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Literature summary extracted from
- Development of a synthetic 3-ketosteroid DELTA1-dehydrogenase for the generation of a novel catabolic pathway enabling cholesterol degradation in human cells (2019), Sci. Rep., 9, 5969 .
Application
| EC Number | Application | Comment | Organism |
|---|---|---|---|
| 1.3.99.4 | molecular biology | development of a synthetic 3-ketosteroid DELTA1-dehydrogenase for the generation of a catabolic pathway enabling cholesterol degradation in human cells | Rhodococcus erythropolis |
| 1.3.99.4 | molecular biology | development of a synthetic 3-ketosteroid DELTA1-dehydrogenase for the generation of a catabolic pathway enabling cholesterol degradation in human cells | Sterolibacterium denitrificans |
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 1.3.99.4 | gene acmB, a synthetic DELTA1-KstD, developed based on the AcmB gene of Sterolibacterium denitrificans, stable recombinant expression in Escherichia coli and in human Hep-3B and U-937 cells. The enzyme, when expressed in human cells, introduces a double bond between the C-1 and C-2 atoms of 3-oxosteroids. This represents an integral step needed for cholestane A-ring aromatization and B-ring opening for which humans have no known orthologue, coexpression with cholesterol-3-hydroxy-dehydrogenase (CholD) and 3-oxosteroid-9alpha-hydroxylase (Kst-9alphaH) | Sterolibacterium denitrificans |
| 1.3.99.4 | gene kstD1, a synthetic DELTA1-KstD, developed based on the KstD1 gene of Rhodococcus erythropolis, stable recombinant expression of the enzyme as an N-terminal FLAG His-patch thioredoxin fusion protein in Escherichia coli and in human Hep-3B and U-937 cells. The enzyme, when expressed in human cells, introduces a double bond between the C-1 and C-2 atoms of 3-oxosteroids. This represents an integral step needed for cholestane A-ring aromatization and B-ring opening for which humans have no known orthologue, coexpression with cholesterol-3-hydroxy-dehydrogenase (CholD) and 3-oxosteroid-9alpha-hydroxylase (Kst-9alphaH) | Rhodococcus erythropolis |
Protein Variants
| EC Number | Protein Variants | Comment | Organism |
|---|---|---|---|
| 1.3.99.4 | additional information | development of a synthetic 3-ketosteroid DELTA1-dehydrogenase (DELTA1-KstDA) for the generation of a catabolic pathway enabling cholesterol degradation in human cells. Coexpression with cholesterol-3-hydroxy-dehydrogenase (CholD) and 3-oxosteroid-9alpha-hydroxylase (Kst-9alphaH). CholD activity catalyzes the generation of a C-3 ketone, which is needed for DELTA1-KstD activity, which introduces a double bond between the C-1 and C-2 atoms of 3-oxosteroids. Cholesterol catabolism pathway in Actinomycetes, overview | Sterolibacterium denitrificans |
| 1.3.99.4 | additional information | development of a synthetic 3-ketosteroid DELTA1-dehydrogenase (DELTA1-KstDR) for the generation of a catabolic pathway enabling cholesterol degradation in human cells. Coexpression with cholesterol-3-hydroxy-dehydrogenase (CholD) and 3-oxosteroid-9alpha-hydroxylase (Kst-9alphaH). CholD activity catalyzes the generation of a C-3 ketone, which is needed for DELTA1-KstD activity, which introduces a double bond between the C-1 and C-2 atoms of 3-oxosteroids. Cholesterol catabolism pathway in Actinomycetes, overview. The downstream enzymes need to have equal or greater activity when compared to the enzymes acting upstream to allow the pathway to flow and to prevent the accumulation of toxic intermediates, kinetic study | Rhodococcus erythropolis |
Natural Substrates/ Products (Substrates)
| EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 1.3.99.4 | a 3-oxosteroid + acceptor | Rhodococcus erythropolis | - |
a 3-oxo-delta1-steroid + reduced acceptor | - |
? |  |
| 1.3.99.4 | a 3-oxosteroid + acceptor | Sterolibacterium denitrificans | - |
a 3-oxo-delta1-steroid + reduced acceptor | - |
? | |
| 1.3.99.4 | androst-4-ene-3,17-dione + acceptor | Rhodococcus erythropolis | - |
androsta-1,4-diene-3,17-dione + reduced acceptor | - |
? | |
| 1.3.99.4 | androst-4-ene-3,17-dione + acceptor | Sterolibacterium denitrificans | - |
androsta-1,4-diene-3,17-dione + reduced acceptor | - |
? | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 1.3.99.4 | no activity in Homo sapiens | - |
- |
- |
| 1.3.99.4 | Rhodococcus erythropolis | Q9RA02 | Arthrobacter picolinophilus | - |
| 1.3.99.4 | Sterolibacterium denitrificans | A9XWD7 | - |
- |
Purification (Commentary)
| EC Number | Purification (Comment) | Organism |
|---|---|---|
| 1.3.99.4 | recombinant N-terminally FLAG and His-tagged synthetic enzyme from Escherichia coli by nickel affinity chromatography | Rhodococcus erythropolis |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 1.3.99.4 | 11-deoxycorticosterone + acceptor | - |
Rhodococcus erythropolis | 21-hydroxypregna-1,4-diene-3,20-dione + reduced acceptor | - |
? | |
| 1.3.99.4 | 11beta,17alpha,21-trihydroxypregn-4-ene-3,20-dione + acceptor | hydrocortisone | Rhodococcus erythropolis | 1,4-pregnene-17alpha,21-diol-3,11,20-trione + reduced acceptor | - |
? | |
| 1.3.99.4 | 17alpha-hydroxyprogesterone + acceptor | - |
Rhodococcus erythropolis | 17alpha-hydroxy-pregna-1,4-diene-3,20-dione + reduced acceptor | - |
? | |
| 1.3.99.4 | a 3-oxosteroid + acceptor | - |
Rhodococcus erythropolis | a 3-oxo-delta1-steroid + reduced acceptor | - |
? | |
| 1.3.99.4 | a 3-oxosteroid + acceptor | - |
Sterolibacterium denitrificans | a 3-oxo-delta1-steroid + reduced acceptor | - |
? | |
| 1.3.99.4 | androst-4-ene-3,17-dione + acceptor | - |
Rhodococcus erythropolis | androsta-1,4-diene-3,17-dione + reduced acceptor | - |
? | |
| 1.3.99.4 | androst-4-ene-3,17-dione + acceptor | - |
Sterolibacterium denitrificans | androsta-1,4-diene-3,17-dione + reduced acceptor | - |
? | |
| 1.3.99.4 | cortisone + acceptor | - |
Rhodococcus erythropolis | prednisone + reduced acceptor | - |
? | |
| 1.3.99.4 | dihydrotestosterone + acceptor | - |
Rhodococcus erythropolis | ? + reduced acceptor | - |
? | |
| 1.3.99.4 | hydrocortisone 21-hemisuccinate + acceptor | - |
Rhodococcus erythropolis | ? + reduced acceptor | - |
? | |
| 1.3.99.4 | additional information | 3-ketosteroid DELTA1-dehydrogenase (DELTA1-KstD) catalyzes the C-1 and C-2 desaturation of ring A | Sterolibacterium denitrificans | ? | - |
- |
|
| 1.3.99.4 | additional information | 3-ketosteroid DELTA1-dehydrogenase (DELTA1-KstD) catalyzes the C-1 and C-2 desaturation of ring A. A direct-coupled flurometric assay is developed based on resazurin, which is a weakly fluorescent redox dye that is irreversibly reduced when it accepts protons released from a donor molecule. Reduction of resazurin results in the formation of the highly fluorescent product, resorufin. In this assay, DELTA1-KstDR removes and donates two protons and two electrons from C-1 and C-2 of ring-A directly to resazurin, resulting in the formation of resorufin and water. This reduction can be measured by monitoring the increase in fluorescence intensity with time, allowing the assessment of the initial rates of DELTA1-KstDR substrate conversion. Substrate specificity analysis of the recombinant enzyme DELTA1-KstDR, overview. Poor or no activity with mifepristone, cholesterol, pregnenolone, dehydroepiandrosterone, corticosterone, 7beta-hydroxycholestenone, prednisolone, cholestenone, and aldosterone | Rhodococcus erythropolis | ? | - |
- |
|
| 1.3.99.4 | prednisolone + acceptor | - |
Rhodococcus erythropolis | ? + reduced acceptor | - |
? | |
| 1.3.99.4 | pregn-4-ene-3,20-dione + acceptor | - |
Sterolibacterium denitrificans | pregn-1,4-diene-3,20-dione + reduced acceptor | - |
? | |
| 1.3.99.4 | pregn-4-ene-3,20-dione + acceptor | preferred substrate by synthetic recombinant enzyme DELTA1-KstDR | Rhodococcus erythropolis | pregn-1,4-diene-3,20-dione + reduced acceptor | - |
? | |
| 1.3.99.4 | progesterone + acceptor | - |
Rhodococcus erythropolis | pregna-1,4-diene-3,20-dione + reduced acceptor | - |
? | |
| 1.3.99.4 | spironolactone + acceptor | - |
Rhodococcus erythropolis | ? + reduced acceptor | - |
? | |
| 1.3.99.4 | testosterone + acceptor | - |
Rhodococcus erythropolis | 17beta-hydroxyandrost-1,4-diene-3-one + reduced acceptor | - |
? | |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 1.3.99.4 | 3-ketosteroid delta1-dehydrogenase | - |
Rhodococcus erythropolis |
| 1.3.99.4 | 3-ketosteroid delta1-dehydrogenase | - |
Sterolibacterium denitrificans |
| 1.3.99.4 | AcmB | - |
Sterolibacterium denitrificans |
| 1.3.99.4 | DELTA1-KstD | - |
Rhodococcus erythropolis |
| 1.3.99.4 | DELTA1-KstD | - |
Sterolibacterium denitrificans |
| 1.3.99.4 | DELTA1-KstDA | - |
Sterolibacterium denitrificans |
| 1.3.99.4 | DELTA1-KstDR | - |
Rhodococcus erythropolis |
| 1.3.99.4 | KSTD1 | - |
Rhodococcus erythropolis |
Temperature Optimum [°C]
| EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|---|
| 1.3.99.4 | 37 | - |
assay at | Rhodococcus erythropolis |
pH Optimum
| EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|---|
| 1.3.99.4 | 7.5 | - |
assay at | Rhodococcus erythropolis |
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