Information on EC 5.3.3.1 - steroid DELTA-isomerase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY
5.3.3.1
-
RECOMMENDED NAME
GeneOntology No.
steroid DELTA-isomerase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
a 3-oxo-DELTA5-steroid = a 3-oxo-DELTA4-steroid
show the reaction diagram
-
-
-
-
a 3-oxo-DELTA5-steroid = a 3-oxo-DELTA4-steroid
show the reaction diagram
'flip-flop' mechanism may be involved
-
a 3-oxo-DELTA5-steroid = a 3-oxo-DELTA4-steroid
show the reaction diagram
mechanism in which the transition state for enolization is dienolate-like, characterized by relatively little proton transfer from Tyr14 in the transition state, and the intermediate in the overall reaction is dienol-like. An alternative mechanism in which the intermediate is stabilized by a short, strong hydrogen bond can also be consistent with the data
-
a 3-oxo-DELTA5-steroid = a 3-oxo-DELTA4-steroid
show the reaction diagram
catalytic mechanism, phenolate binding to the oxyanion hole of ketosteroid isomerase via hydrogen bonding, electrostatic contributions and geometric and electrostatic changes, overview, the KSI reaction involves changes in both geometry and charge distribution as the reaction proceeds from its ground state to its intermediate, Tyr16 and Asp103 are important in catalysis
-
a 3-oxo-DELTA5-steroid = a 3-oxo-DELTA4-steroid
show the reaction diagram
catalytic mechanism, phenolate binding to the oxyanion hole of ketosteroid isomerase via hydrogen bonding, electrostatic contributions and geometric and electrostatic changes, overview, the KSI reaction involves changes in both geometry and charge distribution as the reaction proceeds from its ground state to its intermediate
-
a 3-oxo-DELTA5-steroid = a 3-oxo-DELTA4-steroid
show the reaction diagram
two-step reaction first, Asp38 acts as a base, abstracting the 4beta-H atom from C-4 of the steroid to form a dienolate as the intermediate, next, the intermediate is reketonized by proton transfer to the 6beta-position, each step goes through its own transition state, catalytic mechanism, theoretical model involving residues Tyr14, Asp38 and Asp99
-
a 3-oxo-DELTA5-steroid = a 3-oxo-DELTA4-steroid
show the reaction diagram
enzyme enhances the coupled motion/hydrogen tunneling contribution to the rate acceleration over the solution reaction
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
intramolecular oxidoreduction
-
-
-
-
isomerization
-
-
-
-
isomerization
-
catalysis of the allylic isomerization of a variety of DELTA5-3-ketosteroids by intramolecular transfer of the C4-beta proton to the C6-beta position
isomerization
Q16772
-
PATHWAY
KEGG Link
MetaCyc Link
androgen biosynthesis
-
brassinosteroid biosynthesis I
-
brassinosteroid biosynthesis II
-
cholesterol degradation to androstenedione I (cholesterol oxidase)
-
cholesterol degradation to androstenedione II (cholesterol dehydrogenase)
-
Metabolic pathways
-
Microbial metabolism in diverse environments
-
progesterone biosynthesis
-
sitosterol degradation to androstenedione
-
Steroid degradation
-
Steroid hormone biosynthesis
-
SYSTEMATIC NAME
IUBMB Comments
3-oxosteroid DELTA5-DELTA4-isomerase
This activity is catalysed by several distinct enzymes (cf. EC 1.1.3.6, cholesterol oxidase and EC 1.1.1.145, 3-hydroxy-5-steroid dehydrogenase).
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
3-Keto-DELTA5-steroid isomerase
-
-
-
-
3-Ketosteroid DELTA5-->DELTA4-isomerase
-
-
-
-
3-Oxo steroid DELTA4-DELTA5-isomerase
-
-
-
-
3-Oxo-delta5 steroid isomerase
-
-
-
-
3-oxo-DELTA5-steroid isomerase
-
-
3-Oxosteroid DELTA4-DELTA5-isomerase
-
-
-
-
3-Oxosteroid DELTA5-DELTA4-isomerase
-
-
-
-
3-Oxosteroid DELTA5-DELTA4-isomerase
Q16772
-
3-Oxosteroid isomerase
-
-
-
-
3beta-HSD
P14060, P26439
-
3beta-HSD
Q16772
-
3beta-HSD
-
-
3beta-HSD/isomerase
-
-
3beta-HSD/isomerase
-
-
3beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase
P22071
-
3beta-hydroxysteroid dehydrogenase/isomerase
-
-
3beta-hydroxysteroid dehydrogenase/isomerase type 1
P14060
-
3beta-hydroxysteroid dehydrogenase/isomerase type 2
P26439
-
5-Ene-4-ene isomerase
-
-
-
-
5-Pregnene-3,20-dione isomerase
-
-
-
-
delta 5-3-ketosteroid isomerase
-
-
-
-
DELTA-3-ketosteroid isomerase
-
-
-
-
Delta-5-3-ketosteroid isomerase
-
-
-
-
DELTA5(or DELTA4)-3-keto steroid isomerase
-
-
-
-
DELTA5-3-keto steroid isomerase
-
-
-
-
DELTA5-3-ketosteroid isomerase
-
-
-
-
DELTA5-3-ketosteroid isomerase
-
-
DELTA5-3-ketosteroid isomerase
P00947
-
DELTA5-3-ketosteroid isomerase
Q16772
-
DELTA5-3-ketosteroid isomerase
-
-
DELTA5-3-ketosteroid isomerase
P07445
-
DELTA5-3-ketosteroid isomerase
-
-
DELTA5-3-oxosteroid isomerase
-
-
-
-
DELTA5-ketosteroid isomerase
-
-
-
-
DELTA5-ketosteroid isomerase
-
-
DELTA5-steroid isomerase
-
-
-
-
glutathione transferase A3-3
Q16772
-
GST A3-3
Q16772
-
Hydroxysteroid isomerase
-
-
-
-
Isomerase, steroid DELTA
-
-
-
-
ketosteroid isomerase
-
-
ketosteroid isomerase
P00947
-
ketosteroid isomerase
Commamonas testosteroni
P00947
-
ketosteroid isomerase
P07445
-
KSI
Commamonas testosteroni
P00947
-
Steroid 5-->4-isomerase
-
-
-
-
Steroid isomerase
-
-
-
-
type I 3beta-hydroxysteroid dehydrogenase/isomerase
-
-
additional information
-
3beta-hydroxy-DELTA5-steroid dehydrogenase/3-keto-DELTA5-steroid isomerase
additional information
-
3betaHSD/I; 3beta-hydroxy-5-ene steroid dehydrogenase/isomerase enzyme system
additional information
-
3beta-HSD; 3beta-hydroxysteroid dehydrogenase/5-ene--4-ene isomerase
additional information
-
beta-hydroxysteroid dehydrogenase/DELTA5-DELTA4 isomerase
additional information
P14060, P26439
the enzyme is a member of the short chain dehydrogenase/reductase family, cf. EC 1.1.1.51
additional information
-
3beta-HSD; beta-hydroxysteroid dehydrogenase/DELTA5-DELTA4 isomerase; enzyme complex 3beta-hydroxysteroid dehydrogenase/DELTA5-DELTA4 isomerase
additional information
-
DELTA5-3-beta-hydroxysteroid dehydrogenase-isomerase system
additional information
P22071
cf. EC 1.1.1.51 and EC 1.1.1.145
additional information
-
cf. EC 1.1.1.51 and EC 1.1.1.145
CAS REGISTRY NUMBER
COMMENTARY
9031-36-1
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
strain KITA
-
-
Manually annotated by BRENDA team
Aspergillus tamarii KITA
strain KITA
-
-
Manually annotated by BRENDA team
3beta-hydroxy-5-ene steroid dehydrogenase/steroid DELTA-isomerase enzyme system
-
-
Manually annotated by BRENDA team
3beta-hydroxy-5-ene-steroid dehydrogenase/steroid DELTA5-DELTA4-ene isomerase enzyme complex
-
-
Manually annotated by BRENDA team
i.e. Pseudomonas testosteroni
SwissProt
Manually annotated by BRENDA team
Commamonas testosteroni
-
UniProt
Manually annotated by BRENDA team
bifunctional 3beta-hydroxysteroid dehydrogenase/DELTA5-DELTA4-isomerase, isoform 3betaHSD1; bifunctional 3beta-hydroxysteroid dehydrogenase/DELTA5-DELTA4-isomerase, isoform 3betaHSD2
-
-
Manually annotated by BRENDA team
bifunctional 3beta-hydroxysteroid dehydrogenase/isomerase, isoforms type 1 and 2
-
-
Manually annotated by BRENDA team
enzyme complex of 3beta-hydroxy-5-ene-steroid dehydrogenase and Steroid DELTA-isomerase
-
-
Manually annotated by BRENDA team
isozyme type 1; isozyme type 1
SwissProt
Manually annotated by BRENDA team
isozyme type 2; isozyme type 2
SwissProt
Manually annotated by BRENDA team
type I 3beta-hydroxysteroid dehydrogenase/isomerase
-
-
Manually annotated by BRENDA team
Nocardia erythropolis
-
-
-
Manually annotated by BRENDA team
Nocardia rhodochrous
-
-
-
Manually annotated by BRENDA team
DELTA5-3-beta-hydroxysteroid dehydrogenase-isomerase system
-
-
Manually annotated by BRENDA team
Sprague-Dawley rats
UniProt
Manually annotated by BRENDA team
type I enzyme isoform, bifunctional 3beta-hydroxysteroid dehydrogenase/D5-D4 isomerase
UniProt
Manually annotated by BRENDA team
bifunctional 3beta-hydroxysteroid dehydrogenase/D5-D4 isomerase
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
17beta-Hydroxy-5(10)-estren-3-one
?
show the reaction diagram
-
-
-
-
-
17beta-Hydroxy-5(10)-estren-3-one
?
show the reaction diagram
-
at 0.25% of the activity compared to 5-androstene-3,17-dione
-
-
-
5(10)-estrene-3,17-dione
estr-4-en-3,17-dione
show the reaction diagram
-
-
-
?
5(10)-estrene-3,17-dione
estr-4-en-3,17-dione
show the reaction diagram
-
nonsticky substrate
-
?
5,10-estrene-3,17-dione
?
show the reaction diagram
-
-
-
-
?
5,10-Seco-19-norpregn-5-yne-3,10,20-trione
?
show the reaction diagram
-
i.e. pregnyne
-
-
-
5,10-Secoestr-5-yne-3,10,17-trione
?
show the reaction diagram
-
i.e. estryne
-
-
-
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
-
-
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
-
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
-
-
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
-
-
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
Nocardia erythropolis
-
-
-
-
-
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
P14060, P26439
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
P00947
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
Q16772
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
Commamonas testosteroni
P00947
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
ir
-
-
-
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
residue Asp38 acts as a base to abstract a proton from C-4 of the substrate to form an intermediate dienolate, which is then reprotonated on C-6
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
the hydrogen-bond network which links the two catalytic residues, Tyr14 and Asp99, to Tyr30, Tyr55, and a water molecule in the highly apolar active site provides the structural support that is needed for the enzyme to maintain the active site geometry optimized for both function and stability
-
?
5-Androstene-3-one
?
show the reaction diagram
-
-
-
-
-
5-Cholesten-3-one
4-Cholesten-3-one
show the reaction diagram
Nocardia rhodochrous
-
-
-
-
5-Cholesten-3-one
4-Cholesten-3-one
show the reaction diagram
Nocardia erythropolis
-
r
-
-
5-Pregnene-3,20-dione
4-Pregnene-3,20-dione
show the reaction diagram
-
-
-
-
-
5-Pregnene-3,20-dione
4-Pregnene-3,20-dione
show the reaction diagram
-
-
-
-
-
5-Pregnene-3,20-dione
4-Pregnene-3,20-dione
show the reaction diagram
Nocardia erythropolis
-
-
-
-
-
a 3-oxo-DELTA5-steroid
a 3-oxo-DELTA4-steroid
show the reaction diagram
-
-
-
-
r
a 3-oxo-DELTA5-steroid
a 3-oxo-DELTA4-steroid
show the reaction diagram
-
phenolates binding to the oxyanion hole of the enzyme via electrostatic interactions, different binding of transition state analogue and substrate, hydrogen bonds shorten with increasing charge localization, overview
-
-
r
a 3-oxo-DELTA5-steroid
a 3-oxo-DELTA4-steroid
show the reaction diagram
-
phenolates binding to the oxyanion hole of the enzyme via hydrogen bonding, different binding of transition state analogue and substrate, hydrogen bonds shorten with increasing charge localization, overview
-
-
r
androst-5-ene-3,17-dione
androst-4-ene-3,17-dione
show the reaction diagram
-
alpha-secondary deuterium kinetic isotope effects at C6 of the steroid. Presence of coupled motion/hydrogen tunneling in the enzyme-catalyzed reaction
-
-
r
dehydroepiandrosterone
3beta-hydroxy-4-androsten-17-one
show the reaction diagram
-
-
-
?
dehydroepiandrosterone
3beta-hydroxy-4-androsten-17-one
show the reaction diagram
-
-
-
-
?
pregn-5-ene-3,20-dione-17alpha-ol
17alpha-hydroxyprogesterone
show the reaction diagram
-
-
-
-
?
DELTA5-pregnen-3,20-dione
?
show the reaction diagram
-
-
-
?
additional information
?
-
-
properties of the steroid-enzyme aduct
-
-
-
additional information
?
-
-, P00947
the 2.3 A structure indicates that Phe101 is not a catalytic residue and that Tyr14 and Asp99 COOH should be directly involved in the stabilization of the dienolate intermediate
-
?
additional information
?
-
-
the aromatic residues Phe54, Phe82 and Trp116 in the hydrophobic substrate-binding pocket of the enzyme contribute differentially to steroid species including substrate, intermediate and product
-
?
additional information
?
-
-
in pregnancy placental 3beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5--4 isomerase produce progesterone from pregnenolone and metabolize fetal dehydroepiandrosterone sulfate to androstenedione, an estrogen precursor
-
-
-
additional information
?
-
-
enzyme can be induced by various steroids
-
-
-
additional information
?
-
-
the conversion of 3beta-hydroxy-5-ene steroids by the enzyme complex 3beta-hydroxysteroid dehydrogenase/DELTA5-DELTA4 isomerase is an obligatory step in the biosynthesis of all classes of hormonal steroids in classical steroidogenic as well as in peripheral tissues
-
-
-
additional information
?
-
-
the conversion of 3beta-hydroxy-5-ene steroids by the enzyme complex 3beta-hydroxysteroid dehydrogenase/DELTA5-DELTA4 isomerase is an obligatory step in the biosynthesis of all classes of hormonal steroids in classical steroidogenic as well as in peripheral tissues
-
-
-
additional information
?
-
P14060, P26439
the bifunctional enzyme possesses 3beta-hydroxysteroid dehydrogenase activity, EC 1.1.1.51, and steroid DELTA-isomerase activity, the enzyme is a key steroidogenic enzyme that catalyzes the first step of the multienzyme pathway conversion of circulating dehydroepiandrosterone and pregnenolone to active steroid hormones
-
-
-
additional information
?
-
-
the enzyme is involved in synthesis of sex steroids influencing the development and function of the songbird brain, overview
-
-
-
additional information
?
-
P22071
bifunctional enzyme possessing 3-beta-hydroxy-DELTA5-steroid dehydrogenase, EC 1.1.1.145, and steroid DELTA-isomerase activity
-
-
-
additional information
?
-
-
bifunctional enzyme possessing 3beta-hydroxysteroid dehydrogenase activity, EC 1.1.1.51, and steroid DELTA-isomerase activity
-
-
-
additional information
?
-
-
equilenin geometrically and electrostatically resembles the dienolate reaction intermediate and transition state, binding structure to enzyme mutant D40N, overview
-
-
-
additional information
?
-
P14060, P26439
structure-function relations, analysis of three dimensional model of a ternary complex of human 3beta-HSD type 1 with an NAD+ cofactor and androstenedione product, reaction of EC 1.1.1.51, to elucidate the key substrate binding residues in the active site as well as the basis for dual function of the 3beta-HSD_1 enzyme, Asn100 and Glu126 residues are key residues that participate in the dehydrogenase and isomerization reactions, respectively, isomerase substrate binding structure, overview
-
-
-
additional information
?
-
-
identification of a putative 3beta-hydroxy-steroid dehydrogenase/DELTA5-DELTA4 isomerase pathway with the ability to handle both dehydroepiansdrosterone as well as C-17 side-chain containing compounds such as pregnenolone and 3beta-hydroxy-16alpha,17alpha-epoxypregn-5-en-20-one. 3beta-HSD/isomerase activity only occurrs following lactonization of the steroidal ring-D. Presence of C-7 allylic hydroxylation, in either epimeric form, inhibits 3beta-HSD/isomerase activity. In Aspergillus tamarii, pregnenolone and 3beta-hydroxy-16alpha,17alpha-epoxypregn-5-en-20-one are metabolized solely through the putative 3beta-HSD/isomerase pathway
-
-
-
additional information
?
-
-
mechanism of KSI-catalyzed steroid isomerization, direct detection of physical coupling between the Y16 and D103 hydrogen bonds, ligand binding, overview
-
-
?
additional information
?
-
-
mechanism of KSI-catalyzed steroid isomerization, overview
-
-
-
additional information
?
-
-
the enzyme catalyzes a C-H bond cleavage and formation through an enolate intermediate. Conversion of the ketone substrate to the enolate intermediate is simulated by a photoacid bound to the active site oxyanion hole. The lack of a significant solvation response in KSI suggests a preorganized active site
-
-
-
additional information
?
-
-
the reaction occurs by a two-step general acid-base mechanism involving a dienolate intermediate. Hybrid quantum/classical molecular dynamics simulations elucidating the geometrical, conformational, and electrostatic changes occurring during the isomerization reaction catalyzed by KSI, calculation of rate constants and modeling of the KSI active site, overview. Electrostaic and conformational changes during the proton transfer reactions, rearrangements, overview
-
-
-
additional information
?
-
Q16772
human GST A3-3 does not stabilize a dienolate by an oxyanion hole in the active site
-
-
-
additional information
?
-
Aspergillus tamarii KITA
-
identification of a putative 3beta-hydroxy-steroid dehydrogenase/DELTA5-DELTA4 isomerase pathway with the ability to handle both dehydroepiansdrosterone as well as C-17 side-chain containing compounds such as pregnenolone and 3beta-hydroxy-16alpha,17alpha-epoxypregn-5-en-20-one. 3beta-HSD/isomerase activity only occurrs following lactonization of the steroidal ring-D. Presence of C-7 allylic hydroxylation, in either epimeric form, inhibits 3beta-HSD/isomerase activity. In Aspergillus tamarii, pregnenolone and 3beta-hydroxy-16alpha,17alpha-epoxypregn-5-en-20-one are metabolized solely through the putative 3beta-HSD/isomerase pathway
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
P00947
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
show the reaction diagram
-
-
-
-
?
a 3-oxo-DELTA5-steroid
a 3-oxo-DELTA4-steroid
show the reaction diagram
-
-
-
-
r
additional information
?
-
-
in pregnancy placental 3beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5--4 isomerase produce progesterone from pregnenolone and metabolize fetal dehydroepiandrosterone sulfate to androstenedione, an estrogen precursor
-
-
-
additional information
?
-
-
enzyme can be induced by various steroids
-
-
-
additional information
?
-
-
the conversion of 3beta-hydroxy-5-ene steroids by the enzyme complex 3beta-hydroxysteroid dehydrogenase/DELTA5-DELTA4 isomerase is an obligatory step in the biosynthesis of all classes of hormonal steroids in classical steroidogenic as well as in peripheral tissues
-
-
-
additional information
?
-
-
the conversion of 3beta-hydroxy-5-ene steroids by the enzyme complex 3beta-hydroxysteroid dehydrogenase/DELTA5-DELTA4 isomerase is an obligatory step in the biosynthesis of all classes of hormonal steroids in classical steroidogenic as well as in peripheral tissues
-
-
-
additional information
?
-
P14060, P26439
the bifunctional enzyme possesses 3beta-hydroxysteroid dehydrogenase activity, EC 1.1.1.51, and steroid DELTA-isomerase activity, the enzyme is a key steroidogenic enzyme that catalyzes the first step of the multienzyme pathway conversion of circulating dehydroepiandrosterone and pregnenolone to active steroid hormones
-
-
-
additional information
?
-
-
the enzyme is involved in synthesis of sex steroids influencing the development and function of the songbird brain, overview
-
-
-
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
NADH
-
the isomerase activity of the wild-type enzyme and the deletion mutant enzyme DELTA283-310 requires allosteric activation by NADH, Km for wild-type enzyme and deletion mutant enzyme is 0.0046 mM
NADH
-
wild-type 1 isomerase is activated only by NADH with NADPH producing no isomerase activity. D36A/K37R mutation shifts the cofactor preference of both 3-beta-hydroxysteroid dehydrogenase and isomerase from NAD(H) to NADP(H)
NADH
-
isomerase activity of enzyme requires NADH, Km- and kcat-value of isoform 1 wild-type are 0.0046 mM and 45 per min, resp. Km- and kcat-value of isoform 2 wild-type are 0.0126 mM and 99.1 per min, resp
NADH
P14060, P26439
the cofactor acts as an allosteric activator; the cofactor acts as an allosteric activator
NADH
-
Km value 0.0126 mM, pH 7.4, 27C; Km value of wild-type 0.0046 mM, pH 7.4, 27C
NADPH
-
D36A/K37R mutation shifts the cofactor preference of both 3-beta-hydroxysteroid dehydrogenase and isomerase from NAD(H) to NADP(H)
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
(17S)-Spiro[5alpha-androstane-17,2'-oxiran]-3-one
-
potent irreversible active-site directed
(17S)-Spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran]-3-ol
-
active-site-directed, irreversible
(17S)-Spiro[estra-1,3,5(10)-triene-17,2'-oxiran]-3-ol
-
potent irreversible active-site directed
(3S)-Spiro-[5alpha-androstane-3,2'-oxiran]-17-one
-
-
10beta-(1-Oxoprop-2-ynyl)oestr-4-ene-3,17-dione
-
irreversible, time-dependent, active-site directed
17-beta-Acetoxy-5alpha-androstan-3-one
-
-
17beta-(1-Oxoprop-2-ynyl)androst-4-en-3-one
-
irreversible, time-dependent, active-site directed
17beta-estradiol
-
competitive
17beta-estradiol
-
IC50: 0.0003 mM
17beta-Hydroxy-5,10-seco-oestr-4-yne-3,10-dione
-
irreversible, time-dependent, active-site directed
19-nortestosterone
-
competitive
19-nortestosterone
-
-
2,6-dihydroxynaphthalene
-
-
2alpha,4alpha-Dibromo-17beta-acetoxy-5alpha-androstan-3-one
-
-
2alpha-Bromodihydrotestosterone
-
-
2alpha-Bromotestosterone
-
-
3-Deoxyestradiol
-
mixed noncompetitive
3-Oxo-4-aza steroids
-
-
4,4'-Dihydroxy-2',7'-dimethyl-7-ethyl-trans-stilbene
-
noncompetitive
4,4'-Dihydroxy-2',7-dimethyl-7'-ethyl-trans-stilbene
-
competitive
4alpha,5alpha-epoxy-testosterone
-
-
4alpha-Bromo-17beta-acetoxy-5alpha-androstan-3-one
-
-
5'-(4-fluorosulfonylbenzoyl)adenosine
-
-
5,10-Seco-19-nor-5-cholestyne-3,10-dione
Nocardia erythropolis
-
-
5,10-Seco-oestr-4-yne-3,10,17-trione
-
irreversible, time-dependent, active-site directed
5,10-Secosteroids
-
irreversible
5-Androstene-3,17-dione
-
competitive towards 5-pregnene-3,20-dione
5-Pregnene-3,20-dione
-
competitive inhibitor towards 5-androstene-3,17-dione
6-bromo-2-naphthol
-
-
6-cyano-2-naphthol
-
-
6-methoxy-2-naphthol
-
-
6alpha-Bromotestosterone
-
-
Biochanin A
-
IC50: 0.0004 mM
CaCl2
-
mild
CdCl2
-
moderate
Conjugated allenic 3-oxo-5,10-secosteroids
-
irreversible
-
coumarin 183
-
binds tightly in the oxyanion hole of the enzyme and chemically resemble the intermediate in the catalytic cycle. Upon photoexcitation, the pKa of the hydroxyl group changes substantially. This light-activated change in electron density around the photoacid hydroxyl group simulates the change in substrate pKa during the catalytic cycle. When the light-driven reaction analog occurs in the KSI active site, the electrostatic environment changes little
coumestrol
-
mixed noncompetitive
CuCl2
-
strong
daidzein
-
IC50: 0.0038 mM
Diethylstilbestrol
-
competitive
equilenin
-
reaction intermediate analogue, binds to the active site, binding structure, overview
equilenin
-
binds tightly in the oxyanion hole of the enzyme and chemically resemble the intermediate in the catalytic cycle. Upon photoexcitation, the pKa of the hydroxyl group changes substantially. This light-activated change in electron density around the photoacid hydroxyl group simulates the change in substrate pKa during the catalytic cycle. When the light-driven reaction analog occurs in the KSI active site, the electrostatic environment changes little
estradiol
-
competitive
estrone
-
and derivatives, noncompetitive
FeCl2
-
moderate
formononetin
-
IC50: 0.004 mM
genistein
-
IC50: 0.0023 mM
N,N-Dimethyl-4-methyl-3-oxo-4-aza-5alpha-androstane-17beta-carboxamide
-
non-competitive inhibition of isomerase, competitive inhibition of 3beta-hydroxysteroid dehydrogenase
Spiro-17beta-oxiranyl-DELTA4-androsten-3-one(4beta)
-
active site-directed, irreversible
-
Spiro-17beta-oxiranylestra-1,3,5(10),6,8-pentaene-3-ol
-
active site-directed, irreversible
Testosterone acetate
-
-
Urea
-
noncompetitive
Urea
-
enzymatic activity is affected significantly by urea and decreases exponentially as urea concentration increases
ZnCl2
-
moderate
Halothane
-
allosteric modulation of dynamics-function relationship without direct competition, halothane occupancy at the dimer interface disrupts intersubunit hydrogen bonding
additional information
-
overview: interaction of the enzyme with steroids
-
additional information
-
overview: inactivation by active-site directed acetylenic steroids
-
additional information
-
inhibitor development, a series of substituted naphthol inhibitors shows substantial variations in the contributions of the protonated and deprotonated forms when bound to the active site. Method of quantifying the contributions of each protonation state show the oxyanion hole in the active site of wild-type DELTA5-3-ketosteroid isomerase to have a proton affinity equal to a solution pKa of 10.05, which is similar to the measured pKa of 10.0 of the reaction intermediate
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
NADH
P14060, P26439
the cofactor acts as an allosteric activator; the cofactor acts as an allosteric activator
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0223
-
17beta-Hydroxy-5(10)-estren-3-one
-
-
0.0328
-
5(10)-estrene-3,17-dione
-
25C, pH 7.0, mutant enzyme F82A
0.0651
-
5(10)-estrene-3,17-dione
-
25C, pH 7.0, mutant enzyme F82L
0.0982
-
5(10)-estrene-3,17-dione
-
25C, pH 7.0, mutant enzyme F54L
0.11
-
5(10)-estrene-3,17-dione
-
25C, pH 7.0, wild-type enzyme
0.256
-
5(10)-estrene-3,17-dione
-
25C, pH 7.0, mutant enzyme F54A
0.048
-
5,10-Seco-19-norpregn-5-yne-3,10,20-trione
-
-
0.413
-
5,10-secoestr-5-yne-3,10,20,trione
-
-
-
0.0001
-
5-Androstene-3,17-dione
-
wild-type, 27C, pH 7.4
0.00041
-
5-Androstene-3,17-dione
-
mutant S124T, 27C, pH 7.4
0.00083
-
5-Androstene-3,17-dione
-
mutant M187T, 27C, pH 7.4
0.0024
-
5-Androstene-3,17-dione
-
27C, pH 7.4, wild-type enzyme
0.0033
-
5-Androstene-3,17-dione
-
27C, pH 7.4, mutant enzyme D241N
0.0044
-
5-Androstene-3,17-dione
-
27C, pH 7.4, mutant enzyme D265N
0.0149
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme F82A
0.0237
-
5-Androstene-3,17-dione
-
-
0.024
-
5-Androstene-3,17-dione
-
-
0.0252
-
5-Androstene-3,17-dione
-
22C, pH 7.4, truncated cytosolic enzyme DEALTA283-310
0.0269
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme Y14F
0.0276
-
5-Androstene-3,17-dione
-
-
0.0279
-
5-Androstene-3,17-dione
-
22C, pH 7.4, wild-type enzyme
0.0287
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y14F/Y55F
0.033
-
5-Androstene-3,17-dione
-
mutant D38E, pH 7.0, 5.4 C
0.036
-
5-Androstene-3,17-dione
-
-
0.037
-
5-Androstene-3,17-dione
-
mutant D38E, pH 7.0, 10.3 C
0.04
-
5-Androstene-3,17-dione
-
mutant D38E, pH 7.0, 15.3 C; mutant D38E, pH 7.0, 20.1 C
0.0475
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme Y14F/D99N
0.0481
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme Y14F/D99E
0.0486
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme F116W
0.0499
-
5-Androstene-3,17-dione
-
25C, pH 7.0, wild-type enzyme
0.0499
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, wild-type enzyme
0.0499
-
5-Androstene-3,17-dione
-
wild-type, 25C, pH 7.0
0.0503
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, wild-type enzyme
0.0503
-
5-Androstene-3,17-dione
-
25C, pH 7.0, wild-type enzyme
0.0513
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F
0.0539
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme F82L
0.055
-
5-Androstene-3,17-dione
-
mutant D38E, pH 7.0, 25.0 C
0.0578
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F/D99N
0.0579
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme F54A
0.0616
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme D99E
0.063
-
5-Androstene-3,17-dione
-
mutant D38E, pH 7.0, 29.9 C
0.067
-
5-Androstene-3,17-dione
-
25C, pH 6.5, solvent D2O, mutant enzyme D38A
0.073
-
5-Androstene-3,17-dione
-
25C, pH 8.5, solvent D2O, mutant enzyme D38A
0.077
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme F54L
0.0787
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y14F/Y30F
0.0795
-
5-Androstene-3,17-dione
-
mutant L125F/V127F, 25C, pH 7.0
0.085
-
5-Androstene-3,17-dione
-
25C, pH 7.5, solvent D2O, mutant enzyme D38A
0.088
-
5-Androstene-3,17-dione
-
27C, pH 7.4
0.0918
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F/D99L
0.094
-
5-Androstene-3,17-dione
-
25C, pH 8.5, solvent H2O, mutant enzyme D38A
0.096
-
5-Androstene-3,17-dione
-
25C, pH 7.5, solvent H2O, mutant enzyme D38A
0.0982
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme Y14F/D99L
0.099
-
5-Androstene-3,17-dione
-
25C, pH 6.5, solvent H2O, mutant enzyme D38A
0.0995
-
5-Androstene-3,17-dione
-
mutant W92A, 25C, pH 7.0
0.1185
-
5-Androstene-3,17-dione
-
mutant W92A/L125A/V127A, 25C, pH 7.0
0.1375
-
5-Androstene-3,17-dione
-
mutant L125A/V127A, 25C, pH 7.0
0.149
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme W116F
0.1528
-
5-Androstene-3,17-dione
-
25C, pH 7.0, wild-type enzyme
0.173
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme E118A
0.174
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme W116A
0.258
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme D99L
0.258
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme N120A
0.261
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme E118A/N120A
0.31
0.33
5-Androstene-3,17-dione
-
-
0.38
-
5-Androstene-3,17-dione
-
-
0.548
-
5-Androstene-3,17-dione
Nocardia erythropolis
-
-
0.007
-
5-Cholesten-3-one
Nocardia erythropolis
-
-
0.0093
-
5-Pregnene-3,20-dione
-
-
0.0097
-
5-Pregnene-3,20-dione
-
-
0.011
-
5-Pregnene-3,20-dione
-
-
0.0125
-
5-Pregnene-3,20-dione
Nocardia erythropolis
-
-
0.068
-
5-Pregnene-3,20-dione
-
-
0.023
-
androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y55F
0.0258
-
androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme D99L
0.0502
-
androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y30F/Y55F
0.0552
-
androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y30F
0.0619
-
androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y55F/D99L
0.0735
-
androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y30F/D99L
0.0175
-
dehydroepiandrosterone
-
mutant R240Q, isoform 2, 27C, pH 7.4
0.0279
-
dehydroepiandrosterone
-
wild-type, isoform 1, 27C, pH 7.4
0.033
-
dehydroepiandrosterone
-
mutant Q105M, isoform 1, 27C, pH 7.4
0.0623
-
dehydroepiandrosterone
-
mutant Q240R, isoform 1, 27C, pH 7.4
0.0808
-
dehydroepiandrosterone
-
mutant Q105M, isoform 2, 27C, pH 7.4
0.0884
-
dehydroepiandrosterone
-
wild-type, isoform 2, 27C, pH 7.4
0.01
-
DELTA5-pregnen-3,20-dione
-
25C, pH 7.5
additional information
-
additional information
-
-
-
additional information
-
additional information
-
Km-values of mutant enzymes Y16F and D40N
-
additional information
-
additional information
-
thermodynamic parameters and rate constants for isomerization of 5-androstene-3,17-dione
-
additional information
-
additional information
P14060, P26439
kinetics of wild-type and mutant enzymes; kinetics of wild-type and mutant enzymes
-
additional information
-
additional information
-
kinetics
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
70.8
-
17beta-Hydroxy-5(10)-estren-3-one
-
-
0.25
-
5(10)-estrene-3,17-dione
-
25C, pH 7.0, mutant enzyme F82A
1.7
-
5(10)-estrene-3,17-dione
-
25C, pH 7.0, mutant enzyme F54A
4.3
-
5(10)-estrene-3,17-dione
-
25C, pH 7.0, mutant enzyme F54L
7.5
-
5(10)-estrene-3,17-dione
-
25C, pH 7.0, mutant enzyme F82L
11.1
-
5(10)-estrene-3,17-dione
-
25C, pH 7.0, wild-type enzyme
0.052
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F/D99L
0.295
-
5-Androstene-3,17-dione
-
27C, pH 7.4, mutant enzyme D265N
0.48
-
5-Androstene-3,17-dione
-
27C, pH 7.4, mutant enzyme D241N
0.557
-
5-Androstene-3,17-dione
-
27C, pH 7.4, wild-type enzyme
0.67
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme Y14F/D99L
0.7
1
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme Y14F/D99N
0.71
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme Y14F/D99N
0.83
-
5-Androstene-3,17-dione
-
wild-type, 27C, pH 7.4
1.1
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F/D99N
1.46
-
5-Androstene-3,17-dione
-
mutant M187T, 27C, pH 7.4
1.56
-
5-Androstene-3,17-dione
-
mutant S124T, 27C, pH 7.4
3
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme Y14F/D99E
6.08
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme Y14F/D99L
6.08
-
5-Androstene-3,17-dione
-
27C, pH 7.4, wild-type enzyme
11.5
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme Y14F
13.6
-
5-Androstene-3,17-dione
-
27C, pH 7.4
53
-
5-Androstene-3,17-dione
-
mutant D38E, pH 7.0, 5.4 C
71
-
5-Androstene-3,17-dione
-
mutant D38E, pH 7.0, 10.3 C
101
-
5-Androstene-3,17-dione
-
mutant D38E, pH 7.0, 15.3 C
145
-
5-Androstene-3,17-dione
-
mutant D38E, pH 7.0, 20.1 C
180
-
5-Androstene-3,17-dione
-
mutant D38E, pH 7.0, 25.0 C
220
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme D99L
230
-
5-Androstene-3,17-dione
-
25C, pH 6.5, solvent D2O, mutant enzyme D38A
250
-
5-Androstene-3,17-dione
-
mutant D38E, pH 7.0, 29.9 C
260
-
5-Androstene-3,17-dione
-
25C, pH 8.5, solvent D2O, mutant enzyme D38A
279
-
5-Androstene-3,17-dione
-
25C, pH 6.5, solvent H2O, mutant enzyme D38A
302
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y14F/Y30F
311
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme W116A
340
-
5-Androstene-3,17-dione
-
25C, pH 6.5, solvent H2O, mutant enzyme D38A
360
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y14F/Y55F
412
-
5-Androstene-3,17-dione
-
25C, pH 7.5 or 8.5, solvent H2O, mutant enzyme D38A
539
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme F82A
587
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F
1180
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme E118A/N120A
2540
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme F82L
3630
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme F54A
3710
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme N120A
4420
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme W116Y
4760
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme W116F
4870
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme R72A
6920
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme E118A
7155
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme F116W
7160
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme F116W
8900
-
5-Androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme F54L
12000
-
5-Androstene-3,17-dione
-
mutant W92A/L125A/V127A, 25C, pH 7.0
12700
-
5-Androstene-3,17-dione
-
mutant W92A, 25C, pH 7.0
12900
-
5-Androstene-3,17-dione
-
mutant L125A/V127A, 25C, pH 7.0
17600
-
5-Androstene-3,17-dione
-
mutant L125F/V127F, 25C, pH 7.0
19000
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, mutant enzyme D99E
21200
-
5-Androstene-3,17-dione
-
25C, pH 7.0, wild-type enzyme
21200
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, wild-type enzyme
21200
-
5-Androstene-3,17-dione
-
wild-type, 25C, pH 7.0
21230
-
5-Androstene-3,17-dione
-
25C, pH 7.0, wild-type enzyme
27900
-
5-Androstene-3,17-dione
-
pH 7.0, 25C, wild-type enzyme
27900
-
5-Androstene-3,17-dione
-
25C, pH 7.0, wild-type enzyme
31680
-
5-Androstene-3,17-dione
-
25C, pH 7.0, wild-type enzyme
31700
-
5-Androstene-3,17-dione
-
25C, pH 7.0, wild-type enzyme
288000
-
5-Androstene-3,17-dione
-
-
1.2
-
androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y55F/D99L
40.7
-
androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y30F/D99L
220
-
androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme D99L
3510
-
androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y55F
10680
-
androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y30F/Y55F
10700
-
androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y30F/Y55F
17800
-
androstene-3,17-dione
-
25C, pH 7.0, mutant enzyme Y30F
1990
-
dehydroepiandrosterone
-
mutant Q240R, isoform 1, 27C, pH 7.4
2064
-
dehydroepiandrosterone
-
mutant R240Q, isoform 2, 27C, pH 7.4
2088
-
dehydroepiandrosterone
-
mutant Q105M, isoform 1, 27C, pH 7.4
3012
-
dehydroepiandrosterone
-
wild-type, isoform 1, 27C, pH 7.4
4002
-
dehydroepiandrosterone
-
mutant Q105M, isoform 2, 27C, pH 7.4
4890
-
dehydroepiandrosterone
-
wild-type, isoform 2, 27C, pH 7.4
0.303
-
DELTA5-pregnen-3,20-dione
-
25C, pH 7.5
additional information
-
additional information
-
turnover number of mutant enzymes Y16F and D40N
-
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0028
-
4alpha,5alpha-epoxy-testosterone
-
mutant M187T, 27C, pH 7.4
0.00375
-
4alpha,5alpha-epoxy-testosterone
-
27C, pH 7.4
0.0041
-
4alpha,5alpha-epoxy-testosterone
-
wild-type, 27C, pH 7.4
0.0043
-
4alpha,5alpha-epoxy-testosterone
-
mutant S124T, 27C, pH 7.4
7e-05
-
epostane
-
mutant Q240R, isoform 1, 27C, pH 7.4; wild-type, isoform 1, 27C, pH 7.4
0.00021
-
epostane
-
mutant R240Q, isoform 2, 27C, pH 7.4
0.00098
-
epostane
-
wild-type, isoform 2, 27C, pH 7.4
0.00164
-
epostane
-
mutant Q105M, isoform 2, 27C, pH 7.4
0.0001
-
trilostane
-
wild-type, 27C, pH 7.4
0.00012
-
trilostane
-
mutant R240Q, isoform 2, 27C, pH 7.4
0.00013
-
trilostane
-
mutant Q240R, isoform 1, 27C, pH 7.4
0.00041
-
trilostane
-
mutant S124T, 27C, pH 7.4
0.00083
-
trilostane
-
mutant M187T, 27C, pH 7.4
0.0016
-
trilostane
-
27C, pH 7.4
0.00166
-
trilostane
-
wild-type, isoform 2, 27C, pH 7.4
0.00207
-
trilostane
-
mutant Q105M, isoform 2, 27C, pH 7.4
0.00208
-
trilostane
-
mutant Q105M, isoform 1, 27C, pH 7.4
0.00184
-
epostane
-
mutant Q105M, isoform 1, 27C, pH 7.4
additional information
-
additional information
-
Ki-values for equilenin and 19-nortestosterone, wild-type enzyme and mutant enzyme D99E
-
additional information
-
additional information
-
dissociation constants of inhibitors
-
IC50 VALUE [mM]
IC50 VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0003
-
17beta-estradiol
-
IC50: 0.0003 mM
0.0004
-
Biochanin A
-
IC50: 0.0004 mM
0.0038
-
daidzein
-
IC50: 0.0038 mM
0.004
-
formononetin
-
IC50: 0.004 mM
0.0023
-
genistein
-
IC50: 0.0023 mM
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.622
-
-
reaction with 5-androsten-3,17-dione, truncated cytosolic enzyme DELTA283-310
3.5
-
-
-
30600
-
-
purified diluted recombinant enzyme
additional information
-
-
-
additional information
-
-
-
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
6
7.5
Nocardia rhodochrous
-
-
7.4
-
P14060, P26439
assay at; assay at
7.5
-
-
-
7.5
-
-
wild-type 1 isomerase and mutant enzymes D241N and D265N
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
6.5
8
-
pH 6.5: about 60% of maximal activity, pH 8.0: about 40% of maximal activity, pH 6.3: no activity
6.5
8
-
pH 6.5: about 55% of maximal activity, wild-type enzyme and mutant enzyme D265N, about 65% of maximal activity, mutant enzyme D241N, about 85% of maximal activity mutant enzyme D257L and D258L. pH 8.0: about 50% of maximal activity, wild-type enzyme and mutant enzymes D241N, 95% of maximal activity, mutant enzyme D258L
7.5
9
-
pH 7.5: about 40% of maximal activity, pH 9.0: about 60% of maximal activity
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
27
-
P14060, P26439
assay at; assay at
37
-
-
-
TEMPERATURE RANGE
TEMPERATURE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
22
42
-
22C: about 60% of maximal activity, 40C: about 40% of maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
P14060, P26439
type 2 isozyme
Manually annotated by BRENDA team
-
brain region differences in dehydroepiandrosterone metabolism by enzyme, sex differences in enzyme distribution and regulation by stress
Manually annotated by BRENDA team
P22071
enzyme expression analysis in male and pseudopregnant female rat brain, in situ hybridization, overview, strong expression in olfactory bulb, striatum, cortex, thalamus, hypothalamus, septum, habenula, hippocampus and cerebellum, pseudopregnancy has no effect on enzyme expression in brain, effect of lesions, overview
Manually annotated by BRENDA team
P22071
in ovariectomized-adrenalectomized animals, estrogen treatment increases enzyme mRNA in the hypothalamus as well as enzyme activity
Manually annotated by BRENDA team
P14060, P26439
type 1 isozyme
Manually annotated by BRENDA team
P14060, P26439
type 2 isozyme
Manually annotated by BRENDA team
P14060, P26439
type 1 isozyme
Manually annotated by BRENDA team
-
of embryo. Levels of bifunctional 3beta-hydroxysteroid-dehydrogenase/DELTA4-DELTA5-isomerase are sexually dimorphic and developmental age-dependent. MRNA levels are higher in females than in males at E7, whereas this dimorphism is reversed at E9 and E15. In females, the highest mRNA level is at E7, whereas in males the levels are significantly higher at E9 and E17 than at E7 and E11. Sexual dimorphism can only be detcted in prosencephalon, can not be observed before gonadal sexual differentiation, and is not paralleled by a dimorphism in brain content of progesterone
Manually annotated by BRENDA team
P14060, P26439
type 1 isozyme
Manually annotated by BRENDA team
P14060, P26439
type 2 isozyme
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
modified enzyme in which the membrane-spanning domain, residues 283-310, of the enzyme protein is deleted in the cDNA is expressed by baculovirus in the cytosol instead of in the microsomes and mitochondria of the Sf9 cells
Manually annotated by BRENDA team
-
integral membrane protein
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
14520
-
-
mutant enzyme D40N, electrospray mass spectrometry; mutant enzyme Y16F, electrospray mass spectrometry
18700
-
-
protein concentration 2 ng/ml, gel filtration
20900
-
-
protein concentration 20 ng/ml, gel filtration
23100
-
-
protein concentration 200 ng/ml, gel filtration
24900
-
-
protein concentration 0.002 mg/ml, gel filtration
26000
68000
-
protein concentration 0.043 mg/ml-15.6 mg/ml. At concentrations below 1 mg/ml the enzyme exists as a dimer of MW 26800, determined by gel filtration and ultracentrifugation studies. Concentration-dependent association occurs at higher enzyme concentrations. The latter value represents the weight avarage molecular weight of two or more polymerization species in rapid equilibrium, rather than a discrete polymeric form of the enzyme
26000
-
-
equilibrium sedimentation at 20 C
26800
-
-
protein concentration 0.02 mg/ml, gel filtration
27700
-
-
equilibrium sedimentation at 4 C
40800
-
-
low speed sedimentation without reaching equilibrium
76000
-
-
enzyme complex of 3beta-hydroxy-5-ene-steroid dehydrogenase and steroid DELTA-isomerase, gel filtration
77000
-
-
enzyme complex of 3beta-hydroxy-5-ene-steroid dehydrogenase and steroid DELTA-isomerase gel filtration
81000
-
-
3beta-hydroxy-5-ene steroid dehydrogenase/steroid DELTA-isomerase system, gel filtration
300000
-
-
DELTA5-3-beta-hydroxysteroid dehydrogenase-isomerase system, gel filtration
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 40000, DELTA5-3-beta-hydroxysteroid dehydrogenase-isomerase system, SDS-PAGE
?
-
x * 41874, calculation from nucleotide sequence
?
-
x * 46800, SDS-PAGE, enzyme with steroid DELTA-isomerase activity and 3beta-hydroxy-5-ene-steroid dehydrogenase activity
?
-
x * 38800, truncated soluble enzyme DEALTA283-310; x * 42000, wild-type enzyme
dimer
-
2 * 11000, SDS-PAGE. At concentrations below 1 mg/ml the enzyme exists as a dimer of MW 26800, determined by gel filtration and ultracentrifugation studies. Concentration-dependent association occurs at higher enzyme concentrations
dimer
-
2 * 41000, SDS-PAGE
dimer
-
2 * 45000, 3beta-hydroxy-5-ene steroid dehydrogenase/isomerase enzyme system, SDS-PAGE
dimer
-
2 * 14000, mutant enzymnes R72A, E118A, N120A and E18A/N120A, SDS-PAGE
dimer
P14060, P26439
active enzyme, interface is composed of two pairs of helices related by a 2fold symmetry axis, involved residues, overview; active enzyme, interface is composed of two pairs of helices related by a 2fold symmetry axis, involved residues, overview
homodimer
-
determined by 3D NMR, the secondary and tertiary structures of KSI are not affected significantly by urea up to 3.5 M. The chemical shift analysis of 1H-15N HSQC spectra at various urea concentrations reveales that the residues in the dimeric interface region, particularly around the beta5-strand, are significantly perturbed by urea at low concentrations, while the line-width analysis indicates the possibility of conformational exchange at the interface region around the beta6-strand. The interface region primarily around the beta5- and beta6-strands could play an important role as the starting positions in the unfolding process of KSI.
tetramer
-
4 * 19000, enzyme complex of 3beta-hydroxy-5-ene-steroid dehydrogenase and steroid DELTA-isomerase, SDS-PAGE
homodimer
-
131 amino acid residues per monomer, xenon is employed as an indirect probe for multidimensional NMR to investigate the structural changes in the urea-induced equilibrium unfolding
additional information
-
detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase, NMR spectroscopy, circular dichroism and fluorescence spectroscopies, and small angle X-ray scattering and analytical ultracentrifugation of native and fully unfolded enzymes, overview, the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
1.2-1.5 A resolution X-ray crystallography, 1H and 19F NMR spectroscopy, quantum mechanical calculations, and transition-state analogue binding measurements of the active site. Packing and binding interactions within the KSI active site can constrain local side-chain reorientation and prevent hydrogen bond shortening by 0.1 A or less. This constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. Structural features of the oxyanion hole suggest that hydrogen bond formation to the reacting substrate is geometrically optimal in the transition state but not in the ground state. During steroid isomerization, the hybridization of the substrate oxygen changes from a planar sp2 carbonyl to a tetrahedral sp3 dienolate, altering the spatial distribution of its lone pair electrons. This reorientation of atomic orbitals about the substrate oxygen alters its geometric preference for accepting hydrogen bonds
-
; crystals are grown at 12C from1.3 M ammonium sulfate, 3-5% poly(ethylene glycol)400 and 0.1 M HEPES, pH 7.5 in hanging drops. The crystal structure at 2.3 resolution reveals that the active site environment of the Comamonas testosteroni enzyme is nearly identical to that of Pseudomonas putida enzyme
-
crystals of mutant enzyme F116W are grown by hanging-drop method
-
study on backbone dynamics in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate. Mutation Y14F induces a substantial decrease in the order parameters in free enzyme, indicating that the backbone structures become significantly mobile by mutation, while the chemical shift analysis indicates that the structural perturbations are more profound than those of wild-type upon 19-nortestosterone hemisuccinate binding. In the 19-nortestosterone hemisuccinate complexed mutant, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remain flexible with significantly reduced S2 values, whereas the S2 values for many of the residues in the mutant enzyme become even greater than those of wild-type
-
The 2.26 A crystal structure of the enzyme in complex with a reaction intermediate analogue equilenin reveals that both the Tyr14 OH and the Asp99 COOH provide direct hydrogen bonds to the oxyanion of equilenin
-
GST A3-3 in complex with DELTA5-androstene-3,17-dione, using 100 mM Tris-HCl pH 7.8, 18% (v/v) PEG 4000, and 2 mM dithiothreitol
Q16772
hanging-drop vapor diffusion method. The inability to crystallize the detergent-solubilized, wild-type microsomal enzyme is overcome by engineering a cytosolic form of this protein. Modified enzyme in which the membrane-spanning domain, residues 283-310 of the enzyme protein is deleted in the cDNA is expressed by baculovirus in the cytosol instead of in the microsomes and mitochondria of the Sf9 cells
-
identification of potentially critical residues M187 and S124 by docking of trilostane or 4alpha,5alpha-epoxy-testosterone into the active site
-
1.2-1.5 A resolution X-ray crystallography, 1H and 19F NMR spectroscopy, quantum mechanical calculations, and transition-state analogue binding measurements of the active site. Packing and binding interactions within the KSI active site can constrain local side-chain reorientation and prevent hydrogen bond shortening by 0.1 A or less. This constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. Structural features of the oxyanion hole suggest that hydrogen bond formation to the reacting substrate is geometrically optimal in the transition state but not in the ground state. During steroid isomerization, the hybridization of the substrate oxygen changes from a planar sp2 carbonyl to a tetrahedral sp3 dienolate, altering the spatial distribution of its lone pair electrons. This reorientation of atomic orbitals about the substrate oxygen alters its geometric preference for accepting hydrogen bonds
-
crystal structure of mutant enzyme F82A is determined to 2.1 A resolution. Crystals are grown in a solution containing 1.0 M sodium acetate and 0.1 M ammonioum acetate, pH 4.6, by the hanging drop method of vapor diffusion at 22 C. The crystals belong to the space group c2221 with unit cell dimensions of a = 36.24 A, b = 96.13 A and c = 74.30 A
-
crystal structure of the enzyme in complex with equilenin, an analogue of the reaction intermediate at 1.9 and 2.5 A resolution
-
crystal structure of the R72A mutant enzyme determined at 2.5 A resolution belongs to the space group C2221 with cell dimensions of a = 36.37 A, b = 74.44 A and c = 96.06 A. Crystals are grown from a solution containing 2.0 M ammonioum acetate and 0.1 M sodium acetate at pH 4.6 by hanging drop vapor-diffusion method at 22C
-
crystals of Y30F, Y55F, and Y30F/Y55F are grown in the solution containing 1.0 M sodium acetate and 0.1 M ammonium acetate, pH 4.6 by hanging drop method of vapor diffusion at 22C. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30
-
enzyme mutant D40N bound to phenolate, X-ray diffraction structure determination and analysis at 1.25 A resolution
-
hanging drop vapor diffusion method. Crystal structures of Y14F and Y14F/Y30F/Y55F are determined at 1.8 and 2.0 A resolution, respectively
-
mutant D103N/D40N bound to inhibitor equilenin, hanging drop vapor diffusion method, 0.002 ml of 25 mg/ml protein with equilenin in a molar ratio of 1:1.2 in 40 mM potassium phosphate, pH 7.2, are mixed with 0.002 ml of reservoir solution conraining 1.4 M ammonium sulfate, and 6.5% v/v 2-propanol, pH 7.0, room temperature, 1 week, X-ray diffraction structure determination and analysis at 1.1 A resolution
-
mutant enzyme D99E/D38N complexed with equilenin, an intermediate analogue, crystals of the complexes are grown from 1.1 M ammonium acetate and 0.1 M sodium acetate, pH 4.6, by the hanging drop vapor diffusion method at 22C. The resulting crystals have C2 space group symmetry with unit cell dimensions of a = 89.04 A, b = 72.42 A, c = 51.24 A and beta = 90.9
-
mutant W92A, in complex with d-equilenin, decrease in conformational stability results from destabilization of surface hydrophobic layer
-
mutant Y14F/D99L, increase in hydrophobic interaction while disrupting the hydrogen bond network, mutants Y30F/D99L and Y55F/D99L, disruption of hydrogen bond network
-
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
22
-
-
31.5% loss of activity after 4 days
25
-
-
the free-energy change for unfolding in the absence of urea at 25C is 24.4 kcal/mol for the wild-type enzyme
80
-
Nocardia rhodochrous
-
5 min, about 70% loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
active-site-directed photoinactivation is sensitized by unsaturated steroid ketone photoaffinity reagents
-
in the equilibrium unfolding process the enzyme stability increases by 2.5 kcal/mol in presence of 5% 2,2,2-trifluoroethanol, the increase in entropy by 2,2,2-trifluoroethanol is partially responsible for the increase in stability
-
photoinactivation by ultraviolet irradiation in the presence of the solid-phase photoaffinity reagent DELTA6-testosterone agarose
-
ultraviolet light-dependent photoinactivation is stimulated by DELTA4-3-ketosteroids. The loss of enzymic activity can be correlated with destruction of a single Asp or Asn
-
urea-induced equilibrium unfolding
-
ORGANIC SOLVENT
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
urea
-
the recovery of the activity by refolding is not diminished even after a prolonged time, 24 h of exposure to 7 M urea. Reversibility of the folding is assessed by a 100fold dilution of the denatured protein and subsequent determination of enzyme activity. The refolding kinetics as monitored by fluorescence intensity can be described as a fast first-order process followed by a second-order and a subsequent slow first-order processes, there may be a monomeric folding intermediate
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-20C, 4.3% loss of activity after 2 weeks
-
-70C, 6 months, no loss of activity
-
22C, 31.5% loss of activity after 4 days
-
4C, 12.5% loss of activity after 7 days
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
3beta-hydroxy-5-ene steroid dehydrogenase/isomerase enzyme system
-
3beta-hydroxy-5-ene-steroid dehydrogenase/steroid DELTA5-DELTA4-ene isomerase enzyme complex
-
affinity chromatography
-
recombinant C-terminally His6-tagged enzyme from Escherichia coli strain BLR(DE3) to homogeneity by expanded bed adsorption column utilizing immobilized metal affinity chromatography with denaturant concentration in the feed stream from 8 to 0 M urea, chemical extraction from cells, method optimization and validation, overview
-
recombinant C-terminally His6-tagged enzyme from Escherichia coli strain BLR(DE3) to homogeneity by expanded bed adsorption column utilizing immobilized metal affinity chromatography, this integrated process greatly simplifies the recovery and purification of inclusion body proteins by removing the need for mechanical cell disruption, repeated inclusion body centrifugation, and difficult clarification operations, chemical extraction is as effective as homogenization at releasing the inclusion body proteins from the bacterial cells, over 99% purity of the enzyme, method optimization and validation, overview
-
recombinant enzyme from Escherichia coli strain BLR(DE3) to homogeneity by nickel affinity chromatography after dilution refolding, method optimization, overview
-
recombinant wild-type and mutant enzymes from Escherichia coli strain DH5alpha to over 99% purity
-
enzyme complex of 3beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5 to 4 isomerase
-
glutathione-Sepharose column chromatography
Q16772
modified enzyme in which the membrane-spanning domain, residues 283-310, of the enzyme protein is deleted in the cDNA is expressed by baculovirus in the cytosol instead of in the microsomes and mitochondria of the Sf9 cells
-
mutant enzymes D36A/K37R, D241N, D257L, D258L and D265N are expressed in a baculovirus/Sf9 cell system
-
recombinant type 1 isozyme, wild-type and mutant enzymes, from Spodoptera frugiperda Sf9 cells
P14060, P26439
copurifies with 3beta-hydroxysteroid oxidase activity
Nocardia rhodochrous
-
DELTA5-3-beta-hydroxysteroid dehydrogenase-isomerase system
-
15N labelled KSI is purified from Escherichia coli BL21 DE3 grown in minimal medium (M9) containing 15N ammonium chloride as the nitrogen source.
-
mutant enzymes D40N and Y16F
-
recombinant wild-type and mutant enzymes from Escherichia coli strain DH5alpha to over 99% purity
-
the 3beta-hydroxysteroid dehydrogenase cannot be separated from the isomerase
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
D38A, D38A/Y14F, D38A/Y55F and D38A/D99N proteins are expressed in Epicurian Coli XL1-Blue cells
-
expression of C-terminally His6-tagged enzyme as insoluble protein in inclusion bodies in Escherichia coli strain BLR(DE3)
P00947
expression of wild-type and mutant enzymes in Escherichia coli strain DH5alpha
-
-
Q16772
expressed in HeLa cells
-
expression of type 1 isozyme, wild-type and mutant enzymes, in Spodoptera frugiperda Sf9 cells via baculovirus infection
P14060, P26439
insect Sf9 cells transfected with recombinant baculovirus
-
mutant enzymes D36A/K37R, D241N, D257L, D258L and D265N are expressed in a baculovirus/Sf9 cell system
-
expression of mutant enzymes D40N and Y16F in Escherichia col BL21
-
expression of wild-type and mutant enzymes in Escherichia coli strain DH5alpha
-
overexpression in Escherichia coli
-
overexpression in Escherichia coli strain BL21 (DE3)
-
Overexpression of recombinant 15N-KSI from Escherichia coli BL21 DE3
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
D103A
-
site-directed mutagenesis of the catalytic residue, the mutant shows 5000fold reduced activity compared tot he wild-type enzyme
D38A
-
the catalytic turnover number is 140fold less than that for the wild-type
D38A/D99N
-
no detectable activity
D38A/Y14F
-
no detectable activity
D38A/Y55F
-
no detectable activity
D38E
-
compared to wild-type, 200fold reduction in kcat-value. Analysis of thermodynamic parameters
D40N
-
site-directed mutagenesis, the mutation mimics the protonated aspartate found in the intermediate and equilenin complexes and leads to tighter binding of phenolate and other intermediate analogs
D99A
-
secondary kinetic isotope effects similar to wild-type. Mutation does not significantly decrease the contribution of coupled motion/hydrogen tunneling to the enzymatic reaction
D99L
-
the mutant shows about 10000fold decreased kcat compared to the wild type enzyme
F116W
-
the turnover-number for 5-androstene-3,17-dione is lowered 4.42fold, the KM-value is 3.1fold lower than the Km-value of the wild-type enzyme
Y14F
-
secondary kinetic isotope effects similar to wild-type. Mutation does not significantly decrease the contribution of coupled motion/hydrogen tunneling to the enzymatic reaction
Y14F
-
study on backbone dynamics in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate. Mutation induces a substantial decrease in the order parameters in free enzyme, indicating that the backbone structures become significantly mobile by mutation, while the chemical shift analysis indicates that the structural perturbations are more profound than those of wild-type upon 19-nortestosterone hemisuccinate binding. In the 19-nortestosterone hemisuccinate complexed mutant, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remain flexible with significantly reduced S2 values, whereas the S2 values for many of the residues in the mutant enzyme become even greater than those of wild-type
Y14F
-
the mutant shows 100000fold decreased kcat compared to the wild type enzyme
Y14F/D99L
-
the mutant shows 1000000fold decreased kcat compared to the wild type enzyme
Y16F
-
site-directed mutagenesis of the catalytic residue, the mutant shows 50000fold reduced activity compared tot he wild-type enzyme
Y55F/Y88F
-
replacement of Tyr14 by 3-fluorotyrosine in the Y55,88F modified form of the isomerase results in a 4-fold decrease in turnover number
Y57F/D40N
-
site-directed mutagenesis, 3,4,5-trifluorophenol bind as ionized phenolate to KSI containing the Y57F mutation
D99L
Commamonas testosteroni
P00947
the mutant shows about 10000fold decreased kcat compared to the wild type enzyme
Y14F
Commamonas testosteroni
P00947
the mutant shows 100000fold decreased kcat compared to the wild type enzyme
Y14F/D99L
Commamonas testosteroni
P00947
the mutant shows 1000000fold decreased kcat compared to the wild type enzyme
D241N
-
mutant enzyme nearly has full isomerase activity, substantial decrease in 3beta-hydroxysteroid dehydrogenase activity. Mutant enzyme has a basal isomerase activity in the absence of coenzyme that is 10% of the NADH-stimulated turnover number
D257L
-
complete lack of isomerase activity in absence of coenzyme, substantial decrease in 3beta-hydroxysteroid dehydrogenase activity
D258L
-
complete lack of isomerase activity in absence of coenzyme, substantial decrease in 3beta-hydroxysteroid dehydrogenase activity
D265N
-
complete lack of isomerase activity in absence of coenzyme, substantial decrease in 3beta-hydroxysteroid dehydrogenase activity
D36A/K37R
-
mutation shifts the cofactor preference of both 3-beta-hydroxysteroid dehydrogenase and isomerase from NAD(H) to NADP(H)
DELTA283-310
-
the molecular weight of the subunit is 38800 Da compared to 42000 Da for the wild-type enzyme
E126L
P14060, P26439
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
H156Y
P14060, P26439
site-directed mutagenesis, leads to destabilization of interactions at the dimer interface and a dramatic increase in the substrate Km and inhibitor Ki values of 3beta-HSD isozyme 1 to equal those measured for 3beta-HSD isozyme 2; site-directed mutagenesis, leads to destabilization of interactions at the dimer interface and a dramatic increase in the substrate Km and inhibitor Ki values of 3beta-HSD isozyme 1 to equal those measured for 3beta-HSD isozyme 2
H232A
P14060, P26439
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
K158Q
P14060, P26439
site-directed mutagenesis, results in the complete loss of dehydrogenase activity and a reduction in isomerase activity, probably due to partial destabilization of cofactor binding, in which the catalytic triad is actively involved, and associated substrate binding
M187T
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increase in affinity for NADH as an allosteric activator
N100A
P14060, P26439
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
N100S
P14060, P26439
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
N323L
P14060, P26439
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
Q105M
P14060, P26439
site-directed mutagenesis, leads to destabilization of interactions at the dimer interface and a dramatic increase in the substrate Km and inhibitor Ki values of 3beta-HSD isozyme 1 to equal those measured for 3beta-HSD isozyme 2; site-directed mutagenesis, leads to destabilization of interactions at the dimer interface and a dramatic increase in the substrate Km and inhibitor Ki values of 3beta-HSD isozyme 1 to equal those measured for 3beta-HSD isozyme 2
S124A
P14060, P26439
site-directed mutagenesis, results in the complete loss of dehydrogenase activity and a reduction in isomerase activity, probably due to partial destabilization of cofactor binding, in which the catalytic triad is actively involved, and associated substrate binding
S124T
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11fold decrease in affinity to substrate
S322A
P14060, P26439
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
Y154F
P14060, P26439
site-directed mutagenesis, results in the complete loss of dehydrogenase activity and a reduction in isomerase activity, probably due to partial destabilization of cofactor binding, in which the catalytic triad is actively involved, and associated substrate binding
D103N/D40N
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site-directed mutagenesis, 3,4,5-trifluorophenol bind as ionized phenolate to KSI containing the D103N mutation
D40N
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1484555fold decrease in turnover number, 4.46fold decrease in KM-value as compared to wild-type enzyme
D40N
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site-directed mutagenesis, the mutation mimics the protonated aspartate found in the intermediate and equilenin complexes and leads to tighter binding of phenolate and other intermediate analogs
D40N
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inhibition compared to wild-type enzyme, overview
D99E
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turnover-number for 5-androstene-3,17-dione is 68.1% of the turnover number of the wild-type enzyme, the Km-value is 1.22fold higher than the Km-value of the wild-type enzyme
D99L
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the turnover number for 5-androstene-3,17-dione is 96.5fold lower than that of the wild-type enzyme, the KM-value is 1.9fold lower than that of the wild-type enzyme. Mutation results in a loss of conformational stability of 3.8 kcal/mol, at 25C, pH 7.0
D99L
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turnover-number for 5-androstene-3,17-dione is 125fold lower than the turnover number of the wild-type enzyme, the Km-value is 1.95fold lower than the Km-value of the wild-type enzyme
E118A
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the free-energy change for unfolding in the absence of urea at 25C is decreased by about 3.9 kcal/mol compared to wild-type value. Mutation increasex the dissociation constant for (+)-equilenin, a reaction intermediate analogue. 50% of the protein is unfolded at 4.46 M urea compared to 5.22 M for the wild-type enzyme. The turnover-number for 5-androstene-3,17-dione is 33% of that of the wild-type enzyme, the Km-value is 348% of that of the wild-type enzyme
E118A/N120A
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the free-energy change for unfolding in the absence of urea at 25C is decreased by about 9.5 kcal/mol compared to wild-type value. Mutation increases the dissociation constant for (+)-equilenin, a reaction intermediate analogue. 50% of the protein is unfolded at 3.89 M urea compared to 5.22 M for the wild-type enzyme. The turnover-number for 5-androstene-3,17-dione is 6% of that of the wild-type enzyme, the Km-value is 523% of that of the wild-type enzyme
F54A
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turnover number for 5-androstene-3,17-dione is 17.1% of the turnover number for the wild-type enzyme, turnover number for 5(10)-estrene-3,17-dione is 15.3% of the turnover number of the wild-type enzyme,the KM-value for 5-androstene-3,17-dione is 116% of the KM-value of the wild-type enzyme, the KM-value for 5(10)-estrene-3,17-dione is 233% of the KM-value of the wild-type enzyme
F54L
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turnover number for 5-androstene-3,17-dione is 41.9% of the turnover number of the wild-type enzyme, turnover number for 5(10)-estrene-3,17-dione is 38.7% of the turnover number of the wild-type enzyme, the KM-value for 5-androstene-3,17-dione is 154% of the KM-value of the wild-type enzyme, the KM-value for 5(10)-estrene-3,17-dione is 89.3% of the KM-value of the wild-type enzyme
F82A
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turnover number for 5-androstene-3,17-dione is 2.5% of the turnover number of the wild-type enzyme, turnover number for 5(10)-estrene-3,17-dione is 2.3% of the turnover number of the wild-type enzyme,the KM-value for 5-androstene-3,17-dione is 29.9% of the KM-value of the wild-type enzyme, the KM-value for 5(10)-estrene-3,17-dione is 29.8% of the KM-value of the wild-type enzyme
F82L
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turnover number for 5-androstene-3,17-dione is 12% of the turnover number of the wild-type enzyme, turnover number for 5(10)-estrene-3,17-dione is 67.6% of the turnover number of the wild-type enzyme, the KM-value for 5-androstene-3,17-dione is 108% of the KM-value of the wild-type enzyme, the KM-value for 5(10)-estrene-3,17-dione is 130% of the KM-value of the wild-type enzyme
L125A/V127A
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mutation in small exterior hydrophobic cluster, decrease in conformational stability
L125F/V127F
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mutation in small exterior hydrophobic cluster, slight increase in stability
N120A
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the free-energy change for unfolding in the absence of urea at 25C is decreased by about 7.8 kcal/mol compared to wild-type value. Mutation increasex the dissociation constant for (+)-equilenin, a reaction intermediate analogue. 50% of the protein is unfolded at 3.95 M urea compared to 5.22 M for the wild-type enzyme. The turnover-number for 5-androstene-3,17-dione is 17% of that of the wild-type enzyme, the Km-value is 516% of that of the wild-type enzyme
R72A
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the free-energy change for unfolding in the absence of urea at 25C is decreased by about 3.8 kcal/mol compared to wild-type value. Mutation increasex the dissociation constant for (+)-equilenin, a reaction intermediate analogue. 50% of the protein is unfolded at 4.74 M urea compared to 5.22 M for the wild-type enzyme. The turnover-number for 5-androstene-3,17-dione is 23% of that of the wild-type enzyme
W116A
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turnover number for 5-androstene-3,17-dione is 1.5% of the turnover number of the wild-type enzyme, turnover number for 5(10)-estrene-3,17-dione is below 12%% of the turnover number of the wild-type enzyme, the KM-value for 5-androstene-3,17-dione is 348% of the KM-value of the wild-type enzyme
W116F
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turnover number for 5-androstene-3,17-dione is 22.4% of the turnover number of the wild-type enzyme, the KM-value for 5(10)-estrene-3,17-dione is 299% of the KM-value of the wild-type enzyme
W116Y
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turnover number for 5-androstene-3,17-dione is 21% of the turnover number of the wild-type enzyme
W92A
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mutation in small exterior hydrophobic cluster, decrease in conformational stability. Crystallization data
W92A/L125A/V127A
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mutation in small exterior hydrophobic cluster, decrease in conformational stability
Y14F
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mutation results in a loss of conformational stability of 4.4 kcal/mol, at 25C, pH 7.0
Y14F
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turnover-number for 5-androstene-3,17-dione is less than 1% of the turnover number of the wild-type enzyme, the Km-value is 1.87fold lower than the Km-value of the wild-type enzyme
Y14F
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the hydrogen bond between Asp99 Odelta2 and C3-O of the steroid, which is perturbed by the Y14F mutation, can be partially restored to that in the wild-type enzyme by additional Y30F/Y55F mutations
Y14F/D99E
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turnover-number for 5-androstene-3,17-dione is less than 1% of the turnover number of the wild-type enzyme, the Km-value is 1.05fold lower than the Km-value of the wild-type enzyme
Y14F/D99L
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turnover-number for 5-androstene-3,17-dione is less than 1% of the turnover number of the wild-type enzyme, the Km-value is 1.95fold higher than the Km-value of the wild-type enzyme
Y14F/D99L
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partly additive effect of mutations for both, catalysis and stability, increase in hydrophobic interaction while disrupting the hydrogen bond network, crystallization data
Y14F/D99N
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turnover-number for 5-androstene-3,17-dione is less than 1% of the turnover number of the wild-type enzyme, the Km-value is 1.06fold lower than the Km-value of the wild-type enzyme
Y14F/Y30F
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the turnover-number for 5-androstene-3,17-dione is about 1% of that of the wild-type enzyme, the Km-value is 1.57fold higher than that of the wild-type enzyme
Y14F/Y30F/Y55F
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the turnover-number for 5-androstene-3,17-dione is about 2.1% of that of the wild-type enzyme, the Km-value is comparable to the Km-value of the wild-type enzyme. The hydrogen bond between Asp99 Odelta2 and C3-O of the steroid, which is perturbed by the Y14F mutation, can be partially restored to that in the wild-type enzyme by additional Y30F/Y55F mutations. The improvement in the catalytic activity of Y14F by the additional Y30f/Y55F mutation is due to the changes in the structural integrity at the catalytic site and the resulting restoration of the proton-transfer mechanism in Y14F/Y30F/Y55F
Y14F/Y30F/Y55F/D99L
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the turnover-number for 5-androstene-3,17-dione is less than 1.2% of that of the wild-type enzyme, the Km-value is 1.8fold higher than that of the wild-type enzyme
Y14F/Y30F/Y55F/D99N
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the turnover-number for 5-androstene-3,17-dione is less than 1% of that of the wild-type enzyme, the Km-value is 1.14fold higher than that of the wild-type enzyme
Y14F/Y55F
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the turnover-number for 5-androstene-3,17-dione is about 1.2% of that of the wild-type enzyme, the Km-value is 1.75fold higher than that of the wild-type enzyme
Y16F
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2009fold decrease in turnover number, 3.5fold decrease in KM-value as compared to wild-type enzyme
Y16F
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the number of water molecules directly hydrogen bonded to the ligand oxygen is one in the Y16F mutant
Y16F/Y32F/Y57F
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the number of water molecules directly hydrogen bonded to the ligand oxygen is one in the Y16F/Y32F/Y57F mutant
Y16S
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the number of water molecules directly hydrogen bonded to the ligand oxygen is approximately two in the Y16S mutant
Y30F
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the turnover number for 5-androstene-3,17-dione is 1.2fold lower than that of the wild-type enzyme, the KM-value is 1.1fold lower than that of the wild-type enzyme
Y30F/D99L
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the turnover number for 5-androstene-3,17-dione is 521.6fold lower than that of the wild-type enzyme, the KM-value is 1.47fold lower than that of the wild-type enzyme. Mutation results in a loss of conformational stability of 6.5 kcal/mol, at 25C, pH 7.0
Y30F/D99L
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disruption of hydrogen bond network, crystallization data
Y30F/Y55F
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the turnover number for 5-androstene-3,17-dione is 1.99fold lower than that of the wild-type enzyme, the KM-value is comparable to that of the wild-type enzyme
Y55F
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the turnover number for 5-androstene-3,17-dione is 6fold lower than that of the wild-type enzyme, the KM-value is 2.17fold lower than that of the wild-type enzyme. mutation results in a loss of conformational stability of 3.5 kcal/mol, at 25C, pH 7.0. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30
Y55F/D99L
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the turnover number for 5-androstene-3,17-dione is 17692fold lower than that of the wild-type enzyme, the KM-value is 1.24fold lower than that of the wild-type enzyme. Mutation results in a loss of conformational stability of 7.9 kcal/mol, at 25C, pH 7.0
Y55F/D99L
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disruption of hydrogen bond network, crystallization data
Renatured/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
70-87% refolding of recombinant C-terminally His6-tagged enzyme after expression in Escherichia coli as insoluble protein and solubilization by denaturation, detergent treatment, and 40fold dilution by a single-step column-based refolding step with an elution gradient, dilution with buffer 1% bovine serum albumin, 20 mM phosphate, 150 mM NaCl, optimal at pH 6.8, method optimization, overview
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dilution refolding of recombinant enzyme from inclusion bodies, denatured recombinant C-terminally His6-tagged enzyme 10fold with refolding buffer containing 50 mM potassium phosphate, pH 7.0, 4C, incubation at 4C for 12 h, urea 0.8 M
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repeated adsorptive refolding in an expanded bed with gradient change in the feed-stream composition from denaturing buffer containing 8 M urea and 50 mM potassium phosphate, pH 6.8 to the refolding buffer containing 50 mM potassium phosphate, pH 6.8, constant liquid feed rate, method evaluation, overview
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the recovery of the activity by refolding is not diminished even after a prolonged time, 24 h of exposure to 7 M urea
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50% of the wild-type enzyme is unfolded at 5.22 M urea. Mutant enzymes R72A, E118A, N120A and E118A/N120A are unfolded at lower concentrations
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detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase, NMR spectroscopy, circular dichroism and fluorescence spectroscopies, and small angle X-ray scattering and analytical ultracentrifugation of native and fully unfolded enzymes, overview
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secondary structure is not significantly affected by urea up to 3.5 M, but the residues in the dimeric interface region are significantly perturbed by urea at low concentrations. The interface region primarily around the beta5- and beta6-strands may play an important role as the starting positions in the unfolding process
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urea-induced equilibrium unfolding, two-state mechanism involving only the native dimer and the unfolded monomer. The enzymatic activity is affected significantly by urea and decreases exponentially as urea concentration increases, suggesting that the active site is less stable than the tertiary structure as a whole. Dilution of the unfolded protein in 8 M urea to lower urea concentrations in a reducing condition, the activity of the refolded protein is recovered up to over 95% of that of the native protein. The protein folds into a monomer containing most of the alpha-helical structures before dimerization
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