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 (Comment) | Organism |
---|---|
phylogenetic analysis, recombinant exxpression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)pLysS | Pseudomonas sp. |
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 | 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 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 | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas sp. | - |
- |
- |
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 | Comment | Organism |
---|---|---|
cephalosporin C acylase | - |
Pseudomonas sp. |
glutaryl acylase | - |
Pseudomonas sp. |
VAC acylase | - |
Pseudomonas sp. |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Pseudomonas sp. |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Pseudomonas sp. |
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 | 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. |