephalosporin C substrate inhibition Pseudomonas sp.

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information kinetics of wild-type and mutant enzymes with cephalosporin C, overview Pseudomonas sp.
0.4
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/F58betaD, pH 8.0, 37°C Pseudomonas sp.
0.41
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/M165alphaS/I176betaS, pH 8.0, 37°C Pseudomonas sp.
0.6
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/L176betaK, pH 8.0, 37°C Pseudomonas sp.
1.5
-
cephalosporin C recombinant mutant H57betaS/H70betaS/F58betaD, pH 8.0, 37°C Pseudomonas sp.
1.5
-
cephalosporin C recombinant mutant H57betaS/H70betaS/L176betaC, pH 8.0, 37°C Pseudomonas sp.
1.5
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant wild-type enzyme, pH 8.0, 25°C Pseudomonas sp.
1.8
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/L176betaT, pH 8.0, 37°C Pseudomonas sp.
1.8
-
cephalosporin C recombinant mutant H57betaS/H70betaS/M165alphaS/I176betaS, pH 8.0, 37°C Pseudomonas sp.
2
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/F72betaR, pH 8.0, 37°C Pseudomonas sp.
2
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/L154betaY, pH 8.0, 37°C Pseudomonas sp.
2
-
cephalosporin C recombinant mutant H57betaS/H70betaS/R24betaP, pH 8.0, 37°C Pseudomonas sp.
2.3
-
cephalosporin C recombinant mutant H57betaS/H70betaS/V25betaR, pH 8.0, 37°C Pseudomonas sp.
3.1
-
cephalosporin C recombinant mutant H57betaS/H70betaS/L154betaY/M165alphaS, pH 8.0, 37°C Pseudomonas sp.
3.2
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/L176betaQ, pH 8.0, 37°C Pseudomonas sp.
3.6
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/V25betaR, pH 8.0, 37°C Pseudomonas sp.
3.8
-
cephalosporin C recombinant mutant H57betaS/H70betaS/L175betaT, pH 8.0, 37°C Pseudomonas sp.
3.8
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/L175betaT, pH 8.0, 37°C Pseudomonas sp.
4.5
-
cephalosporin C recombinant mutant H57betaS/H70betaS/L154betaY, pH 8.0, 37°C Pseudomonas sp.
5.4
-
cephalosporin C recombinant mutant H57betaS/H70betaS/L176betaT, pH 8.0, 37°C Pseudomonas sp.
6.3
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/F58betaI, pH 8.0, 37°C Pseudomonas sp.
6.6
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/L154betaY/M165alphaS, pH 8.0, 37°C Pseudomonas sp.
6.9
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS, pH 8.0, 25°C Pseudomonas sp.
7.5
-
cephalosporin C recombinant mutant H57betaS/H70betaS/L176betaK, pH 8.0, 37°C Pseudomonas sp.
9.2
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/R24betaP, pH 8.0, 37°C Pseudomonas sp.
9.5
-
cephalosporin C recombinant mutant wild-type enzyme, pH 8.0, 25°C Pseudomonas sp.
12.2
-
cephalosporin C recombinant mutant H57betaS/H70betaS, pH 8.0, 25°C Pseudomonas sp.
16.4
-
cephalosporin C recombinant mutant H57betaS/H70betaS/F72betaR, pH 8.0, 37°C Pseudomonas sp.
21.9
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/M165alphaS, pH 8.0, 37°C Pseudomonas sp.
27.7
-
cephalosporin C recombinant mutant H57betaS/H70betaS/M165alphaS, pH 8.0, 37°C Pseudomonas sp.
33.7
-
cephalosporin C recombinant mutant H57betaS/H70betaS/F58betaI, pH 8.0, 37°C Pseudomonas sp.

Organism

Organism UniProt Comment Textmining
Pseudomonas sp.
-
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(7R)-7-(4-carboxybutanamido)cephalosporanate + H2O
-
Pseudomonas sp. (7R)-7-aminocephalosporanate + glutarate
-
?
cephalosporin C + H2O the wild-type enzyme shows weak activity with cephalosporin C, but some enzyme mutants show increased activity Pseudomonas sp. (7R)-7-aminocephalosporanate + 2-aminoadipate
-
?
additional information the amino acid moiety of cephalosporin C that are potentially involved in the enzyme substrate selectivity are Met165alpha, His23beta, Arg24beta, Val25beta, Tyr32beta, Phe55beta, Phe58beta, Phe72beta, Leu154beta, Leu175beta, Ile176beta and His178beta, substrate docking in the active site, overview Pseudomonas sp. ?
-
?

Synonyms

Synonyms Comment Organism
cephalosporin C acylase
-
Pseudomonas sp.
glutaryl acylase
-
Pseudomonas sp.
VAC acylase
-
Pseudomonas sp.

Temperature Optimum [°C]

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

pH Optimum

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

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1
-
(7R)-7-aminocephalosporanate recombinant mutant H57betaS/H70betaS/I176betaT, pH 8.0, 25°C Pseudomonas sp.
2
-
(7R)-7-aminocephalosporanate recombinant mutant H57betaS/H70betaS/R24betaP, pH 8.0, 25°C Pseudomonas sp.
6.3
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/F72betaR, pH 8.0, 25°C Pseudomonas sp.
6.9
-
cephalosporin C recombinant mutant H57betaS/H70betaS/L154betaY/M165alphaS, pH 8.0, 25°C Pseudomonas sp.
21
-
(7R)-7-(4-carboxybutanamido)cephalosporanate wild-type enzyme, pH 8.0, 25°C Pseudomonas sp.
21
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/I176betaT, pH 8.0, 25°C Pseudomonas sp.
33
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/I176betaQ, pH 8.0, 25°C Pseudomonas sp.
65
-
(7R)-7-(4-carboxybutanamido)cephalosporanate recombinant mutant H57betaS/H70betaS/I176betaK, pH 8.0, 25°C Pseudomonas sp.
77
-
cephalosporin C recombinant mutant H57betaS/H70betaS/I176betaT, pH 8.0, 25°C Pseudomonas sp.

General Information

General Information Comment Organism
evolution phylogenetic analysis and active site evolution concerning cephalosporin C binding, overview Pseudomonas sp.
additional information substrate binding, molecular modeling study, overview Pseudomonas sp.