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 (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 | 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 | 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 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 | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
cytoplasm | - |
Pimelobacter simplex | 5737 | - |
extracellular | - |
Pimelobacter simplex | - |
- |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
additional information | no significant effects by 1 mM of EDTA, Mg2+, K+, and Na+ | Pimelobacter simplex |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Pimelobacter simplex | - |
- |
- |
Pimelobacter simplex CGMCC 14539 | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged isozyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and ultrafiltration | Pimelobacter simplex |
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 | 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 | Comment | Organism |
---|---|---|
More | secondary structure analysis and comparison of KsdD isozymes from Arthrobacter simplex, overview | Pimelobacter simplex |
Synonyms | Comment | Organism |
---|---|---|
3-ketosteroid delta1-dehydrogenase | - |
Pimelobacter simplex |
KsdD | - |
Pimelobacter simplex |
KsdD3 | - |
Pimelobacter simplex |
KsdD5 | - |
Pimelobacter simplex |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
isozyme KsdD3 | Pimelobacter simplex |
35 | - |
isozyme KsdD5 | Pimelobacter simplex |
Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
---|---|---|---|
15 | 45 | over 40% of maximal activity within this range | Pimelobacter simplex |
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 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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
isozyme KsdD3 | Pimelobacter simplex |
7.5 | - |
isozyme KsdD5 | Pimelobacter simplex |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
6 | 9 | over 40% of maximal activity within this range | Pimelobacter simplex |
pH Stability | pH Stability Maximum | Comment | Organism |
---|---|---|---|
5.5 | 9 | over 40% of activity retained within this range | Pimelobacter simplex |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
2,6-dichlorophenolindophenol | DCPIP | Pimelobacter simplex | |
FAD | - |
Pimelobacter simplex |
Organism | Comment | pI Value Maximum | pI Value |
---|---|---|---|
Pimelobacter simplex | sequence calculation | - |
5.43 |
Pimelobacter simplex | sequence calculation | - |
5.79 |
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 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 |