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Literature summary for 3.4.16.4 extracted from

  • Rhazi, N.; Charlier, P.; Dehareng, D.; Engher, D.; Vermeire, M.; Frere, J.M.; Nguyen-Disteche, M.; Fonze, E.
    Catalytic mechanism of the Streptomyces K15 DD-transpeptidase/penicillin-binding protein probed by site-directed mutagenesis and structural analysis (2003), Biochemistry, 42, 2895-2906.
    View publication on PubMed
Search for more literature for 3.4.16.4

Cloned(Commentary)

Cloned (Comment) Organism
mutant enzyme S96A is produced in cytoplasm of Escherichia coli. Mutant enzymes K38H, C98A and C98N are produced in culture medium of Streptomyces lividans Streptomyces sp.

Crystallization (Commentary)

Crystallization (Comment) Organism
hanging drop vapor diffusion method. Mutant enzymes S96A, K38H, C98A and C98N are produced in culture medium of Streptomyces lividans Streptomyces sp.

Protein Variants

Protein Variants Comment Organism
C98A half-life at 72°C increases to 13 min, compared to 10 min for the wild-type enzyme. Half-life at 60°C increases to 220 min compared to 170 min for the wild-type enzyme. The value of the second-order acylation rate constant is not very different from those of the wild-type enzyme with the exception of the cephalotin, oxacillin and piperacillin values which are increased 5-10fold Streptomyces sp.
C98N half-life at 72°C decreases to 6 min, compared to 10 min for the wild-type enzyme. Half-life at 60°C decreases to 30 min compared to 170 min for the wild-type enzyme. The acylation rate constants by most of the beta-lactams are increased 10-70fold compared to the wild-type enzyme. The deacylation rate constant values als increases Streptomyces sp.
K38H half-life at 72°C decreases to 3.5 min, compared to 10 min for the wild-type enzyme. The mutant enzyme fails to bind beta-lactams Streptomyces sp.
S96A half-life at 72°C decreases to 8 min, compared to 10 min for the wild-type enzyme. Half-life at 60°C decreases to 120 min compared to 170 min for the wild-type enzyme. The mutant enzyme is inactive in terms of beta-lactam binding with the exception of cefoxitin, for which a residual activity is detected.The value of the second-order acylation rate constant is decreased by a factor of 3000, and that of the deacylation rate constant is decreased 120fold Streptomyces sp.

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.22
-
 benzoyl-Gly-thiolactate pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
0.3
-
 benzoyl-Gly-thiolactate pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
0.35
-
 Nalpha,Nepsilon-Diacetyl-L-Lys-D-Ala-D-Ala above, pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
0.4
-
 N-alpha-acetyl-L-Lys-D-Ala-thiolactate pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
0.6
-
 Nalpha-acetyl-L-Lys-D-Ala-thiolactate pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
0.8
-
 benzoyl-Gly-thioglycolate pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
1
-
 benzoyl-Gly-thiolactate pH 7.2, 37°C, mutant enzyme K38H Streptomyces sp.
1.2
-
 phenylacetyl-D-Ala-thiolactate pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
1.2
-
 phenylacetyl-D-Ala-thiolactate pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
1.3
-
 benzoyl-D-Ala-thioglycolate pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
1.5
-
 benzoyl-D-Ala-thioglycolate pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
2
-
 Nalpha,Nepsilon-diacetyl-L-Lys-D-Ala-thiolactate pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
2.4
-
 phenylacetyl-D-Ala-thioglycolate pH 7.2, 37°C, mutant enzyme K38H Streptomyces sp.
3
-
 phenylacetyl-D-Ala-thioglycolate above, pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
3
-
 phenylacetyl-D-Ala-thioglycolate above, pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
3
-
 Nalpha,Nepsilon-diacetyl-L-Lys-D-Ala-thiolactate above, pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
3
-
 benzoyl-Gly-thioglycolate above, pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
6.2
-
 Nalpha,Nepsilon-Diacetyl-L-Lys-D-Ala-D-Ala pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
9.4
-
 Nalpha-Acetyl-L-Lys-D-Ala-D-Ala pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
30
-
 Nalpha-Acetyl-L-Lys-D-Ala-D-Ala pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.

Localization

Localization Comment Organism GeneOntology No. Textmining
cytoplasm mutant enzyme S96A is produced in cytoplasm of Escherichia coli Streptomyces sp. 5737
-
extracellular mutant enzymes K38H, C98A and C98N are produced in culture medium of Streptomyces lividans Streptomyces sp.
-
-

Organism

Organism UniProt Comment Textmining
Streptomyces sp. P39042 K15
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
benzoyl-D-Ala-thioglycolate + H2O
-
 Streptomyces sp. ?
-
 ?
benzoyl-Gly-thioglycolate + H2O
-
 Streptomyces sp. ?
-
 ?
benzoyl-Gly-thiolactate + H2O
-
 Streptomyces sp. ?
-
 ?
N-alpha-acetyl-L-Lys-D-Ala-thiolactate + H2O
-
 Streptomyces sp. ?
-
 ?
Nalpha,Nepsilon-diacetyl-L-Lys-D-Ala-D-Ala + H2O
-
 Streptomyces sp. Nalpha,Nepsilon-diacetyl-L-Lys-D-Ala + D-Ala
-
 ?
Nalpha,Nepsilon-diacetyl-L-Lys-D-Ala-thiolactate + H2O
-
 Streptomyces sp. ?
-
 ?
Nalpha-Acetyl-L-Lys-D-Ala-D-Ala + H2O
-
 Streptomyces sp. Nalpha-Acetyl-L-Lys-D-Ala + D-Ala
-
 ?
phenylacetyl-D-Ala-thioglycolate + H2O
-
 Streptomyces sp. ?
-
 ?
phenylacetyl-D-Ala-thiolactate + H2O
-
 Streptomyces sp. ?
-
 ?

Synonyms

Synonyms Comment Organism
DD-transpeptidase/penicillin-binding protein
-
 Streptomyces sp.

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
60
-
 half-lives: 120 min for mutant S96A, 220 min for mutant C98A, 30 min for mutant C98N and 170 min for the wild-type enzyme Streptomyces sp.
72
-
 half-lives: 3.5 min for mutant K38H, 8 min for mutant S96A, 6 min for mutant C98A, 13 min for mutant enzyme C98A and 10 min for the wild-type enzyme Streptomyces sp.

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.006
-
 benzoyl-Gly-thiolactate pH 7.2, 37°C, mutant enzyme K38H Streptomyces sp.
0.015
-
 phenylacetyl-D-Ala-thioglycolate pH 7.2, 37°C, mutant enzyme K38H Streptomyces sp.
0.017
-
 benzoyl-Gly-thiolactate pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
0.024
-
 benzoyl-Gly-thioglycolate above, pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
0.08
-
 benzoyl-Gly-thiolactate pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
0.08
-
 benzoyl-Gly-thioglycolate pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
0.1
-
 N-alpha-acetyl-L-Lys-D-Ala-thiolactate pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
0.11
-
 benzoyl-D-Ala-thioglycolate pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
0.13
-
 Nalpha-Acetyl-L-Lys-D-Ala-D-Ala pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
0.2
-
 Nalpha,Nepsilon-Diacetyl-L-Lys-D-Ala-D-Ala above, pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
0.3
-
 benzoyl-D-Ala-thioglycolate pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
0.3
-
 Nalpha,Nepsilon-Diacetyl-L-Lys-D-Ala-D-Ala pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
0.43
-
 Nalpha,Nepsilon-diacetyl-L-Lys-D-Ala-thiolactate above, pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
0.45
-
 Nalpha-Acetyl-L-Lys-D-Ala-D-Ala pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
0.5
-
 phenylacetyl-D-Ala-thioglycolate above, pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
0.6
-
 phenylacetyl-D-Ala-thiolactate pH 7.2, 37°C, wild-type enzyme Streptomyces sp.
0.8
-
 N-alpha-acetyl-L-Lys-D-Ala-thiolactate pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
0.87
-
 phenylacetyl-D-Ala-thioglycolate above, pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
1.1
-
 Nalpha,Nepsilon-diacetyl-L-Lys-D-Ala-thiolactate pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
2.2
-
 phenylacetyl-D-Ala-thiolactate pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.
6.08
-
 phenylacetyl-D-Ala-thioglycolate above, pH 7.2, 37°C, mutant enzyme C98A Streptomyces sp.