Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary for 1.3.99.4 extracted from

  • Luo, J.M.; Cui, H.L.; Jia, H.C.; Li, F.; Cheng, H.J.; Shen, Y.B.; Wang, M.
    Identification, biological characteristics, and active site residues of 3-ketosteroid DELTA1-dehydrogenase homologues from Arthrobacter simplex (2020), J. Agric. Food Chem., 68, 9496-9512 .
    View publication on PubMed

Activating Compound

Activating Compound Comment Organism Structure
acetone activates at up to 25% v/v, inhibits slightly at 50% v/v Pimelobacter simplex
DMSO activates at up to 50% v/v Pimelobacter simplex
ethanol activates at up to 25% v/v, inhibits slightly at 50% v/v Pimelobacter simplex
glycerol activates at up to 25% v/v, inhibits slightly at 50% v/v Pimelobacter simplex
isopropylalcohol activates at up to 25% v/v, inhibits slightly at 50% v/v Pimelobacter simplex
methanol activates at up to 25% v/v, inhibits slightly at 50% v/v Pimelobacter simplex
Tween 60 activates at up to 25% v/v, inhibits slightly at 50% v/v Pimelobacter simplex

Cloned(Commentary)

Cloned (Comment) Organism
isozyme KsdD3, sequence comparisons and phylogenetic analysis, genetic organization of the five KsdD homologues and in silico analysis of ksdD promoter regions, the putative promoter sequence for ksdD3 ORF lies in the intergenic region between bphE3 and ksdD3 ORFs, quantitative real time PCR enzyme expression analysis, recombinant expression of codon-optimized His-tagged enzyme in Escherichia coli strain BL21(DE3) Pimelobacter simplex
isozyme KsdD5, sequence comparisons and phylogenetic analysis, genetic organization of the five KsdD homologues in silico analysis of ksdD promoter regions, quantitative real time PCR enzyme expression analysis, recombinant expression of codon-optimized His-tagged enzyme in Escherichia coli strain BL21(DE3) Pimelobacter simplex

Protein Variants

Protein Variants Comment Organism
A255F site-directed mutagenesis, inactive mutant Pimelobacter simplex
A255G site-directed mutagenesis, inactive mutant Pimelobacter simplex
F266A site-directed mutagenesis, inactive mutant Pimelobacter simplex
G258A site-directed mutagenesis, inactive mutant Pimelobacter simplex
G532A site-directed mutagenesis, the mutant shows over 70% reduced activity compare to wild-type enzyme Pimelobacter simplex
L261A site-directed mutagenesis, inactive mutant Pimelobacter simplex
R256A site-directed mutagenesis, almost inactive mutant Pimelobacter simplex
R256H site-directed mutagenesis, the mutant shows over 70% reduced activity compare to wild-type enzyme Pimelobacter simplex
R273A site-directed mutagenesis, inactive mutant Pimelobacter simplex
V259A site-directed mutagenesis, inactive mutant Pimelobacter simplex
V259F site-directed mutagenesis, inactive mutant Pimelobacter simplex
Y118A site-directed mutagenesis, almost inactive mutant Pimelobacter simplex
Y355A site-directed mutagenesis, the mutant shows over 70% reduced activity compare to wild-type enzyme Pimelobacter simplex
Y528A site-directed mutagenesis, inactive mutant Pimelobacter simplex

Inhibitors

Inhibitors Comment Organism Structure
acetone activates at up to 25% v/v, inhibits slightly at 50% v/v Pimelobacter simplex
Ca2+ 70% inhibition at 1 mM Pimelobacter simplex
Cu2+ over 95% inhibition at 1 mM Pimelobacter simplex
ethanol activates at up to 25% v/v, inhibits slightly at 50% v/v Pimelobacter simplex
Fe3+ 50% inhibition at 1 mM Pimelobacter simplex
glycerol activates at up to 25% v/v, inhibits slightly at 50% v/v Pimelobacter simplex
isopropylalcohol activates at up to 25% v/v, inhibits slightly at 50% v/v Pimelobacter simplex
methanol activates at up to 25% v/v, inhibits slightly at 50% v/v Pimelobacter simplex
Mn2+ 72% inhibition at 1 mM Pimelobacter simplex
NH4+ 10% inhibition at 1 mM Pimelobacter simplex
Ni2+ 90% inhibition at 1 mM Pimelobacter simplex
Tween 60 activates at up to 25% v/v, inhibits slightly at 50% v/v Pimelobacter simplex
Zn2+ 55% inhibition at 1 mM Pimelobacter simplex

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0596
-
progesterone pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex
0.0623
-
testosterone pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex
0.0837
-
canrenone pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex
0.0885
-
17-methyl-testosterone pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex
0.0888
-
cortisone acetate pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
0.0952
-
4-Cholesten-3-one pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex
0.1264
-
4-androstene-3,17-dione pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex
0.1512
-
17alpha,21-dihydroxyprogesterone 21-acetate pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
0.1527
-
17-methyl-testosterone pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
0.1552
-
progesterone pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
0.1552
-
17alpha-hydroxyprogesterone pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
0.159
-
4-androstene-3,17-dione pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
0.1736
-
testosterone pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex

Localization

Localization Comment Organism GeneOntology No. Textmining
cytoplasm
-
Pimelobacter simplex 5737
-
extracellular
-
Pimelobacter simplex
-
-

Metals/Ions

Metals/Ions Comment Organism Structure
additional information no significant effects by 1 mM of EDTA, Mg2+, K+, and Na+ Pimelobacter simplex

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
a 3-oxosteroid + acceptor Pimelobacter simplex
-
a 3-oxo-delta1-steroid + reduced acceptor
-
?

Organism

Organism UniProt Comment Textmining
Pimelobacter simplex
-
-
-
Pimelobacter simplex CGMCC 14539
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged isozyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and ultrafiltration Pimelobacter simplex

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
15700
-
purified recombinant wild-type enzyme, pH 7.0, 30°C, substrate progesterone Pimelobacter simplex
23100
-
purified recombinant wild-type enzyme, pH 7.0, 30°C, substrate testosterone Pimelobacter simplex
40900
-
purified recombinant wild-type enzyme, pH 7.0, 30°C, substrate 4-cholesten-3-one Pimelobacter simplex
51300
-
purified recombinant wild-type enzyme, pH 7.5, 35°C, substrate testosterone Pimelobacter simplex
51600
-
purified recombinant wild-type enzyme, pH 7.5, 35°C, substrate progesterone Pimelobacter simplex
59200
-
purified recombinant wild-type enzyme, pH 7.5, 35°C, substrate cortisone acetate Pimelobacter simplex
66200
-
purified recombinant wild-type enzyme, pH 7.0, 30°C, substrate 17-methyl-testosterone Pimelobacter simplex
72600
-
purified recombinant wild-type enzyme, pH 7.5, 35°C, substrate 4-androstene-3,17-dione Pimelobacter simplex
80600
-
purified recombinant wild-type enzyme, pH 7.0, 30°C, substrate 4-androstene-3,17-dione Pimelobacter simplex
81700
-
purified recombinant wild-type enzyme, pH 7.5, 35°C, substrate 17alpha-hydroxyprogesterone Pimelobacter simplex
90700
-
purified recombinant wild-type enzyme, pH 7.5, 35°C, substrate 17-methyl-testosterone Pimelobacter simplex
91500
-
purified recombinant wild-type enzyme, pH 7.0, 30°C, substrate canrenone Pimelobacter simplex
109200
-
purified recombinant wild-type enzyme, pH 7.5, 35°C, substrate 17alpha,21-dihydroxyprogesterone 21-acetate Pimelobacter simplex

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
17-methyl-testosterone + 2,6-dichlorophenol-indophenol
-
Pimelobacter simplex ? + reduced 2,6-dichlorophenol-indophenol
-
?
17alpha,21-dihydroxyprogesterone 21-acetate + 2,6-dichlorophenol-indophenol Reichstein substance S 21-acetate (RSA), best substrate Pimelobacter simplex ? + reduced 2,6-dichlorophenol-indophenol
-
?
17alpha-hydroxyprogesterone + 2,6-dichlorophenol-indophenol
-
Pimelobacter simplex 17alpha-hydroxy-pregna-1,4-diene-3,20-dione + reduced 2,6-dichlorophenol-indophenol
-
?
4-androstene-3,17-dione + 2,6-dichlorophenol-indophenol
-
Pimelobacter simplex 1,4-androstadiene-3,17-dione + reduced 2,6-dichlorophenol-indophenol
-
?
4-androstene-3,17-dione + 2,6-dichlorophenol-indophenol
-
Pimelobacter simplex CGMCC 14539 1,4-androstadiene-3,17-dione + reduced 2,6-dichlorophenol-indophenol
-
?
4-cholesten-3-one + 2,6-dichlorophenol-indophenol
-
Pimelobacter simplex ? + reduced 2,6-dichlorophenol-indophenol
-
?
a 3-oxosteroid + acceptor
-
Pimelobacter simplex a 3-oxo-delta1-steroid + reduced acceptor
-
?
canrenone + 2,6-dichlorophenol-indophenol best substrate Pimelobacter simplex ? + reduced 2,6-dichlorophenol-indophenol
-
?
cortisone acetate + 2,6-dichlorophenol-indophenol
-
Pimelobacter simplex prednisolone acetate + reduced 2,6-dichlorophenol-indophenol
-
?
additional information assay and detection with phenazine methosulfate (PMS) and 2,6-dichlorophenloindophenol (DCPIP). Catalytic activity and substrate profile of isozyme KsdD3, each KsdD isozyme exhibits distinct substrate profile, overview. KsdD3 is most active and versatile isozyme from Arthrobacter simplex. No activity of KsdD3 with 9alpha-hydroxy-4-androstene-3,17-dione, 17alpha-hydroxyprogesterone, 17alpha,21-dihydroxyprogesterone 21-acetate (RSA), dehydroepiandrosterone, 11beta,17alpha,21-trihydroxypregn-4-ene-3,20-dione, 11beta,17alpha,21-trihydroxypregn-4-ene-3,20-dione acetate, cortisone acetate, 5alpha-androstane-17beta-ol-3-one, and 5alpha-pregnane-3,20-dione Pimelobacter simplex ?
-
-
additional information assay with phenazine methosulfate (PMS) and 2,6-dichlorophenloindophenol (DCPIP). Catalytic activity and substrate profile of isozyme KsdD5, each KsdD isozyme exhibits distinct substrate profile, overview. No activity of KsdD5 with 9alpha-hydroxy-4-androstene-3,17-dione, canrenone, 4-cholest-3-one, dehydroepiandrosterone, 11beta,17alpha,21-trihydroxypregn-4-ene-3,20-dione, 11beta,17alpha,21-trihydroxypregn-4-ene-3,20-dione acetate, 5alpha-androstane-17beta-ol-3-one, and 5alpha-pregnane-3,20-dione Pimelobacter simplex ?
-
-
additional information assay and detection with phenazine methosulfate (PMS) and 2,6-dichlorophenloindophenol (DCPIP). Catalytic activity and substrate profile of isozyme KsdD3, each KsdD isozyme exhibits distinct substrate profile, overview. KsdD3 is most active and versatile isozyme from Arthrobacter simplex. No activity of KsdD3 with 9alpha-hydroxy-4-androstene-3,17-dione, 17alpha-hydroxyprogesterone, 17alpha,21-dihydroxyprogesterone 21-acetate (RSA), dehydroepiandrosterone, 11beta,17alpha,21-trihydroxypregn-4-ene-3,20-dione, 11beta,17alpha,21-trihydroxypregn-4-ene-3,20-dione acetate, cortisone acetate, 5alpha-androstane-17beta-ol-3-one, and 5alpha-pregnane-3,20-dione Pimelobacter simplex CGMCC 14539 ?
-
-
additional information assay with phenazine methosulfate (PMS) and 2,6-dichlorophenloindophenol (DCPIP). Catalytic activity and substrate profile of isozyme KsdD5, each KsdD isozyme exhibits distinct substrate profile, overview. No activity of KsdD5 with 9alpha-hydroxy-4-androstene-3,17-dione, canrenone, 4-cholest-3-one, dehydroepiandrosterone, 11beta,17alpha,21-trihydroxypregn-4-ene-3,20-dione, 11beta,17alpha,21-trihydroxypregn-4-ene-3,20-dione acetate, 5alpha-androstane-17beta-ol-3-one, and 5alpha-pregnane-3,20-dione Pimelobacter simplex CGMCC 14539 ?
-
-
progesterone + 2,6-dichlorophenol-indophenol
-
Pimelobacter simplex pregna-1,4-diene-3,20-dione + reduced 2,6-dichlorophenol-indophenol
-
?
progesterone + 2,6-dichlorophenol-indophenol
-
Pimelobacter simplex CGMCC 14539 pregna-1,4-diene-3,20-dione + reduced 2,6-dichlorophenol-indophenol
-
?
testosterone + 2,6-dichlorophenol-indophenol
-
Pimelobacter simplex 17beta-hydroxyandrost-1,4-diene-3-one + reduced 2,6-dichlorophenol-indophenol
-
?
testosterone + 2,6-dichlorophenol-indophenol
-
Pimelobacter simplex CGMCC 14539 17beta-hydroxyandrost-1,4-diene-3-one + reduced 2,6-dichlorophenol-indophenol
-
?

Subunits

Subunits Comment Organism
More secondary structure analysis and comparison of KsdD isozymes from Arthrobacter simplex, overview Pimelobacter simplex

Synonyms

Synonyms Comment Organism
3-ketosteroid delta1-dehydrogenase
-
Pimelobacter simplex
KsdD
-
Pimelobacter simplex
KsdD3
-
Pimelobacter simplex
KsdD5
-
Pimelobacter simplex

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
isozyme KsdD3 Pimelobacter simplex
35
-
isozyme KsdD5 Pimelobacter simplex

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
15 45 over 40% of maximal activity within this range Pimelobacter simplex

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
20
-
purified recombinant enzyme, completely stable at Pimelobacter simplex
35
-
purified recombinant enzyme, loss of 40% activity Pimelobacter simplex
40
-
purified recombinant enzyme, loss of 80% activity Pimelobacter simplex
55
-
purified recombinant enzyme, inactivation Pimelobacter simplex

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
15.7
-
progesterone pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex
24.7
-
testosterone pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex
27.8
-
4-Cholesten-3-one pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex
45.4
-
17-methyl-testosterone pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex
57.7
-
progesterone pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
59.2
-
testosterone pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
67.4
-
cortisone acetate pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
79.7
-
4-androstene-3,17-dione pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
86.1
-
4-androstene-3,17-dione pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex
92.8
-
17alpha-hydroxyprogesterone pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
95.8
-
canrenone pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex
103.1
-
17-methyl-testosterone pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
126.1
-
17alpha,21-dihydroxyprogesterone 21-acetate pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
isozyme KsdD3 Pimelobacter simplex
7.5
-
isozyme KsdD5 Pimelobacter simplex

pH Range

pH Minimum pH Maximum Comment Organism
6 9 over 40% of maximal activity within this range Pimelobacter simplex

pH Stability

pH Stability pH Stability Maximum Comment Organism
5.5 9 over 40% of activity retained within this range Pimelobacter simplex

Cofactor

Cofactor Comment Organism Structure
2,6-dichlorophenolindophenol DCPIP Pimelobacter simplex
FAD
-
Pimelobacter simplex

pI Value

Organism Comment pI Value Maximum pI Value
Pimelobacter simplex sequence calculation
-
5.43
Pimelobacter simplex sequence calculation
-
5.79

General Information

General Information Comment Organism
malfunction all ksdD-overexpressing strains (isozymes KsdD1-5) not only exhibit a faster initial conversion rate (increased by 22-102%) but also achieve a higher conversion ratio (increased by 15-66%) than that of the control strain during the conversion of 4-androstene-3,17-dione to 1,4-androstadiene-3,17-dione. Apparently, KsdD3 and KsdD2 have more effect on DELTA1-dehydrogenation Pimelobacter simplex
additional information the key residues of isozyme KsdD5 with more versatility are revealed by structural modeling and site-directed mutagenesis. Enzyme residues Y528 and G532 act as an electrophile to promote labilization of the C2 hydrogen atoms of the 3-oxosteroid substrate. With the assistance of Y118, the general base Y355 captured the axial beta-hydrogen from the C2 atom as a proton, whereas FAD accepts the axial alpha-hydrogen from the C1 atom of the substrate as a hydride ion. Finally, a double bond between the C1 and C2 atoms is formed. Local models of the active site residues Y118, Y355, Y528, and G532, A255, R256, G258, G259, and L261, and F266 and R273 for KsdD5 with cofactor FAD Pimelobacter simplex
physiological function 3-ketosteroid DELTA1-dehydrogenase (KsdD) is the key enzyme responsible for DELTA1-dehydrogenation, which is one of the most valuable reactions for steroid catabolism, roles of the putative KsdD homologues in DELTA1-dehydrogenation Pimelobacter simplex

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
263.4
-
progesterone pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex
341
-
testosterone pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
371.8
-
progesterone pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
396.5
-
testosterone pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex
501.3
-
4-androstene-3,17-dione pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
513
-
17-methyl-testosterone pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex
597.9
-
17alpha-hydroxyprogesterone pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
675.2
-
17-methyl-testosterone pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
681.2
-
4-androstene-3,17-dione pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex
759
-
cortisone acetate pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
834
-
17alpha,21-dihydroxyprogesterone 21-acetate pH 7.5, 35°C, recombinant isozyme KsdD3 Pimelobacter simplex
1144.6
-
canrenone pH 7.0, 30°C, recombinant isozyme KsdD3 Pimelobacter simplex