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 hide
5.3.3.1
-
RECOMMENDED NAME
GeneOntology No.
steroid DELTA-isomerase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
a 3-oxo-DELTA5-steroid = a 3-oxo-DELTA4-steroid
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
intramolecular oxidoreduction
-
-
-
-
isomerization
PATHWAY
BRENDA Link
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).
CAS REGISTRY NUMBER
COMMENTARY hide
9031-36-1
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain KITA
-
-
Manually annotated by BRENDA team
strain KITA
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
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
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
-
the enzyme is responsible for progesterone and androstenedione production
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
17beta-Hydroxy-5(10)-estren-3-one
?
show the reaction diagram
5(10)-estrene-3,17-dione
estr-4-en-3,17-dione
show the reaction diagram
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-one
?
show the reaction diagram
-
-
-
-
-
5-Cholesten-3-one
4-Cholesten-3-one
show the reaction diagram
5-Pregnene-3,20-dione
4-Pregnene-3,20-dione
show the reaction diagram
a 3-oxo-DELTA5-steroid
a 3-oxo-DELTA4-steroid
show the reaction diagram
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
androstenedione
show the reaction diagram
-
-
-
-
?
DELTA5-pregnen-3,20-dione
?
show the reaction diagram
-
-
-
?
pregn-5-ene-3,20-dione-17alpha-ol
17alpha-hydroxyprogesterone
show the reaction diagram
-
-
-
-
?
pregnenolone
progesterone
show the reaction diagram
-
-
-
-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
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
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
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
COMMENTARY hide
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
17beta-Hydroxy-5,10-seco-oestr-4-yne-3,10-dione
-
irreversible, time-dependent, active-site directed
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
5alpha-androstran-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
epostane
-
-
equilenin
estradiol
-
competitive
estrone
-
and derivatives, noncompetitive
FeCl2
-
moderate
formononetin
-
IC50: 0.004 mM
genistein
-
IC50: 0.0023 mM
Halothane
-
allosteric modulation of dynamics-function relationship without direct competition, halothane occupancy at the dimer interface disrupts intersubunit hydrogen bonding
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
-
-
Testosterone acetate
-
-
trilostane
-
-
ZnCl2
-
moderate
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADH
the cofactor acts as an allosteric activator; the cofactor acts as an allosteric activator
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0223
17beta-Hydroxy-5(10)-estren-3-one
-
-
0.0328 - 0.256
5(10)-estrene-3,17-dione
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 - 0.548
5-androstene-3,17-dione
0.007
5-Cholesten-3-one
Nocardia erythropolis
-
-
0.0093 - 0.068
5-Pregnene-3,20-dione
0.023 - 0.0735
androstene-3,17-dione
0.0175 - 0.0884
dehydroepiandrosterone
0.01
DELTA5-pregnen-3,20-dione
-
25C, pH 7.5
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
70.8
17beta-Hydroxy-5(10)-estren-3-one
Comamonas testosteroni
-
-
0.25 - 11.1
5(10)-estrene-3,17-dione
0.052 - 288000
5-androstene-3,17-dione
1.2 - 17800
androstene-3,17-dione
1990 - 4890
dehydroepiandrosterone
0.303
DELTA5-pregnen-3,20-dione
Bos taurus
-
25C, pH 7.5
additional information
additional information
Pseudomonas putida
-
turnover number of mutant enzymes Y16F and D40N
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0028 - 0.0043
4alpha,5alpha-epoxy-testosterone
0.00007 - 0.00184
epostane
0.0001 - 0.00208
trilostane
additional information
additional information
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0003
17beta-estradiol
Bos taurus
-
IC50: 0.0003 mM
0.0004
Biochanin A
Bos taurus
-
IC50: 0.0004 mM
0.0038
daidzein
Bos taurus
-
IC50: 0.0038 mM
0.004
formononetin
Bos taurus
-
IC50: 0.004 mM
0.0023
genistein
Bos taurus
-
IC50: 0.0023 mM
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.622
-
reaction with 5-androsten-3,17-dione, truncated cytosolic enzyme DELTA283-310
3.5
-
-
30600
-
purified diluted recombinant enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 7.5
Nocardia rhodochrous
-
-
7.4
assay at; assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8
7.5 - 9
-
pH 7.5: about 40% of maximal activity, pH 9.0: about 60% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27
assay at; assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22 - 42
-
22C: about 60% of maximal activity, 40C: about 40% of maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
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
type 1 isozyme
Manually annotated by BRENDA team
-
-
Manually annotated by BRENDA team
-
-
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
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
UNIPROT
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
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
COMMENTARY hide
LITERATURE
homodimer
tetramer
-
4 * 19000, enzyme complex of 3beta-hydroxy-5-ene-steroid dehydrogenase and steroid DELTA-isomerase, SDS-PAGE
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
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
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
ORGANISM
UNIPROT
COMMENTARY hide
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
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
COMMENTARY hide
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
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
LITERATURE
15N labelled KSI is purified from Escherichia coli BL21 DE3 grown in minimal medium (M9) containing 15N ammonium chloride as the nitrogen source.
-
3beta-hydroxy-5-ene steroid dehydrogenase/isomerase enzyme system
-
3beta-hydroxy-5-ene-steroid dehydrogenase/steroid DELTA5-DELTA4-ene isomerase enzyme complex
-
affinity chromatography
-
copurifies with 3beta-hydroxysteroid oxidase activity
Nocardia rhodochrous
-
DELTA5-3-beta-hydroxysteroid dehydrogenase-isomerase system
-
enzyme complex of 3beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5 to 4 isomerase
-
glutathione-Sepharose column chromatography
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
-
mutant enzymes D40N and Y16F
-
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
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recombinant type 1 isozyme, wild-type and mutant enzymes, from Spodoptera frugiperda Sf9 cells
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
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
D38A, D38A/Y14F, D38A/Y55F and D38A/D99N proteins are expressed in Epicurian Coli XL1-Blue cells
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expressed in HeLa cells
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expression of C-terminally His6-tagged enzyme as insoluble protein in inclusion bodies in Escherichia coli strain BLR(DE3)
expression of mutant enzymes D40N and Y16F in Escherichia col BL21
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expression of type 1 isozyme, wild-type and mutant enzymes, in Spodoptera frugiperda Sf9 cells via baculovirus infection
expression of wild-type and mutant enzymes in Escherichia coli strain DH5alpha
insect Sf9 cells transfected with recombinant baculovirus
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mutant enzymes D36A/K37R, D241N, D257L, D258L and D265N are expressed in a baculovirus/Sf9 cell system
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overexpression in Escherichia coli
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overexpression in Escherichia coli strain BL21 (DE3)
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Overexpression of recombinant 15N-KSI from Escherichia coli BL21 DE3
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
during the estrous cycle, the endometrial enzyme activity is higher on days 10 to 11 and decreases on days 12 to 13 and days 15 to 16
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D103A
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site-directed mutagenesis of the catalytic residue, the mutant shows 5000fold reduced activity compared tot he wild-type enzyme
D38A
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the catalytic turnover number is 140fold less than that for the wild-type
D38A/D99N
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no detectable activity
D38A/Y14F
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no detectable activity
D38A/Y55F
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no detectable activity
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
D99A
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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
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the mutant shows about 10000fold decreased kcat compared to the wild type enzyme
F116W
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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/D99L
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the mutant shows 1000000fold decreased kcat compared to the wild type enzyme
Y16F
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site-directed mutagenesis of the catalytic residue, the mutant shows 50000fold reduced activity compared tot he wild-type enzyme
Y55F/Y88F
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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
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site-directed mutagenesis, 3,4,5-trifluorophenol bind as ionized phenolate to KSI containing the Y57F mutation
D241N
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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
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complete lack of isomerase activity in absence of coenzyme, substantial decrease in 3beta-hydroxysteroid dehydrogenase activity
D258L
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complete lack of isomerase activity in absence of coenzyme, substantial decrease in 3beta-hydroxysteroid dehydrogenase activity
D265N
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complete lack of isomerase activity in absence of coenzyme, substantial decrease in 3beta-hydroxysteroid dehydrogenase activity
D36A/K37R
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mutation shifts the cofactor preference of both 3-beta-hydroxysteroid dehydrogenase and isomerase from NAD(H) to NADP(H)
DELTA283-310
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the molecular weight of the subunit is 38800 Da compared to 42000 Da for the wild-type enzyme
E126L
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
H156Y
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
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
K158Q
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
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
N100S
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
N323L
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
Q105M
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
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
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
Y154F
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
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
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/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
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/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
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
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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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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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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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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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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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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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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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Show AA Sequence (162 entries)
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