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Literature summary for 5.3.3.1 extracted from

  • Choi, G.; Ha, N.C.; Kim, M.S.; Hong, B.H.; Oh, B.H.; Choi, K.Y.
    Pseudoreversion of the catalytic activity of Y14F by the additional substitution(s) of tyrosine with phenylalanine in the hydrogen bond network of DELTA 5-3-ketosteroid isomerase from Pseudomonas putida biotype B (2001), Biochemistry, 40, 6828-6835.
    View publication on PubMed

Crystallization (Commentary)

Crystallization (Comment) Organism
hanging drop vapor diffusion method. Crystal structures of Y14F and Y14F/Y30F/Y55F are determined at 1.8 and 2.0 A resolution, respectively Pseudomonas putida

Protein Variants

Protein Variants Comment Organism
Y14F the hydrogen bond between Asp99 Odelta2 and C3-O of the steroid, which is perturbed by the Y14F mutation, can be partially restored to that in the wild-type enzyme by additional Y30F/Y55F mutations Pseudomonas putida
Y14F/Y30F the turnover-number for 5-androstene-3,17-dione is about 1% of that of the wild-type enzyme, the Km-value is 1.57fold higher than that of the wild-type enzyme Pseudomonas putida
Y14F/Y30F/Y55F the turnover-number for 5-androstene-3,17-dione is about 2.1% of that of the wild-type enzyme, the Km-value is comparable to the Km-value of the wild-type enzyme. The hydrogen bond between Asp99 Odelta2 and C3-O of the steroid, which is perturbed by the Y14F mutation, can be partially restored to that in the wild-type enzyme by additional Y30F/Y55F mutations. The improvement in the catalytic activity of Y14F by the additional Y30f/Y55F mutation is due to the changes in the structural integrity at the catalytic site and the resulting restoration of the proton-transfer mechanism in Y14F/Y30F/Y55F Pseudomonas putida
Y14F/Y30F/Y55F/D99L the turnover-number for 5-androstene-3,17-dione is less than 1.2% of that of the wild-type enzyme, the Km-value is 1.8fold higher than that of the wild-type enzyme Pseudomonas putida
Y14F/Y30F/Y55F/D99N the turnover-number for 5-androstene-3,17-dione is less than 1% of that of the wild-type enzyme, the Km-value is 1.14fold higher than that of the wild-type enzyme Pseudomonas putida
Y14F/Y55F the turnover-number for 5-androstene-3,17-dione is about 1.2% of that of the wild-type enzyme, the Km-value is 1.75fold higher than that of the wild-type enzyme Pseudomonas putida

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0287
-
5-androstene-3,17-dione 25°C, pH 7.0, mutant enzyme Y14F/Y55F Pseudomonas putida
0.0503
-
5-androstene-3,17-dione 25°C, pH 7.0, wild-type enzyme Pseudomonas putida
0.0513
-
5-androstene-3,17-dione 25°C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F Pseudomonas putida
0.0578
-
5-androstene-3,17-dione 25°C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F/D99N Pseudomonas putida
0.0787
-
5-androstene-3,17-dione 25°C, pH 7.0, mutant enzyme Y14F/Y30F Pseudomonas putida
0.0918
-
5-androstene-3,17-dione 25°C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F/D99L Pseudomonas putida

Organism

Organism UniProt Comment Textmining
Pseudomonas putida
-
biotype B
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5-Androstene-3,17-dione
-
Pseudomonas putida 4-Androstene-3,17-dione
-
?

Synonyms

Synonyms Comment Organism
DELTA5-ketosteroid isomerase
-
Pseudomonas putida
KSI
-
Pseudomonas putida

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.052
-
5-androstene-3,17-dione 25°C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F/D99L Pseudomonas putida
1.1
-
5-androstene-3,17-dione 25°C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F/D99N Pseudomonas putida
302
-
5-androstene-3,17-dione 25°C, pH 7.0, mutant enzyme Y14F/Y30F Pseudomonas putida
360
-
5-androstene-3,17-dione 25°C, pH 7.0, mutant enzyme Y14F/Y55F Pseudomonas putida
587
-
5-androstene-3,17-dione 25°C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F Pseudomonas putida
27900
-
5-androstene-3,17-dione 25°C, pH 7.0, wild-type enzyme Pseudomonas putida