Cloned (Comment) | Organism |
---|---|
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 (Comment) | Organism |
---|---|
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 | Comment | Organism |
---|---|---|
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. |
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 | Comment | Organism | Structure |
---|---|---|---|
(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] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.5 | - |
(7R)-7-(4-carboxybutanamido)cephalosporanate | recombinant wild-type enzyme, pH 8.0, 25°C | Pseudomonas sp. | |
6.9 | - |
(7R)-7-(4-carboxybutanamido)cephalosporanate | recombinant mutant H57betaS/H70betaS, pH 8.0, 25°C | Pseudomonas sp. | |
9.5 | - |
cephalosporin C | recombinant 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. | |
21.9 | - |
(7R)-7-(4-carboxybutanamido)cephalosporanate | recombinant mutant M165alphaS/H57betaS/H70betaS, pH 8.0, 25°C | Pseudomonas sp. | |
27.7 | - |
cephalosporin C | recombinant mutant M165alphaS/H57betaS/H70betaS, pH 8.0, 25°C | Pseudomonas sp. |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
(7R)-7-(4-carboxybutanamido)cephalosporanate + H2O | Pseudomonas sp. | - |
(7R)-7-aminocephalosporanate + glutarate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas sp. | - |
- |
- |
Posttranslational Modification | Comment | Organism |
---|---|---|
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 (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)pLysS | 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, while the activity of double mutant H57betaS/H70betaS with this substrate is enhanced | Pseudomonas sp. | (7R)-7-aminocephalosporanate + 2-aminoadipate | - |
? |
Subunits | Comment | Organism |
---|---|---|
heterodimer | alphabeta | Pseudomonas sp. |
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 | Comment | Organism |
---|---|---|
cephalosporin acylase | - |
Pseudomonas sp. |
class III acylase | - |
Pseudomonas sp. |
class III GA | - |
Pseudomonas sp. |
class III glutaryl acylase | - |
Pseudomonas sp. |
glutaryl acylase | - |
Pseudomonas sp. |
glutaryl-7-(7-aminocephalosporanic acid) acylase | - |
Pseudomonas sp. |
glutaryl-7-ACA acylase | - |
Pseudomonas sp. |
VAC | - |
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-(4-carboxybutanamido)cephalosporanate | recombinant mutant M165alphaS/H57betaS/H70betaS, pH 8.0, 25°C | Pseudomonas sp. |
General Information | Comment | Organism |
---|---|---|
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. |