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Information on EC 5.3.3.1 - steroid DELTA-isomerase and Organism(s) Pseudomonas putida and UniProt Accession P07445
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5.3.3.1 steroid DELTA-isomeraseIUBMB Comments
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).
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Word Map
- 5.3.3.1
-
steroidogenic
- progesterone
- adrenal
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steroidogenesis
- androgen
- p450scc
- leydig
- star
- testicular
- pregnenolone
- 17beta-hsd
- lh
- aromatase
-
5-3-ketosteroids
- gonad
- testosteroni
-
granulosa
- dehydroepiandrosterone
- luteal
- androstenedione
-
17beta-hydroxysteroid
- dheas
-
theca
- p450arom
- 17alpha-hydroxylase
-
neurosteroids
- cyp11a1
- trilostane
- 19-nortestosterone
-
dienolate
-
5alpha-reductase
- equilenin
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low-barrier
- 17alpha-hydroxylase/17,20-lyase
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interna
- 3betahsd
- delta5-3beta-hydroxysteroid
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intracrine
-
double-bond
- hsd3b1
-
talalay
- 20alpha-hsd
-
beta-proton
The taxonomic range for the selected organisms is: Pseudomonas putida
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
3beta-hsd, ketosteroid isomerase, delta 5-3-ketosteroid isomerase, steroid isomerase, 3beta-hydroxysteroid dehydrogenase/isomerase, steroid delta-isomerase, gst a3-3, 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase, 3beta-hsd/isomerase, 5-ene-4-ene isomerase, 
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topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
3-Keto-DELTA5-steroid isomerase
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-
-
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3-Ketosteroid DELTA5-->DELTA4-isomerase
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-
-
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3-Oxo steroid DELTA4-DELTA5-isomerase
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-
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3-Oxo-delta5 steroid isomerase
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-
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3-Oxosteroid DELTA4-DELTA5-isomerase
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-
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3-Oxosteroid DELTA5-DELTA4-isomerase
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-
-
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3-Oxosteroid isomerase
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-
-
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5-Ene-4-ene isomerase
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-
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5-Pregnene-3,20-dione isomerase
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-
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delta 5-3-ketosteroid isomerase
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-
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DELTA-3-ketosteroid isomerase
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Delta-5-3-ketosteroid isomerase
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DELTA5(or DELTA4)-3-keto steroid isomerase
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-
-
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DELTA5-3-keto steroid isomerase
DELTA5-3-ketosteroid isomerase
DELTA5-3-oxosteroid isomerase
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-
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DELTA5-ketosteroid isomerase
DELTA5-steroid isomerase
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-
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Hydroxysteroid isomerase
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-
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Isomerase, steroid DELTA
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Steroid 5-->4-isomerase
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-
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Steroid isomerase
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DELTA5-3-keto steroid isomerase
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DELTA5-3-ketosteroid isomerase
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DELTA5-ketosteroid isomerase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a 3-oxo-DELTA5-steroid = a 3-oxo-DELTA4-steroid
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
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
isomerization
intramolecular oxidoreduction
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-
-
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isomerization
-
-
-
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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
PATHWAY SOURCE
PATHWAYS
KEGG
-
-, -, -, -, -, -, -, -, -, -
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
9031-36-1
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(+)-cis-isopulegone
(+)-pulegone
the enzyme also exhibits (+)-cis-isopulegone isomerase activity
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
-
-
-
?
5-Androstene-3,17-dione
4-Androstene-3,17-dione
-
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
-
?
a 3-oxo-DELTA5-steroid
a 3-oxo-DELTA4-steroid
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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
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
?
-
-
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
?
-
-
enzyme can be induced by various steroids
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-
?
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
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
-
-
enzyme can be induced by various steroids
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-
?
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
coumarin 183
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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
Urea
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enzymatic activity is affected significantly by urea and decreases exponentially as urea concentration increases
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
-
equilenin
reaction intermediate analogue, binds to the active site, binding structure, overview
equilenin
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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
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0171
5-androstene-3,17-dione
mutant enzyme Y14F, at pH 7.0, temperature not specified in the publication
0.023
5-androstene-3,17-dione
mutant enzyme Y55F, at pH 7.0, temperature not specified in the publication
0.0417
5-androstene-3,17-dione
mutant enzyme Y115F, at pH 7.0, temperature not specified in the publication
0.0499
5-androstene-3,17-dione
wild type enzyme, at pH 7.0, temperature not specified in the publication
0.0502
5-androstene-3,17-dione
mutant enzyme Y30F/Y55F, at pH 7.0, temperature not specified in the publication
0.0552
5-androstene-3,17-dione
mutant enzyme Y30F, at pH 7.0, temperature not specified in the publication
0.0773
5-androstene-3,17-dione
mutant enzyme Y30F/Y55F/Y115F, at pH 7.0, temperature not specified in the publication
0.0513
5-androstene-3,17-dione
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25°C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F
0.0578
5-androstene-3,17-dione
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25°C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F/D99N
0.0918
5-androstene-3,17-dione
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25°C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F/D99L
additional information
additional information
-
Km-values of mutant enzymes Y16F and D40N
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
turnover number of mutant enzymes Y16F and D40N
-
13.3
5-androstene-3,17-dione
mutant enzyme Y14F, at pH 7.0, temperature not specified in the publication
3510
5-androstene-3,17-dione
mutant enzyme Y55F, at pH 7.0, temperature not specified in the publication
10700
5-androstene-3,17-dione
mutant enzyme Y30F/Y55F, at pH 7.0, temperature not specified in the publication
12200
5-androstene-3,17-dione
mutant enzyme Y30F/Y55F/Y115F, at pH 7.0, temperature not specified in the publication
14500
5-androstene-3,17-dione
mutant enzyme Y115F, at pH 7.0, temperature not specified in the publication
17800
5-androstene-3,17-dione
mutant enzyme Y30F, at pH 7.0, temperature not specified in the publication
21000
5-androstene-3,17-dione
wild type enzyme, at pH 7.0, temperature not specified in the publication
0.052
5-androstene-3,17-dione
-
25°C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F/D99L
1.1
5-androstene-3,17-dione
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25°C, pH 7.0, mutant enzyme Y14F/Y30F/Y55F/D99N
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
780
5-androstene-3,17-dione
mutant enzyme Y14F, at pH 7.0, temperature not specified in the publication
150000
5-androstene-3,17-dione
mutant enzyme Y55F, at pH 7.0, temperature not specified in the publication
160000
5-androstene-3,17-dione
mutant enzyme Y30F/Y55F/Y115F, at pH 7.0, temperature not specified in the publication
210000
5-androstene-3,17-dione
mutant enzyme Y30F/Y55F, at pH 7.0, temperature not specified in the publication
330000
5-androstene-3,17-dione
mutant enzyme Y30F, at pH 7.0, temperature not specified in the publication
350000
5-androstene-3,17-dione
mutant enzyme Y115F, at pH 7.0, temperature not specified in the publication
430000
5-androstene-3,17-dione
wild type enzyme, at pH 7.0, temperature not specified in the publication
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
14000
-
2 * 14000, mutant enzymnes R72A, E118A, N120A and E18A/N120A, SDS-PAGE
14520
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
homodimer
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
dimer
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2 * 14000, mutant enzymnes R72A, E118A, N120A and E18A/N120A, 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
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.
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
crystal structure of the enzyme in complex with equilenin, an analogue of the reaction intermediate at 1.9 and 2.5 A resolution
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 Y30F/Y55F/Y115F/D38N KSI complexed with equilenin, hanging drop vapor diffusion method, using 0.1 M sodium acetate, pH 4.5, 0.6 M ammonium acetate, and 30% PEG 4000
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
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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 22°C
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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 22°C. 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
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enzyme mutant D40N bound to phenolate, X-ray diffraction structure determination and analysis at 1.25 A resolution
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hanging drop vapor diffusion method. Crystal structures of Y14F and Y14F/Y30F/Y55F are determined at 1.8 and 2.0 A resolution, respectively
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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 22°C. 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°
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mutant W92A, in complex with d-equilenin, decrease in conformational stability results from destabilization of surface hydrophobic layer
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mutant Y14F/D99L, increase in hydrophobic interaction while disrupting the hydrogen bond network, mutants Y30F/D99L and Y55F/D99L, disruption of hydrogen bond network
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D103N/D40N
site-directed mutagenesis, 3,4,5-trifluorophenol bind as ionized phenolate to KSI containing the D103N mutation
Y115F
the mutant shows about 68% activity compared to the wild type enzyme
Y14F
the mutant shows almost no activity compared to the wild type enzyme
Y16F
the number of water molecules directly hydrogen bonded to the ligand oxygen is one in the Y16F mutant
Y16F/Y32F/Y57F
the number of water molecules directly hydrogen bonded to the ligand oxygen is one in the Y16F/Y32F/Y57F mutant
Y16S
the number of water molecules directly hydrogen bonded to the ligand oxygen is approximately two in the Y16S mutant
Y30F
the mutant shows about 84% activity compared to the wild type enzyme
Y30F/Y55F
the mutant shows about 50% activity compared to the wild type enzyme
Y30F/Y55F/Y115F
the mutant shows about 57% activity compared to the wild type enzyme
Y55F
the mutant shows about 17% activity compared to the wild type enzyme
D40N
D99E
-
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
E118A
-
the free-energy change for unfolding in the absence of urea at 25°C 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
-
the free-energy change for unfolding in the absence of urea at 25°C 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
-
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
-
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
-
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
-
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
-
mutation in small exterior hydrophobic cluster, decrease in conformational stability
L125F/V127F
-
mutation in small exterior hydrophobic cluster, slight increase in stability
N120A
-
the free-energy change for unfolding in the absence of urea at 25°C 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
-
the free-energy change for unfolding in the absence of urea at 25°C 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
-
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
-
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
-
turnover number for 5-androstene-3,17-dione is 21% of the turnover number of the wild-type enzyme
W92A
-
mutation in small exterior hydrophobic cluster, decrease in conformational stability. Crystallization data
W92A/L125A/V127A
-
mutation in small exterior hydrophobic cluster, decrease in conformational stability
Y14F
Y14F/D99E
-
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
-
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
-
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
-
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
-
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
-
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
-
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
-
2009fold decrease in turnover number, 3.5fold decrease in KM-value as compared to wild-type enzyme
Y30F
-
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
-
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
-
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 25°C, 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
Y57S
-
the mutation causes a large decrease in the catalytic performance of the enzyme
D40N
-
1484555fold decrease in turnover number, 4.46fold decrease in KM-value as compared to wild-type enzyme
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
D40N
-
the mutation does not perturb the gross tertiary and secondary structure of the enzyme The D40N mutant mimics the charge distribution of the enzyme active site in the intermediate state (protonated base), and is not the active form of the enzyme
D99L
-
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 25°C, pH 7.0
D99L
-
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
Y14F
-
mutation results in a loss of conformational stability of 4.4 kcal/mol, at 25°C, pH 7.0
Y14F
-
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
-
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/D99L
-
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
-
partly additive effect of mutations for both, catalysis and stability, increase in hydrophobic interaction while disrupting the hydrogen bond network, crystallization data
Y30F/D99L
-
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 25°C, pH 7.0
Y55F/D99L
-
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 25°C, pH 7.0
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25
-
the free-energy change for unfolding in the absence of urea at 25°C is 24.4 kcal/mol for the wild-type enzyme
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant wild-type and mutant enzymes from Escherichia coli strain DH5alpha to over 99% purity
15N labelled KSI is purified from Escherichia coli BL21 DE3 grown in minimal medium (M9) containing 15N ammonium chloride as the nitrogen source.
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of wild-type and mutant enzymes in Escherichia coli strain DH5alpha
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
-
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
-
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
-
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
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Smith, S.B.; Richards, J.W.; Benisek, W.F.
The purification and characterization of DELTA5-3-ketosteroid isomerase from Pseudomonas putida, a cysteine-containing isomerase
J. Biol. Chem.
255
2678-2684
1980
Pseudomonas putida
Oh, B.H.; Kim, S.W.; Ryu, S.E.; Kim, S.S.; Choi, K.Y.
Crystallization and preliminary X-ray crystallographic studies of ketosteroid isomerase from Pseudomonas putida biotype B
Proteins
24
514-515
1996
Pseudomonas putida
Kim, S.W.; Cha, S.S.; Cho, H.S.; Kim, J.S.; Ha, N.C.; Cho, M.J.; Joo, S.; Kim, K.K.; Choi, K.Y.; Oh, B.H.
High-resolution crystal structures of delta5-3-ketosteroid isomerase with and without a reaction intermediate analogue [published erratum appears in Biochemistry 1998 May 5;37(18):6614]
Biochemistry
36
14030-14036
1997
Pseudomonas putida (P07445), Pseudomonas putida
Kim, D.H.; Nam, G.H.; Jang, D.S.; Choi, G.; Joo, S.; Kim, J.S.; Oh, B.H.; Choi, K.Y.
Roles of active site aromatic residues in catalysis by ketosteroid isomerase from Pseudomonas putida biotype B
Biochemistry
38
13810-13819
1999
Pseudomonas putida
Kim, D.H.; Jang, D.S.; Nam, G.H.; Choi, G.; Kim, J.S.; Ha, N.C.; Kim, M.S.; Oh, B.H.; Choi, K.Y.
Contribution of the hydrogen-bond network involving a tyrosine triad in the active site to the structure and function of a highly proficient ketosteroid isomerase from Pseudomonas putida biotype B
Biochemistry
39
4581-4589
2000
Pseudomonas putida
Choi, G.; Ha, N.C.; Kim, S.W.; Kim, D.H.; Park, S.; Oh, B.H.; Choi, K.Y.
Asp-99 donates a hydrogen bond not to Tyr-14 but to the steroid directly in the catalytic mechanism of DELTA 5-3-ketosteroid isomerase from Pseudomonas putida biotype B
Biochemistry
39
903-909
2000
Pseudomonas putida
Choi, G.; Ha, N.C.; Kim, M.S.; Hong, B.H.; Oh, B.H.; Choi, K.Y.
Pseudoreversion of the catalytic activity of Y14F by the additional substitution(s) of tyrosine with phenylalanine in the hydrogen bond network of DELTA 5-3-ketosteroid isomerase from Pseudomonas putida biotype B
Biochemistry
40
6828-6835
2001
Pseudomonas putida
Kim, S.W.; Choi, K.Y.
Identification of active site residues by site-directed mutagenesis of DELTA5-3-ketosteroid isomerase from Pseudomonas putida biotype B
J. Bacteriol.
177
2602-2605
1995
Pseudomonas putida
Nam, G.H.; Kim, D.H.; Ha, N.C.; Jang do, S.; Yun, Y.S.; Hong, B.H.; Oh, B.H.; Choi, K.Y.
Contribution of conserved amino acids at the dimeric interface to the conformational stability and the structural integrity of the active site in ketosteroid isomerase from Pseudomonas putida biotype B
J. Biochem.
134
101-110
2003
Pseudomonas putida
Kim, D.H.; Nam, G.H.; Jang, D.S.; Yun, S.; Choi, G.; Lee, H.C.; Choi, K.Y.
Roles of dimerization in folding and stability of ketosteroid isomerase from Pseudomonas putida biotype B
Protein Sci.
10
741-752
2001
Pseudomonas putida
Jang do, S.; Cha, H.J.; Cha, S.S.; Hong, B.H.; Ha, N.C.; Lee, J.Y.; Oh, B.H.; Lee, H.S.; Choi, K.Y.
Structural double-mutant cycle analysis of a hydrogen bond network in ketosteroid isomerase from Pseudomonas putida biotype B
Biochem. J.
382
967-973
2004
Pseudomonas putida
Yun, Y.S.; Nam, G.H.; Kim, Y.G.; Oh, B.H.; Choi, K.Y.
Small exterior hydrophobic cluster contributes to conformational stability and steroid binding in ketosteroid isomerase from Pseudomonas putida biotype B
FEBS J.
272
1999-2011
2005
Pseudomonas putida
Jang, D.S.; Lee, H.J.; Lee, B.; Hong, B.H.; Cha, H.J.; Yoon, J.; Lim, K.; Yoon, Y.J.; Kim, J.; Ree, M.; Lee, H.C.; Choi, K.Y.
Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase
FEBS Lett.
580
4166-4171
2006
Pseudomonas putida
Kraut, D.A.; Sigala, P.A.; Pybus, B.; Liu, C.W.; Ringe, D.; Petsko, G.A.; Herschlag, D.
Testing electrostatic complementarity in enzyme catalysis: hydrogen bonding in the ketosteroid isomerase oxyanion hole
PLoS Biol.
4
501-519
2006
Comamonas testosteroni, Pseudomonas putida
-
Sigala, P.A.; Kraut, D.A.; Caaveiro, J.M.; Pybus, B.; Ruben, E.A.; Ringe, D.; Petsko, G.A.; Herschlag, D.
Testing geometrical discrimination within an enzyme active site: constrained hydrogen bonding in the ketosteroid isomerase oxyanion hole
J. Am. Chem. Soc.
130
13696-13708
2008
Comamonas testosteroni, Pseudomonas putida (P07445)
Lee, H.J.; Jang, do S.; Cha, H.J.; Moon, H.S.; Hong, B.H.; Choi, K.Y.; Lee, H.C.
NMR studies on the equilibrium unfolding of ketosteroid isomerase by urea
J. Biochem.
144
215-221
2008
Pseudomonas putida
Lee, H.J.; Moon, H.S.; Jang, do S.; Cha, H.J.; Hong, B.H.; Choi, K.Y.; Lee, H.C.
Probing the equilibrium unfolding of ketosteroid isomerase through xenon-perturbed (1)H- (15)N multidimensional NMR spectroscopy
J. Biomol. NMR
40
65-70
2008
Pseudomonas putida
Sigala, P.; Caaveiro, J.; Ringe, D.; Petsko, G.; Herschlag, D.
Hydrogen bond coupling in the ketosteroid isomerase active site
Biochemistry
48
6932-6939
2009
Comamonas testosteroni, Pseudomonas putida (P07445)
Childs, W.; Boxer, S.G.
Proton affinity of the oxyanion hole in the active site of ketosteroid isomerase
Biochemistry
49
2725-2731
2010
Pseudomonas putida
Childs, W.; Boxer, S.G.
Solvation response along the reaction coordinate in the active site of ketosteroid isomerase
J. Am. Chem. Soc.
132
6474-6480
2010
Pseudomonas putida
Hanoian, P.; Hammes-Schiffer, S.
Water in the active site of ketosteroid isomerase
Biochemistry
50
6689-6700
2011
Pseudomonas putida (P07445)
Jha, S.K.; Ji, M.; Gaffney, K.J.; Boxer, S.G.
Site-specific measurement of water dynamics in the substrate pocket of ketosteroid isomerase using time-resolved vibrational spectroscopy
J. Phys. Chem. B
116
11414-11421
2012
Pseudomonas putida
Parker, M.; Osuna, S.; Bollot, G.; Vaddypally, S.; Zdilla, M.; Houk, K.; Schafmeister, C.
Acceleration of an aromatic claisen rearrangement via a designed spiroligozyme catalyst that mimics the ketosteroid isomerase catalytic dyad
J. Am. Chem. Soc.
136
3817-3827
2014
Pseudomonas putida (P07445)
Jang, D.S.; Choi, G.; Cha, H.J.; Shin, S.; Hong, B.H.; Lee, H.J.; Lee, H.C.; Choi, K.Y.
Contribution of a low-barrier hydrogen bond to catalysis is not significant in ketosteroid isomerase
Mol. Cells
38
409-415
2015
Pseudomonas putida (P07445)
Cha, H.; Jang, D.; Jin, K.; Lee, H.; Hong, B.; Kim, E.; Kim, J.; Lee, H.; Choi, K.; Ree, M.
Three-dimensional structures of a wild-type ketosteroid isomerase and its single mutant in solution
Sci. Adv. Mat.
6
2325-2333
2014
Pseudomonas putida
-
Currin, A.; Dunstan, M.S.; Johannissen, L.O.; Hollywood, K.A.; Vinaixa, M.; Jervis, A.J.; Swainston, N.; Rattray, N.J.W.; Gardiner, J.M.; Kell, D.B.; Takano, E.; Toogood, H.S.; Scrutton, N.S.
Engineering the missing link in biosynthetic (-)-menthol production bacterial isopulegone isomerase
ACS Catal.
8
2012-2020
2018
Pseudomonas putida (P07445)
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