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Literature summary extracted from

  • Rohman, A.; van Oosterwijk, N.; Thunnissen, A.M.; Dijkstra, B.W.
    Crystal structure and site-directed mutagenesis of 3-ketosteroid Delta1-dehydrogenase from Rhodococcus erythropolis SQ1 explain its catalytic mechanism (2013), J. Biol. Chem., 288, 35559-35568 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.3.99.4
-
 Rhodococcus erythropolis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.3.99.4 in complex with the reaction product 1,4-androstadiene-3,17-dione, to 2.3 A resolution. Key active site residues are Tyr119, Tyr318, Tyr487, and Gly491 Rhodococcus erythropolis

Protein Variants

EC Number Protein Variants Comment Organism
1.3.99.4 Y119F almost complete loss of activity Rhodococcus erythropolis
1.3.99.4 Y318F almost complete loss of activity Rhodococcus erythropolis
1.3.99.4 Y487F almost complete loss of activity Rhodococcus erythropolis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.3.99.4 0.037
-
 4-androstene-3,17-dione wild-type, pH 8.0, 22°C Rhodococcus erythropolis
1.3.99.4 0.193
-
 4-androstene-3,17-dione mutant Y487F, pH 8.0, 22°C Rhodococcus erythropolis
1.3.99.4 0.335
-
 4-androstene-3,17-dione mutant Y119F, pH 8.0, 22°C Rhodococcus erythropolis

Organism

EC Number Organism UniProt Comment Textmining
1.3.99.4 Rhodococcus erythropolis Q9RA02
-
-
1.3.99.4 Rhodococcus erythropolis SQ1 Q9RA02
-
-

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.3.99.4 72.9
-
 pH 8.0, 22°C Rhodococcus erythropolis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.3.99.4 4-androstene-3,17-dione + 2,6-dichlorophenol-indophenol
-
 Rhodococcus erythropolis 1,4-androstadiene-3,17-dione + reduced 2,6-dichlorophenol-indophenol
-
 ?
1.3.99.4 4-androstene-3,17-dione + 2,6-dichlorophenol-indophenol
-
 Rhodococcus erythropolis SQ1 1,4-androstadiene-3,17-dione + reduced 2,6-dichlorophenol-indophenol
-
 ?
1.3.99.4 9alpha-hydroxy-4-androstene-3,17-dione + 2,6-dichlorophenol-indophenol
-
 Rhodococcus erythropolis 9alpha-hydroxy-androst-1,4-diene-3,17-dione + reduced 2,6-dichlorophenol-indophenol
-
 ?
1.3.99.4 9alpha-hydroxy-4-androstene-3,17-dione + 2,6-dichlorophenol-indophenol
-
 Rhodococcus erythropolis SQ1 9alpha-hydroxy-androst-1,4-diene-3,17-dione + reduced 2,6-dichlorophenol-indophenol
-
 ?
1.3.99.4 additional information in catalytic mechanism Tyr487 and Gly491 work in tandem to promote keto-enol tautomerization and increase the acidity of the C2 hydrogen atoms of the substrate. With assistance of Tyr119, the general base Tyr318 abstracts the axialbeta-hydrogen from C2 as a proton, whereas the FAD accepts the axial alpha-hydrogen from the C1 atom of the substrate as a hydride ion Rhodococcus erythropolis ?
-
 ?
1.3.99.4 additional information in catalytic mechanism Tyr487 and Gly491 work in tandem to promote keto-enol tautomerization and increase the acidity of the C2 hydrogen atoms of the substrate. With assistance of Tyr119, the general base Tyr318 abstracts the axialbeta-hydrogen from C2 as a proton, whereas the FAD accepts the axial alpha-hydrogen from the C1 atom of the substrate as a hydride ion Rhodococcus erythropolis SQ1 ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.3.99.4 DELTA1-KSTD1
-
 Rhodococcus erythropolis
1.3.99.4 KSTD1
-
 Rhodococcus erythropolis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.3.99.4 0.036
-
 4-androstene-3,17-dione mutant Y119F, pH 8.0, 22°C Rhodococcus erythropolis
1.3.99.4 1.7
-
 4-androstene-3,17-dione mutant Y487F, pH 8.0, 22°C Rhodococcus erythropolis
1.3.99.4 68.1
-
 4-androstene-3,17-dione wild-type, pH 8.0, 22°C Rhodococcus erythropolis

Cofactor

EC Number Cofactor Comment Organism Structure
1.3.99.4 FAD the C1 and C2 atoms of the substrate are at reaction distance to the N5 atom of the isoalloxazine ring of FAD and the hydroxyl group of Tyr318, respectively, whereas the C3 carbonyl group is at hydrogen bonding distance from the hydroxyl group of Tyr487 and the backbone amide of Gly491 Rhodococcus erythropolis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.3.99.4 0.108
-
 4-androstene-3,17-dione mutant Y119F, pH 8.0, 22°C Rhodococcus erythropolis
1.3.99.4 9.1
-
 4-androstene-3,17-dione mutant Y487F, pH 8.0, 22°C Rhodococcus erythropolis
1.3.99.4 1838
-
 4-androstene-3,17-dione wild-type, pH 8.0, 22°C Rhodococcus erythropolis