Information on EC 5.3.3.2 - isopentenyl-diphosphate DELTA-isomerase

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

EC NUMBER
COMMENTARY hide
5.3.3.2
-
RECOMMENDED NAME
GeneOntology No.
isopentenyl-diphosphate DELTA-isomerase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Isopentenyl diphosphate = dimethylallyl diphosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
intramolecular oxidoreduction
-
-
-
-
isomerization
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
all-trans-farnesol biosynthesis
-
-
Biosynthesis of antibiotics
-
-
Biosynthesis of secondary metabolites
-
-
bisabolene biosynthesis (engineered)
-
-
isoprene biosynthesis II (engineered)
-
-
Metabolic pathways
-
-
methylerythritol phosphate pathway I
-
-
methylerythritol phosphate pathway II
-
-
mevalonate metabolism
-
-
mevalonate pathway I
-
-
mevalonate pathway II (archaea)
-
-
mevalonate pathway III (archaea)
-
-
mono-trans, poly-cis decaprenyl phosphate biosynthesis
-
-
Terpenoid backbone biosynthesis
-
-
trans, trans-farnesyl diphosphate biosynthesis
-
-
SYSTEMATIC NAME
IUBMB Comments
isopentenyl-diphosphate DELTA3-DELTA2-isomerase
The enzyme from Streptomyces sp. strain CL190 requires FMN and NAD(P)H as cofactors. Activity is reduced if FMN is replaced by FAD, but the enzyme becomes inactive when NAD(P)H is replaced by NAD+ or NADP+. That enzyme also requires Mg2+, Mn2+ or Ca2+ for activity.
CAS REGISTRY NUMBER
COMMENTARY hide
9033-27-6
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Cinchona robusta
-
-
-
Manually annotated by BRENDA team
Citrus sp.
-
-
-
Manually annotated by BRENDA team
Claviceps sp.
Claviceps sp. SD58
strain SD58
-
-
Manually annotated by BRENDA team
Cucurbita sp.
-
-
-
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
Uniprot
Manually annotated by BRENDA team
Methanothermobacter thermautotrophicum
type II isopentenyl diphosphate isomerase
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
isoform IDI2
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain IDI-2, type II enzyme
-
-
Manually annotated by BRENDA team
strain PCC 6803 ORF sll 1556
-
-
Manually annotated by BRENDA team
strain HB 27
-
-
Manually annotated by BRENDA team
Vitis vinifera x Vitis riparia
-
-
-
Manually annotated by BRENDA team
Vitis vinifera x Vitis vinifera
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(Z)-3-(difluoromethyl)-2-buten-1-yl diphosphate
?
show the reaction diagram
-
about 2% of the activity with isopenteyl diphosphate
-
-
-
(Z)-3-(fluoromethyl)-2-buten-1-yl diphosphate
?
show the reaction diagram
-
about 2% of the activity with isopenteyl diphosphate
-
-
-
3-(fluoromethyl)-3-buten-1-yl diphosphate
?
show the reaction diagram
-
competition between isomerization and alkylation of the flavin cofactor
-
-
?
3-Ethylbut-3-enyl diphosphate
trans-3-Methylpent-3-enyl diphosphate
show the reaction diagram
-
ir
-
-
3-methylene-4-penten-1-yl diphosphate
?
show the reaction diagram
-
competition between isomerization and alkylation of the flavin cofactor
-
-
?
Isopentenyl diphosphate
?
show the reaction diagram
Isopentenyl diphosphate
Dimethylallyl diphosphate
show the reaction diagram
isopentenyl diphosphate
dimethylallyl phosphate
show the reaction diagram
-
-
-
-
?
pyruvate
lactate
show the reaction diagram
-
-
-
-
?
trans-3-Methylpent-3-enyl diphosphate
trans-3-Methylpent-2-enyl diphosphate
show the reaction diagram
-
r
-
-
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
Isopentenyl diphosphate
?
show the reaction diagram
Isopentenyl diphosphate
Dimethylallyl diphosphate
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NAD(P)H
NADH
-
or NADPH required under aerobic, not under anaerobic conditions
additional information
-
IDI showed almost the same activity in the presence of NADH or dithionite. Data indicate that the reduced form of FMN is sufficient
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cd2+
-
reconstitution of metal-free enzyme with Mg2+, Mn2+, Zn2+, Co2+, Ni2+ or Cd2+ generates active enzyme
Cu2+
-
10mM, relative activity under 0.005%
Zinc
-
essential cofactor. Type I enzyme contains an atom of tinc. The metal is located in an unusual six-coordinate pocket and may facilitate protonation of the unactivated carbon-carbon double bond in isopentenyl diphosphate
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(+/-)-cis/trans-Chrysanthemyl diphosphate
-
-
(+/-)-cis/trans-Chrysanthemyl monophosphate
-
-
(2Z)-3-methylhept-2-en-1-yl trihydrogen diphosphate
(2Z)-3-methylhex-2-en-1-yl trihydrogen diphosphate
(2Z)-3-methyloct-2-en-1-yl trihydrogen diphosphate
(E/Z)-3-cyclopropyl-2-buten-1-yl diphosphate
-
-
(N,N-dimethylamino)-1-ethyl diphosphate
-
transition state analogue
(Z)-3-difluoromethyl-2-buten-1-yl diphosphate
-
reversible
(Z)-3-fluoromethyl-2-buten-1-yl diphosphate
-
reversible
(Z)-4-Fluoro-3-methyl-2-buten-1-yl diphosphate
Claviceps sp.
-
inactivates through an active-site-directed covalent modification
2,2-Diphenyl-1-(beta-dimethylaminoethoxy)pentane hydrochloride
-
-
2,3-butadien-1-yl diphosphate
2,3-cis,6,7-trans-farnesyl diphosphate
-
-
2-(Dimethylamino)ethyl phosphate
2-[ethyl(methyl)amino]ethyl trihydrogen diphosphate
2-[ethyl(propyl)amino]ethyl trihydrogen diphosphate
3,4-Epoxy-1-butenyl diphosphate
-
active-site directed irreversible inhibitor
3,4-epoxy-3-methyl-1-butyl diphosphate
-
-
3,4-epoxy-3-methylbutyl diphosphate
3,4-oxido-3-methyl-1-butyl diphosphate
-
inactivation through formation of covalent adducts with the reduced flavin, modification of the isoalloxazine ring at position N5
3-(Fluoromethyl)-3-buten-1-yl diphosphate
3-Bromo-3-butenyl diphosphate
-
competitive
3-butyn-1-yl diphosphate
3-cyclopropyl-3-buten-1-yl diphosphate
3-cyclopropylbut-3-en-1-yl trihydrogen diphosphate
-
cyclopropyl-derivative of isopentenyl diphosphate, mechanism-based inhibitor
3-Methyl-3,4-epoxybutyl diphosphate
3-Methyl-5-oxo-hexyl diphosphate
-
-
3-Methyl-5-oxo-hexyl diphosphate ethylene glycol acetal
-
-
3-methylene-4-penten-1-yl diphosphate
3-oxiran-2-ylbut-3-en-1-yl trihydrogen diphosphate
-
epoxy-derivative of isopentenyl diphosphate, mechanism-based inhibitor. The enzyme catalyzes formation of a stable covalent adduct between the inhibitor and FMNH2 in a reaction catalyzed by protonation of the epoxide moiety followed by nucleophilic addition of the cofactor
3-oxiranyl-3-buten-1-yl diphosphate
4-aminophenyl-2-ethane-1,1-bisphosphonic acid
-
weak. Due to inhibition of more than one enzyme in the mevalonate pathway may lead to an increase in antiresorptive potency compared to bisphosphonates that only inhibit farnesyl diphosphate synthase
5-deaza-FMN
-
-
ATP
-
partial
beta-Diethylaminoethyldiphenylpropylacetate hydrochloride
-
i.e. SKF-525A
bromohydrin
-
-
dimethylallyl diphosphate
Dimethylallyl diphosphate containing fluorine, epoxy, and ammonium function groups
-
-
-
dimethylammonium diphosphate
diphosphate
EDTA
addditon of 5mM EDTA results in almost complete loss of activity
epoxide of isopentenyl diphosphate
-
-
farnesyl diphosphate
geranyl diphosphate
geranyl monophosphate
-
-
homodimethylallyl diphosphate
homoisopentenyl diphosphate
iodoacetamide
iodoacetic acid
Isoamyl diphosphate
-
competitive
Isopentenyl diphosphates containing fluorine, epoxy, and ammonium functional groups
-
-
-
KH2PO4
-
-
Linaloyl diphosphate
-
-
Linaloyl monophosphate
-
-
Methyl diphosphate
-
competitive
Mg2+
Cinchona robusta
-
isomerase I is activated by concentrations below 1 mM of Mn2+ or Mg2+, increasing concentrations are inhibitory
N,N-dimethyl-2-amino-1-ethyl diphosphate
-
transition state analogue, competitive
N,N-dimethylaminoethyl diphosphate
-
-
NEM
-
-
neryl diphosphate
-
-
Neryl monophosphate
-
-
O2
-
the overall reaction is sensitive to O2
Octyl diphosphate
-
-
p-hydroxymercuribenzoate
-
-
Sodium acetate
-
-
Terpene diphosphate esters
-
-
-
Terpene monophosphate esters
-
-
-
additional information
-
not inhibited by 2-[ethyl(propyl)amino]ethyl trihydrogen diphosphate
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
dithiothreitol
FMN
Methanothermobacter thermautotrophicum
-
required as cofactor. The enzyme requires the combined presence of FMN, NADPH and Mg2+
glutathione
-
activates
L-Cysteine hydrochloride
-
activates
mevalonic acid
-
activates
Mevalonic acid lactone
-
weakly activates
NADPH
Methanothermobacter thermautotrophicum
-
required as cofactor. The enzyme requires the combined presence of FMN, NADPH and Mg2+
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.017
dimethylallyl diphosphate
Cinchona robusta
-
isomerase I
0.0008 - 15.3
isopentenyl diphosphate
0.0874 - 0.0904
NADH
additional information
additional information
-
substrate binds to the inactive oxidized and active reduced form of enzyme with similar affinities
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000197 - 29900
isopentenyl diphosphate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.031 - 4.05
isopentenyl diphosphate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.383
(Z)-3-difluoromethyl-2-buten-1-yl diphosphate
-
pH 7.0, 37C
0.012
(Z)-3-fluoromethyl-2-buten-1-yl diphosphate
-
pH 7.0, 37C
0.031 - 0.036
2,3-butadien-1-yl diphosphate
0.0565
3,4-epoxy-3-methylbutyl diphosphate
-
pH 7.0, 85C
0.0486
3,4-oxido-3-methyl-1-butyl diphosphate
-
pH 7.0, 37C
0.0074
3-(Fluoromethyl)-3-buten-1-yl diphosphate
-
pH 7.0, 37C
0.048 - 0.049
3-butyn-1-yl diphosphate
0.054
3-cyclopropyl-3-buten-1-yl diphosphate
-
pH 7.0, 37C
0.008
3-methylene-4-penten-1-yl diphosphate
-
pH 7.0, 37C
0.0014
3-oxiranyl-3-buten-1-yl diphosphate
-
pH 7.0, 37C
0.00013
5-deaza-FMN
-
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.158
(2Z)-3-methylhept-2-en-1-yl trihydrogen diphosphate
Choristoneura fumiferana
-
in 25 mM Tris-HCl, pH 7.0, at 37C
0.156
(2Z)-3-methylhex-2-en-1-yl trihydrogen diphosphate
Choristoneura fumiferana
-
in 25 mM Tris-HCl, pH 7.0, at 37C
0.234
(2Z)-3-methyloct-2-en-1-yl trihydrogen diphosphate
Choristoneura fumiferana
-
in 25 mM Tris-HCl, pH 7.0, at 37C
0.278
(E/Z)-3-cyclopropyl-2-buten-1-yl diphosphate
Thermus thermophilus
-
25C, pH 8.6
0.003 - 0.0055
2-[ethyl(methyl)amino]ethyl trihydrogen diphosphate
0.007
2-[ethyl(propyl)amino]ethyl trihydrogen diphosphate
Choristoneura fumiferana
-
in 25 mM Tris-HCl, pH 7.0, at 37C
-
0.1
3-cyclopropyl-3-buten-1-yl diphosphate
Thermus thermophilus
-
25C, pH 8.6
0.00114
3-oxiranyl-3-buten-1-yl diphosphate
Thermus thermophilus
-
pH 7.0, 37C
0.092
dimethylallyl diphosphate
Choristoneura fumiferana
-
in 25 mM Tris-HCl, pH 7.0, at 37C
0.0008
dimethylammonium diphosphate
Sus scrofa
-
in 50 mM Tris-HCl (pH 7.0), at 37C
-
0.131
homodimethylallyl diphosphate
Choristoneura fumiferana
-
in 25 mM Tris-HCl, pH 7.0, at 37C
0.066
homoisopentenyl diphosphate
Choristoneura fumiferana
-
in 25 mM Tris-HCl, pH 7.0, at 37C
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.001
-
pyruvate, C13-labeled at positions 2 and 3, aerobic, pH 8, 37C
0.046
Cinchona robusta
-
-
0.08
-
dimethylallyl diphosphate, C13-labeled at positions 3, 4 and 5, aerobic, pH 8, 37C
0.19
-
dimethylallyl diphosphate, anaerobic, pH 8, 37C
0.23
-
dimethylallyl diphosphate, aerobic, pH 8, 37C
0.62
-
isopentenyl diphosphate, anaerobic, pH 8, 37C
0.63
-
isopentenyl diphosphate, aerobic, pH 8, 37C
1.2
-
pH 8.0
5.92
-
-
52
-
isoenzyme type II, isopentenyl diphosphate, pH 7.0, 37 C
3000
Claviceps sp.
-
-
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8.5
Claviceps sp.
-
in presence of Mg2+. A sharper maximum is observed in presence of Mn2+
6.3
-
-
6.5
Methanothermobacter thermautotrophicum
-
-
7 - 7.8
-
isomerase I
7 - 8
-
isomerase II
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 9.5
-
pH 4.5: about 39% of maximal activity, pH 9.5: about 60% of maximal activity
5.5 - 7.5
-
pH 5.5: about 35% of maximal activity, pH 7.5: about 30% of maximal activity
7 - 8.5
Cinchona robusta
-
pH 7.0-7.8: maximal activity, pH 8.5: 20% of maximal activity, isomerase I
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70
Methanothermobacter thermautotrophicum
-
-
80
-
maximum activity
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.8
calculated
6
-
calculated from amino acid sequence
6.4
-
calculated from amino acid sequence
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
Cinchona robusta
-
-
Manually annotated by BRENDA team
-
developing
Manually annotated by BRENDA team
-
in normal fibroblasts and Zellweger fibroblasts, in which the synthesis of cholesterol is impaired
Manually annotated by BRENDA team
Citrus sp.
-
-
Manually annotated by BRENDA team
-
isoform IDI2 is only expressed in skeletal muscle
Manually annotated by BRENDA team
in mature and tender leaf, expression is higher than in tubers
Manually annotated by BRENDA team
additional information
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Streptococcus mutans serotype c (strain ATCC 700610 / UA159)
Streptococcus pneumoniae (strain CGSP14)
Thermus thermophilus (strain HB27 / ATCC BAA-163 / DSM 7039)
Thermus thermophilus (strain HB27 / ATCC BAA-163 / DSM 7039)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27180
theoretical
30000
determined by SDS-PAGE
33500
-
gel filtration
34000
-
gel filtration
38460
-
calculated; mass spectrometry
39000
-
SDS-PAGE
50000
-
SDS-PAGE
82500
-
gel filtration
197000
gel filtration
345000
-
gel filtration chromatography
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
octamer
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting drop vapor diffusion method, ligand-free form of the FMN-bound enzyme form at 2.8 A resolution. The octamer forms a D4 symmetrical open, cage-like structure. The monomers of 45000 Da display a classical TIM barrel fold
comparison of orthorhombic, monoclinic and trigonal crystal forms, up to 2.2 A resolution. Crystallization of free enzyme and in complex wih transition-state analogue N,N-dimethyl-2-amino-1-ethyl diphosphate
-
crystal structure of free and metal-bound C67A mutant enzyme
-
crystal structure of the isomerase-bromohydrin complex
-
crystallographic investigation of phosphoantigen binding
crystals soaked with transition state analogue (N,N-dimethylamino)-1-ethyl diphosphate
-
hanging drop vapor diffusion method, crystal structure of the C67A mutant of isopentenyl diphosphate isomerase complexed with the mechanism-based irreversible inhibitor 3,4-epoxy-3-methyl-1-butyl diphosphate
-
hanging drop vapor diffusion method, crystal structures of complexes with transition state analogue N,N-dimethyl-2-amino-1-ethyl diphosphate and the covalently attached irreversible inhibitors 3,4-epoxy-3-methyl-1-butyl diphosphate at 1.96 A resolution
-
hanging drop vapour diffusion method, selenomethionyl form, crystals display trigonal symmetry, with unit-cell parameters, a = b = 71.3 A, c = 61.7 A, and diffract to 1.45 A resolution
-
of wild-type and mutants Y104A, Y104F
-
enzyme shows a flexible N-terminal alpha-helix covering the active pocket and blocking the entrance. Substrate binding induces conformational change in the active site. A water molecule is the direct proton donor for the substrate
native enzyme at 1.7 A and in complex with substrate at 1.9 A resolution. comparison with Escherichia coli enzyme structure
sitting drop vapor diffusion method, using 0.6 M calcium acetate and 50 mM HEPES pH 7.5
-
molecular modeling of structure and comparison with structures of Streptococcus pneumoniae and Thermus thermophilus enzymes
crystallized at 20C using the hanging-drop vapor diffusion method with a reservoir solution containing 0.1 M Tris-HCl (pH 8.0), 0.2 M sodium citrate, and 30% (vol/vol) polyethylene glycol 400 (PEG 400)
-
the covalent adduct formed between irreversible mechanism based inhibitors, 3-methylene-4-penten-1-yl diphosphate or 3-oxiranyl-3-buten-1-yl diphosphate, and the flavin cofactor are investigated by X-ray crystallography and UV-visible spectroscopy. Both the crystal structures of enzyme binding the flavin-inhibitor adduct and the UV-visible spectra of the adducts indicate that the covalent bond is formed at C4a of flavin rather than at N5. The high-resolution crystal structures of enzyme-substrate complexes and the kinetic studies of new mutants confirm that only the flavin cofactor can catalyze protonation of the substrates and suggest that N5 of flavin is most likely to be involved in proton transfer
-
the crystal structures of the substrate-free enzyme and of the substrate-enzyme complexes, in the oxidized and reduced states, are solved to resolutions between 1.99 and 3.1 A, six distinct types of type 2 IDI crystals are obtained
-
in complex with diphosphate. The diphosphate moiety is located near the conserved residues H10, R97, H152, Q157, E158, and W219, and the flavin cofactor. The putative active site may stabilize a carbocationic intermediate
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
Claviceps sp.
-
half-life in dilute solution: 30 min. In more concentrated solutions of the protein or dilute enzyme in the presence of 0.01% bovine serum albumin, little loss of activity after 1 h
48
-
melting temperature of mutant Y104A
55
-
melting temperature of mutant Y104F
69
-
melting temperature of wild-type
70
-
1h, IDI retained 50% of the activity
80
10 min, more than 90% residual activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
high susceptibility to proteolysis
Claviceps sp.
-
unstable in dilute solutions
Claviceps sp.
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-15C, stable for several months
-
-20C, 0.1 M potassium phosphate buffer, pH 7.0, 2 mM dithiothreitol, stable for several weeks
-
-80C, 0.05 M Tris/HCl, pH 7.5, containing 0.01 mM leupeptin, 2 mM dithiothreitol, 5% glycerol, 55% of the activity is recovered after 3 months
Cinchona robusta
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 isoforms: I and II
Cinchona robusta
-
by affinity chromatography on Ni-NTA resin
-
Co2+ column chromatography, and gel filtration
-
cobalt column chromatography
-
DEAE Sepharose column chromatography and Resource Phe column chromatograpyh
-
Ni-NTA agarose column chromatography
-
nickel-nitrilotriacetic acid affinity chromatography
-
on a HisTrap column, for crystallisation, the enzyme is loaded on a HiLoad 16/60 Superdex 200 column
-
partial
-
partial. Dye-ligand and immobilized metal ion interaction chromatography are efficient techniques for the rapid batchwise fractionation, from crude plant extracts, of a series of enzymes of prenyl diphosphate metabolism
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recombinant enzyme
recombinant protein, enzyme purified under aerobic conditions is inactive until the flavin cofactor is reduced by NADPH or dithionite or photochemically
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TALON metal affinity resin column chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
expressed in Escherichia coli BL21 cells
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expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli BL21(DE3) pLysS cells
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expressed in Escherichia coli Rosetta cells
expression in Escherichia coli
expression in Escherichia coli strain BL21(DE3)
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expression in Escherichia coli strain JMSB0373a
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expression in Escherichia coli, His6-tagged type II enzyme
Methanothermobacter thermautotrophicum
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for expression in Escherichia coli BL21DE3 cells
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into a pET-vector for expression in Escherichia coli Bl21DE3 cells
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into the vector pMD18-T for sequencing, and subsequently into pET-28a+ for expression in Escherichia coli BL21DE3 cells
overexpressed in Escherichia coli
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overexpression in Escherichia coli
recombinantly expressed with a polyhistidine tag at its N terminus in Escherichia coli BL21
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the plasmid pDL11, which contains the genes idi, gps and egsA, is constructed by inserting idi and egsA into pCW3, derived from pCL1920, pDL11 is transformed into Escherichia coli TOP10 cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C113S
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inactive
C113S/114S
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inactive
C114S
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inactive
E169/171Q
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inactive
E169Q
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inactive
E171Q
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inactive
W216F
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10% activity compared to the wild type enzyme
Y105F
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115% activity compared to the wild type enzyme
C67A
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inactive mutant enzyme
K55A
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the ratio of maximal velocity to turnover number is 66% of that of the wild-type enzyme
K55R
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the ratio of maximal velocity to turnover number is 28% of that of the wild-type enzyme
R51K
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the ratio of maximal velocity to turnover number is 4.2% of that of the wild-type enzyme
R83K
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the ratio of maximal velocity to turnover number is 104% of that of the wild-type enzyme
W161F
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the ratio of maximal velocity to turnover number is 0.8% of that of the wild-type enzyme
Y104A
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0.1% of wild-type activity. Crystallization data. The M2+ metal-binding site is absent in the structure, but Mg2+ is still present and bound to C67, E87, and four water molecules
Y104F
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0.1% of wild-type activity. Crystallization data. General fold of enzyme is similar to wild-type
Q154N
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active site mutant
D216A
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mutation of highly conserved residue
E13R/R235E
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the mutant and the wild-type enzyme show similar Vmax values, while the Km of E13R/R235E is smaller than that of the wild type. The mutant is in the tetrameric state even at a concentration where the wild-type enzyme dominantly forms an octamer
E161A
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mutation of highly conserved residue
E194A
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mutation of highly conserved residue
E229A
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mutation of highly conserved residue
H11A
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mutation of highly conserved residue
H155A
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mutation of highly conserved residue
K193A
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mutation of highly conserved residue
K8A
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mutation of highly conserved residue
N125A
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mutation of highly conserved residue
N157A
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mutation of highly conserved residue
Q160A
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mutation of highly conserved residue
Q160E
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10-fold decrease in kcat/Km; the mutant shows a 10fold decrease in kcat/Km
Q160H
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23-fold decrease in kcat/Km; the mutant shows a 23fold decrease in kcat/Km
Q160K
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130-fold decrease in kcat/Km; the mutant shows a 130fold decrease in kcat/Km
Q160L
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28-fold decrease in kcat/Km; the mutant shows a 28fold decrease in kcat/Km
Q160N
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150-fold decrease in kcat, kcat/Km decreases 66-fold; the mutant shows a 150fold decrease in kcat, although kcat/Km only decreases 66fold
R232A
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mutation of highly conserved residue
R7A
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mutation of highly conserved residue
S96A
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mutation of highly conserved residue
T68A
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mutation of highly conserved residue
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme purified under aerobic conditions is inactive until the flavin cofactor is reduced by NADPH or dithionite or photochemically
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reconstitution of apo-enzyme with 5-deaza-FMN results in an inactive enzyme, whereas reconstitution with 1-deaza-FMN leads to full recovery of activity
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reconstitution of metal-free enzyme with Mg2+, Mn2+, Zn2+, Co2+, Ni2+ or Cd2+ generates active enzyme
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
industry
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over-expression of isopentenyl diphosphate isomerase and 1-deoxy-D-xylulose 5-phosphate synthase in combination with heterologous expression of squalene/phytoene synthase and farnesyl diphosphate synthase of Streptomyces peucetius in Escherichia coli facilitates the synthesis of the industrially important compound squalene
synthesis
Show AA Sequence (4971 entries)
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