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Literature summary for 1.3.99.5 extracted from

  • van Oosterwijk, N.; Knol, J.; Dijkhuizen, L.; van der Geize, R.; Dijkstra, B.W.
    Structure and catalytic mechanism of 3-ketosteroid-DELTA4-(5alpha)-dehydrogenase from Rhodococcus jostii RHA1 genome (2012), J. Biol. Chem., 287, 30975-30983.
    View publication on PubMedView publication on EuropePMC

Crystallization (Commentary)

Crystallization (Comment) Organism
to 1.6 A resolution, structure reveals three conserved residues, Y319, Y466, and S468 in a pocket near the isoalloxazine ring system of the FAD co-factor. Crystal structure with bound product 4-androstene-3,17-dione shows that residue S468 is in a position in which it can serve as the base abstracting the 4beta-proton from the C4 atom of the substrate. S468 is assisted by Y319, which possibly is involved in shuttling the proton to the solvent. Y466 is at hydrogen bonding distance to the C3 oxygen atom of the substrate and can stabilize the keto-enol intermediate occurring during the reaction. The FAD N5 atom is in a position to be able to abstract the 5alpha-hydrogen of the substrate as a hydride ion Rhodococcus jostii

Protein Variants

Protein Variants Comment Organism
S468A no residual activity Rhodococcus jostii
S468T about 65% of wild-type catalytic efficiency Rhodococcus jostii
W136A no residual activity Rhodococcus jostii
W136F about 40% of wild-type catalytic efficiency Rhodococcus jostii
Y319F no residual activity Rhodococcus jostii
Y466A no residual activity Rhodococcus jostii
Y466F no residual activity Rhodococcus jostii

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.01
-
(5alpha)-androst-1-en-3,17-dione wild-type, pH 7.4, 30°C Rhodococcus jostii
0.066
-
(5alpha)-androst-1-en-3,17-dione mutant W136F, pH 7.4, 30°C Rhodococcus jostii
0.17
-
(5alpha)-androst-1-en-3,17-dione mutant S468T, pH 7.4, 30°C Rhodococcus jostii

Organism

Organism UniProt Comment Textmining
Rhodococcus jostii Q0S4Q9
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(5alpha)-androst-1-en-3,17-dione + oxidized dichlorophenolindophenol
-
Rhodococcus jostii 1,4-androstadiene-3,17-dione + reduced dichlorophenolindophenol
-
?

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.7
-
(5alpha)-androst-1-en-3,17-dione mutant W136F, pH 7.4, 30°C Rhodococcus jostii
1.6
-
(5alpha)-androst-1-en-3,17-dione mutant S468T, pH 7.4, 30°C Rhodococcus jostii
2.4
-
(5alpha)-androst-1-en-3,17-dione wild-type, pH 7.4, 30°C Rhodococcus jostii

Cofactor

Cofactor Comment Organism Structure
FAD
-
Rhodococcus jostii

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
9
-
(5alpha)-androst-1-en-3,17-dione mutant S468T, pH 7.4, 30°C Rhodococcus jostii
70
-
(5alpha)-androst-1-en-3,17-dione mutant W136F, pH 7.4, 30°C Rhodococcus jostii
2400
-
(5alpha)-androst-1-en-3,17-dione wild-type, pH 7.4, 30°C Rhodococcus jostii