Literature summary for 1.3.99.4 extracted from

D'Arcy, B.M.; Swingle, M.R.; Schambeau, L.; Pannell, L.; Prakash, A.; Honkanen, R.E.
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

Application	Comment	Organism
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
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)

Cloned (Comment)	Organism
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
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

Cloned (Comment)

Organism

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

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

Protein Variants	Comment	Organism
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
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)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
a 3-oxosteroid + acceptor	Rhodococcus erythropolis	-	a 3-oxo-delta1-steroid + reduced acceptor	-	?
a 3-oxosteroid + acceptor	Sterolibacterium denitrificans	-	a 3-oxo-delta1-steroid + reduced acceptor	-	?
androst-4-ene-3,17-dione + acceptor	Rhodococcus erythropolis	-	androsta-1,4-diene-3,17-dione + reduced acceptor	-	?
androst-4-ene-3,17-dione + acceptor	Sterolibacterium denitrificans	-	androsta-1,4-diene-3,17-dione + reduced acceptor	-	?

Organism

Organism	UniProt	Comment	Textmining
no activity in Homo sapiens	-	-	-
Rhodococcus erythropolis	Q9RA02	Arthrobacter picolinophilus	-
Sterolibacterium denitrificans	A9XWD7	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally FLAG and His-tagged synthetic enzyme from Escherichia coli by nickel affinity chromatography	Rhodococcus erythropolis

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
11-deoxycorticosterone + acceptor	-	Rhodococcus erythropolis	21-hydroxypregna-1,4-diene-3,20-dione + reduced acceptor	-	?
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	-	?
17alpha-hydroxyprogesterone + acceptor	-	Rhodococcus erythropolis	17alpha-hydroxy-pregna-1,4-diene-3,20-dione + reduced acceptor	-	?
a 3-oxosteroid + acceptor	-	Rhodococcus erythropolis	a 3-oxo-delta1-steroid + reduced acceptor	-	?
a 3-oxosteroid + acceptor	-	Sterolibacterium denitrificans	a 3-oxo-delta1-steroid + reduced acceptor	-	?
androst-4-ene-3,17-dione + acceptor	-	Rhodococcus erythropolis	androsta-1,4-diene-3,17-dione + reduced acceptor	-	?
androst-4-ene-3,17-dione + acceptor	-	Sterolibacterium denitrificans	androsta-1,4-diene-3,17-dione + reduced acceptor	-	?
cortisone + acceptor	-	Rhodococcus erythropolis	prednisone + reduced acceptor	-	?
dihydrotestosterone + acceptor	-	Rhodococcus erythropolis	? + reduced acceptor	-	?
hydrocortisone 21-hemisuccinate + acceptor	-	Rhodococcus erythropolis	? + reduced acceptor	-	?
additional information	3-ketosteroid DELTA1-dehydrogenase (DELTA1-KstD) catalyzes the C-1 and C-2 desaturation of ring A	Sterolibacterium denitrificans	?	-	-
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	?	-	-
prednisolone + acceptor	-	Rhodococcus erythropolis	? + reduced acceptor	-	?
pregn-4-ene-3,20-dione + acceptor	-	Sterolibacterium denitrificans	pregn-1,4-diene-3,20-dione + reduced acceptor	-	?
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	-	?
progesterone + acceptor	-	Rhodococcus erythropolis	pregna-1,4-diene-3,20-dione + reduced acceptor	-	?
spironolactone + acceptor	-	Rhodococcus erythropolis	? + reduced acceptor	-	?
testosterone + acceptor	-	Rhodococcus erythropolis	17beta-hydroxyandrost-1,4-diene-3-one + reduced acceptor	-	?

Synonyms

Synonyms	Comment	Organism
3-ketosteroid delta1-dehydrogenase	-	Rhodococcus erythropolis
3-ketosteroid delta1-dehydrogenase	-	Sterolibacterium denitrificans
AcmB	-	Sterolibacterium denitrificans
DELTA1-KstD	-	Rhodococcus erythropolis
DELTA1-KstD	-	Sterolibacterium denitrificans
DELTA1-KstDA	-	Sterolibacterium denitrificans
DELTA1-KstDR	-	Rhodococcus erythropolis
KSTD1	-	Rhodococcus erythropolis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Rhodococcus erythropolis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Rhodococcus erythropolis