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Literature summary for 3.5.1.93 extracted from

  • Otten, L.G.; Sio, C.F.; Vrielink, J.; Cool, R.H.; Quax, W.J.
    Altering the substrate specificity of cephalosporin acylase by directed evolution of the beta-subunit (2002), J. Biol. Chem., 277, 42121-42127.
    View publication on PubMed

Protein Variants

Protein Variants Comment Organism
F229L turnover number for adipyl-7-aminodesacetoxycephalosporanic acid is nealy identical to wild-type value, 1.8fold decrease in KM-value for adipyl-7-aminodesacetoxycephalosporanic acid, 1.4fold decrease in turnover number for glutaryl-7-aminocephalosporanic acid, 1.45fold increase in Km-value for glutaryl-7-aminocephalosporanic acid as compared to wild-type enzyme Pseudomonas sp.
F375L 1.5fold increase in turnover number for adipyl-7-aminodesacetoxycephalosporanic acid, 1.2fold decrease in KM-value for adipyl-7-aminodesacetoxycephalosporanic acid, 3.9fold decrease in turnover number for glutaryl-7-aminocephalosporanic acid, 8.2fold increase in Km-value for glutaryl-7-aminocephalosporanic acid as compared to wild-type enzyme. Improved activity ratio for adipyl-7-aminodesacetoxycephalosporanic acid to glutaryl-7-aminocephalosporanic acid of the mutant enzyme is a consequence of a decreased catalytic efficiency towards glutaryl-7-aminocephalosporanic acid Pseudomonas sp.
M271V/Q291K/T374S 1.3fold increase in turnover number for adipyl-7-aminodesacetoxycephalosporanic acid, 1.4fold decrease in KM-value for adipyl-7-aminodesacetoxycephalosporanic acid, 1.2fold decrease in turnover number for glutaryl-7-aminocephalosporanic acid, 1.3fold increase in Km-value for glutaryl-7-aminocephalosporanic acid as compared to wild-type enzyme Pseudomonas sp.
N266H 1.2fold increase in turnover number for adipyl-7-aminodesacetoxycephalosporanic acid, 6.9fold decrease in KM-value for adipyl-7-aminodesacetoxycephalosporanic acid, 1.3fold decrease in turnover number for glutaryl-7-aminocephalosporanic acid, 2fold decrease in Km-value for glutaryl-7-aminocephalosporanic acid as compared to wild-type enzyme. Nearly 10fold improved catalytic efficiency on adipyl-7-aminodesacetoxycephalosporanic acid, resulting from a 50% increase in turnover-number and a 6fold decrease in KM-value without decreasing the catalytic efficiency on glutaryl-7-aminocephalosporanic acid Pseudomonas sp.
N266S 1.4fold decrease in turnover number for adipyl-7-aminodesacetoxycephalosporanic acid, 2.3fold decrease in KM-value for adipyl-7-aminodesacetoxycephalosporanic acid, 1.95fold decrease in turnover number for glutaryl-7-aminocephalosporanic acid, 2.8fold increase in Km-value for glutaryl-7-aminocephalosporanic acid as compared to wild-type enzyme Pseudomonas sp.

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.016
-
glutaryl-7-aminocephalosporanic acid pH 7.5, 37°C, mutant enzyme N266H Pseudomonas sp.
0.033
-
glutaryl-7-aminocephalosporanic acid pH 7.5, 37°C, wild-type enzyme Pseudomonas sp.
0.043
-
glutaryl-7-aminocephalosporanic acid pH 7.5, 37°C, mutant enzyme M271V/Q291K/T374S Pseudomonas sp.
0.048
-
glutaryl-7-aminocephalosporanic acid pH 7.5, 37°C, mutant enzyme F229L Pseudomonas sp.
0.094
-
glutaryl-7-aminocephalosporanic acid pH 7.5, 37°C, mutant enzyme N266S Pseudomonas sp.
0.14
-
adipyl-7-aminodesacetoxycephalosporanic acid pH 7.5, 37°C, mutant enzyme N266H Pseudomonas sp.
0.27
-
glutaryl-7-aminocephalosporanic acid pH 7.5, 37°C, mutant enzyme F375L Pseudomonas sp.
0.42
-
adipyl-7-aminodesacetoxycephalosporanic acid pH 7.5, 37°C, mutant enzyme N266S Pseudomonas sp.
0.52
-
adipyl-7-aminodesacetoxycephalosporanic acid pH 7.5, 37°C, mutant enzyme F229L Pseudomonas sp.
0.7
-
adipyl-7-aminodesacetoxycephalosporanic acid pH 7.5, 37°C, mutant enzyme M271V/Q291K/T374S Pseudomonas sp.
0.82
-
adipyl-7-aminodesacetoxycephalosporanic acid pH 7.5, 37°C, mutant enzyme F375L Pseudomonas sp.
0.96
-
adipyl-7-aminodesacetoxycephalosporanic acid pH 7.5, 37°C, wild-type enzyme Pseudomonas sp.

Organism

Organism UniProt Comment Textmining
Pseudomonas sp. P07662 SY-77
-
Pseudomonas sp. SY-77 P07662 SY-77
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
adipyl-7-aminodesacetoxycephalosporanic acid + H2O
-
Pseudomonas sp. deacetoxycephalosporanic acid + adipate
-
?
adipyl-7-aminodesacetoxycephalosporanic acid + H2O
-
Pseudomonas sp. SY-77 deacetoxycephalosporanic acid + adipate
-
?
glutaryl-7-aminocephalosporanic acid + H2O
-
Pseudomonas sp. 7-aminocephalosporanate + glutarate
-
?
glutaryl-7-aminocephalosporanic acid + H2O
-
Pseudomonas sp. SY-77 7-aminocephalosporanate + glutarate
-
?

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.34
-
adipyl-7-aminodesacetoxycephalosporanic acid pH 7.5, 37°C, mutant enzyme N266S Pseudomonas sp.
0.46
-
adipyl-7-aminodesacetoxycephalosporanic acid pH 7.5, 37°C, wild-type enzyme Pseudomonas sp.
0.47
-
adipyl-7-aminodesacetoxycephalosporanic acid pH 7.5, 37°C, mutant enzyme F229L Pseudomonas sp.
0.55
-
adipyl-7-aminodesacetoxycephalosporanic acid pH 7.5, 37°C, mutant enzyme N266H Pseudomonas sp.
0.61
-
adipyl-7-aminodesacetoxycephalosporanic acid pH 7.5, 37°C, mutant enzyme M271V/Q291K/T374S Pseudomonas sp.
0.67
-
adipyl-7-aminodesacetoxycephalosporanic acid pH 7.5, 37°C, mutant enzyme F375L Pseudomonas sp.
1.1
-
glutaryl-7-aminocephalosporanic acid pH 7.5, 37°C, mutant enzyme F375L Pseudomonas sp.
2.2
-
glutaryl-7-aminocephalosporanic acid pH 7.5, 37°C, mutant enzyme N266S Pseudomonas sp.
3.1
-
glutaryl-7-aminocephalosporanic acid pH 7.5, 37°C, mutant enzyme F229L Pseudomonas sp.
3.3
-
glutaryl-7-aminocephalosporanic acid pH 7.5, 37°C, mutant enzyme N266H Pseudomonas sp.
3.5
-
glutaryl-7-aminocephalosporanic acid pH 7.5, 37°C, mutant enzyme M271V/Q291K/T374S Pseudomonas sp.
4.3
-
glutaryl-7-aminocephalosporanic acid pH 7.5, 37°C, wild-type enzyme Pseudomonas sp.