Information on EC 1.1.1.25 - shikimate dehydrogenase

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

EC NUMBER
COMMENTARY hide
1.1.1.25
-
RECOMMENDED NAME
GeneOntology No.
shikimate dehydrogenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
shikimate + NADP+ = 3-dehydroshikimate + NADPH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
chorismate biosynthesis from 3-dehydroquinate
-
-
chorismate metabolism
-
-
Phenylalanine, tyrosine and tryptophan biosynthesis
-
-
Metabolic pathways
-
-
Biosynthesis of secondary metabolites
-
-
Biosynthesis of antibiotics
-
-
SYSTEMATIC NAME
IUBMB Comments
shikimate:NADP+ 3-oxidoreductase
NAD+ cannot replace NADP+ [3]. In higher organisms, this enzyme forms part of a multienzyme complex with EC 4.2.1.10, 3-dehydroquinate dehydratase [4].
CAS REGISTRY NUMBER
COMMENTARY hide
9026-87-3
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
UniProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Bambusa sp.
bamboo
-
-
Manually annotated by BRENDA team
tea plant
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain IFO 3244
-
-
Manually annotated by BRENDA team
strain IFO 3244
-
-
Manually annotated by BRENDA team
gene aroE; strain SS1, gene aroE
SwissProt
Manually annotated by BRENDA team
gene aroE; strain SS1, gene aroE
SwissProt
Manually annotated by BRENDA team
multienzyme complex
-
-
Manually annotated by BRENDA team
no activity in Homo sapiens
-
-
-
Manually annotated by BRENDA team
enzyme complex of EC 4.2.1.10 and EC 1.1.1.25
-
-
Manually annotated by BRENDA team
pea
-
-
Manually annotated by BRENDA team
homolog ael1; strain KT2240
UniProt
Manually annotated by BRENDA team
tomato
-
-
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
strain HB8
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3-dehydroquinate + NADH + H+
quinate + NAD+
show the reaction diagram
3-dehydroquinate + NADPH + H+
quinate + NADP+
show the reaction diagram
3-dehydroshikimate + NAD(P)H + H+
shikimate + NAD(P)+
show the reaction diagram
3-dehydroshikimate + NADH + H+
shikimate + NAD+
show the reaction diagram
3-dehydroshikimate + NADPH
?
show the reaction diagram
3-dehydroshikimate + NADPH
shikimate + NADP+
show the reaction diagram
3-dehydroshikimate + NADPH + H+
shikimate + NADP+
show the reaction diagram
L-quinate + NADP+
3-dehydroquinate + NADPH + H+
show the reaction diagram
-
-
-
r
quinate + NAD(P)+
3-dehydroquinate + NAD(P)H + H+
show the reaction diagram
-
-
-
?
quinate + NAD+
3-dehydroquinate + NADH + H+
show the reaction diagram
quinate + NADP+
3-dehydroquinate + NADPH + H+
show the reaction diagram
shikimate + NAD(P)+
3-dehydroshikimate + NAD(P)H + H+
show the reaction diagram
shikimate + NAD+
3-dehydroshikimate + NADH + H+
show the reaction diagram
shikimate + NADP+
3-dehydroshikimate + NADPH
show the reaction diagram
-
assay at 24C, pH 9.0
-
-
?
shikimate + NADP+
3-dehydroshikimate + NADPH + H+
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
3-dehydroshikimate + NAD(P)H + H+
shikimate + NAD(P)+
show the reaction diagram
3-dehydroshikimate + NADPH
?
show the reaction diagram
3-dehydroshikimate + NADPH
shikimate + NADP+
show the reaction diagram
3-dehydroshikimate + NADPH + H+
shikimate + NADP+
show the reaction diagram
P95001
-
-
-
r
L-quinate + NADP+
3-dehydroquinate + NADPH + H+
show the reaction diagram
Q6PUF9, Q6PUG0
-
-
-
r
quinate + NAD+
3-dehydroquinate + NADH + H+
show the reaction diagram
Q88GF6, Q88IJ7, Q88JP1, Q88K85, Q88RQ5
the fourth enzyme in the shikimate pathway
-
-
r
quinate + NADP+
3-dehydroquinate + NADPH + H+
show the reaction diagram
-
reversible activity of YdiB, no activity with paralogue HI0607
-
-
r
shikimate + NAD+
3-dehydroshikimate + NADH + H+
show the reaction diagram
shikimate + NADP+
3-dehydroshikimate + NADPH + H+
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NAD(P)+
additional information
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,4-Dichlorophenoxyacetic acid
-
-
2-([2-([2-([2-(2,3-dimethylanilino)-2-oxoethyl]sulfanyl)-1,3-benzothiazol-6-yl]amino)2-oxoethyl]sulfanyl)-N-(2-naphthyl)acetamide
IC50: 0.0029 mM, competitive inhibition with respect to shikimate, noncompetitive to NADP+, potent antibacterial activity
3,5-Dihydroxybenzoate
-
moderate
3-(2-naphthyloxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7-yl 3-chlorobenzoate
IC50: 0.0039 mM, noncompetitive inhibition with respect to shikimate, competitive to NADP+
arsenite
-
-
butyl 2-([3-(2-naphthyloxy)4-oxo-2-(trifluoromethyl)4H-chromen-7-yl]oxy)propanoate
IC50: 0.0134 mM, noncompetitive inhibition with respect to shikimate, competitive to NADP+, potent antibacterial activity
Cd2+
-
500 nM
curcumin
IC50: 0.0154 mM, noncompetitive inhibition with respect to shikimate and NADP+
guaiacol
-
-
HgCl2
-
complete inhibition at concentration 0.05 mM
iodoacetate
maesaquinone diacetate
IC50: 0.0035 mM, noncompetitive inhibition with respect to shikimate and NADP+
Metal ions
-
-
-
N-ethylmaleimide
-
-
NADP+
product inhibition, competitive versus NADPH, noncompetitive versus 3-dehydroshikimate
p-chloromercuribenzoate
p-hydroxymercuribenzoate
-
moderate
protocatechuic acid
SDS
-
nearly complete inactivation of AroE at 0.02%
shikimate
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
benzo-thiadiazole-7-carbothioic acid S-methyl ester
-
activates the enzymes of the anthocyanin metabolism, including the shikimate dehydrogenase, and increases the anthocyanin content in strawberries after harvest, with SKDH playing a crucial role
Nonidet P-40
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120-190% activity of AroE at 0.1-0.5%
S-methyl 1,2,3-benzothiadiazole-7-carbothioate
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treatment of fruits
Triton X-100
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120-190% activity at 0.1-0.5%
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00036 - 0.42
3-dehydroquinate
0.029 - 0.272
3-dehydroshikimate
0.572 - 11.4
NAD+
0.007 - 0.279
NADP+
0.01 - 0.034
NADPH
0.783 - 10.2
quinate
0.0046 - 46.6
shikimate
0.05 - 0.087
shikimic acid
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.36 - 114
3-dehydroquinate
0.078 - 329
3-dehydroshikimate
5.2 - 97.1
NAD+
0.008 - 399
NADP+
45 - 50
NADPH
0.16 - 61.9
quinate
0.013 - 428
shikimate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.143 - 272
3-dehydroquinate
894
9.6 - 1700
3-dehydroshikimate
2260
24 - 4500
NADPH
5
168 - 1670
shikimate
352
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0029
2-([2-([2-([2-(2,3-dimethylanilino)-2-oxoethyl]sulfanyl)-1,3-benzothiazol-6-yl]amino)2-oxoethyl]sulfanyl)-N-(2-naphthyl)acetamide
Helicobacter pylori
Q56S04
IC50: 0.0029 mM, competitive inhibition with respect to shikimate, noncompetitive to NADP+, potent antibacterial activity
0.0039
3-(2-naphthyloxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7-yl 3-chlorobenzoate
Helicobacter pylori
Q56S04
IC50: 0.0039 mM, noncompetitive inhibition with respect to shikimate, competitive to NADP+
0.0134
butyl 2-([3-(2-naphthyloxy)4-oxo-2-(trifluoromethyl)4H-chromen-7-yl]oxy)propanoate
Helicobacter pylori
Q56S04
IC50: 0.0134 mM, noncompetitive inhibition with respect to shikimate, competitive to NADP+, potent antibacterial activity
0.0154
curcumin
Helicobacter pylori
Q56S04
IC50: 0.0154 mM, noncompetitive inhibition with respect to shikimate and NADP+
0.0035
maesaquinone diacetate
Helicobacter pylori
Q56S04
IC50: 0.0035 mM, noncompetitive inhibition with respect to shikimate and NADP+
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.2
-
crude extract
3.5
-
crude extract
3.9
purified recombinant AroE
14.1
-
11.8fold purified enzyme
25.4
-
purified enzyme
95
-
value about, fruits without any treatment
184
-
after treatment with benzo-thiadiaziole-7-carbothioic acid S-methyl ester; pH 9.0, 25C, fruits after harvest
732
-
87C, pH 7.3, cosubstrate NADP+
826
-
purified recombinant AroE
1479
-
87C, pH 7.3, cosubstrate NAD+
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0
-
oxidation of shikimate
6
reduction of 3-dehydroquinate
6.8 - 7.2
formation of shikimate
7.5
-
assay at
7.5 - 9.8
-
paralogue HI0607
7.7
-
Tris-HCl acid buffer
8 - 9
oxidation reaction, recombinant His-tagged enzyme
8.8
assay at; assay at; assay at; assay at
9 - 9.4
formation of 3-dehydroshikimate
9
assay at; assay at
10.1
-
glycine-sodium hydroxide buffer
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 10
-
-
6 - 11
-
no activity below and above
8.5 - 10.5
-
pH 8.5: about 50% of activity maximum, pH 10.5: about 90% of activity maximum
additional information
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
24
-
assay at
35
-
assay at
60
oxidation reaction, recombinant His-tagged enzyme
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 55
-
-
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.55
-
molecular modeling
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
etiolated
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
DHD/SHD-1 containing a plastidic targeting sequence
Manually annotated by BRENDA team
DHD/SHD-2; exclusively cytosolic
Manually annotated by BRENDA team
additional information
-
the freeze-thaw method is able to yield the mutant protein in soluble form, after growth at 37C for 24 h with IPTG induction of Escherichia coli C41 (DE3) cells harboring the recombinant plasmid. Best recombinant protein expression protocol of the K69A mutant in insoluble form is using Escherichia coli C41(DE3) strain, grown at 37C for 24 h after induction with 1 mM IPTG, and cell disruption by sonication. Solubilization of recombinant wild-type and K69A enzymes in inclusion bodies is achieved by either treating the pellet with the denaturant agent urea (3 or 6 M) or by treating with the non ionic detergent zwittergent 3-14 (5%)
-
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Clostridium acetobutylicum (strain ATCC 824 / DSM 792 / JCM 1419 / LMG 5710 / VKM B-1787)
Escherichia coli (strain K12)
Geobacillus kaustophilus (strain HTA426)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Listeria monocytogenes serovar 1/2a (strain ATCC BAA-679 / EGD-e)
Listeria monocytogenes serovar 1/2a (strain ATCC BAA-679 / EGD-e)
Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Neosartorya fumigata (strain CEA10 / CBS 144.89 / FGSC A1163)
Peptoclostridium difficile (strain 630)
Pseudomonas putida (strain KT2440)
Pseudomonas putida (strain KT2440)
Staphylococcus epidermidis (strain ATCC 35984 / RP62A)
Staphylococcus epidermidis (strain ATCC 35984 / RP62A)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27080
recombinant AroE, mass spectrometry
29400
-
mass spectometry and calculation from DNA sequence
29410
-
mass spectrometry
30000
-
dynamic light-scattering
30160
-
molecular modeling
31000
mutant Y211F, mass spectrometry; mutant Y211F, sequence analysis
31010
recombinant SDH, mass spectrometry; recombinant SDH, sequence analysis
31690
recombinant AroE, mass spectrometry
32000
-
gel filtration, SDS-PAGE
45000
-
gel filtration, 2 different isoenzymes with molecular weight about 45000 Da
48000
-
gel filtration
50000
-
gel filtration
54150
recombinant AroE, mass spectrometry
58000
-
gel filtration
58370
recombinant AroE, gel filtration
67000
-
gel filtration, 4 different isoenzymes
73000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
apo-enzyme and in complex with both shikimate and NADP+, which assumes the closed conformation
in complex with shikimate, and in ternary complex with shikimate and NADP+
-
purified native and selenomethionine-labeled DELTA88DHQ-SDH complexed with shikimate, hanging drop vapour diffusion method, 10 mg/ml protein in 1 mM shikimate mixed with equal volume of reservoir solution containing 0.4 M potassium sodium tartrate tetrahydrate, 0.1 M tri-sodium dihydrate, pH 5.6, and 2.8 M ammonium sulfate, X-ray diffraction structure determination and analysi at 1.95-2.2 A resolution
-
crystallized at 23C using ammonium sulfate as a precipitant. Crystals grown in the presence of NADP+ diffract to 2.8 A resolution and belong to the trigonal space group P3(2)21 (or P3(2)21), with unit-cell parameters a = 111.3, b = 111.3, c = 76.2 A; in complex with NADP+, to 2.8 A resolution. Crystal belong to space group P3221
purified recombinant enzyme, sitting drop vapour diffusion method, 3.75 mg/ml protein in 50 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 1 mM NaN3, and 20% v/v glycerol, the reservoir solution contains 0.1 M sodium acetate, pH 4.6, 0.2 M NaCl, and 20% v/v precipitant 2,4-methylenepentanediol, 0.002 ml of protein, reservoir and 0.002 mM NADH solutions are mixed at 20C, about 5 months, formation of rod- and needle-shaped crystals, X-ray diffraction structure determination and analysis at 1.64 A resolution
-
purified recombinant enzyme, 20 mg/ml in 10 mM Tris-HCl, pH 7.6, 0.4 mM DTT, and 2.5 mM NADPH, 20C, sitting-drop vapour-diffusion method, reservoir solution contains 1.65 M ammonium sulfate, 100 mM cacodylate buffer, pH 5.8, 1.5 ml of protein solution is mixed with an equal volume of reservoir solution, 10-12 days, cryoprotection with 20% v/v glycerol, X-ray diffraction structure determination and analysis at 2.3 A resolution
-
catalytic domain with open twisted alpha/beta motif plus NADPH binding domain with typical Rossman fold; wild-type and selenomethionine-labeled enzyme, free and in complex with NADPH, sitting drop vapour diffusion method, 10 mg/m protein, with or without 5 mM NADPH, mixture in a ratio of 1:1 with crystallization solution containing 1.0 M sodium citrate, and 0.1 M 2-(cyclohexylamino)ethanesulfonic acid (CHES), pH 8.8, for the free enzyme, and 12% PEG 8000, 0.15 M calcium acetate, pH 7.8, and 0.1 M imidazole for the selenomethionine-labeled enzyme, 4C, X-ray diffraction structure determination and analysis at 2.4 A and 1.95 A, respectively, structure modeling
purified recombinant wild-type and selenomethionine-labeled paralogue HI0607, hanging drop vapour diffusion method, 0.001 ml protein solution containing 20 mg/ml protein mixed with equal volume of reservoir solution containing 0.1 M sodium acetate, pH 4.6, 2.0 M NaCl, and 10% v/v PEG 400, X-ray diffraction structure determination and analysis at 1.75 A resolution
-
in complex with NADP+, compact alpha/beta sandwich with two domains for binding substrate and cofactor, resp.; purified recombinant AroE bound to cofactor NADP+, hanging drop vapour diffusion method at 25C, 10 mg/ml protein in 20 mM HEPES, pH 7.1, and 0.1 M KCl, is mixed with reservoir solution containing 20 mM HEPES, pH 7.1, 20% PEG 3350, and 0.2 M ammonium fluoride, cryoprotection with 15% glycerol, X-ray diffraction structure determination and analysis at 2.35 A resolution
homology modeling and circular dichroism study. NADP+ binding results in small conformational changes
-
at 4C, using the hanging-drop vapor-diffusion method. SDH both in its ligand-free form and in complex with shikimate. Overall structure of apo-SDH is basically identical to that of the shikimate-SDH complex, both structures contain one molecule per asymmetric unit. Overall folding of SDH comprises the N-terminal alpha/beta domain for substrate binding and the C-terminal Rossmann fold for NADP binding. The active site is within a large groove between the two domains. Residue Tyr211 does not interact with shikimate in the binary SDH-shikimate complex structure. The main function of Tyr211 may be to stabilize the catalytic intermediate during catalysis. The NADP-binding domain of SDH is less conserved. The long helix specifically recognizing the adenine ribose phosphate is substituted with a short 310 helix in the NADP-binding domain. The interdomain angle of SDH is the widest among all known SDH structures, indicating an inactive open state of the SDH structure. Thus, a closing process may occur upon NADP+ binding to bring the cofactor close to the substrate for catalysis
determination of the crystal structure at 1.45 A by molecular replacement. The protein shows a monomeric architecture. The overall structure comprises the N-terminal alpha/beta sandwich domain for substrate binding and the C-terminal domain for NADP binding.the enzyme is in a tightly closed conf ormation, which should be open for catalysis. Four ammonium sulfate ions were identified in the structure. They are located in the active site and appear to mimic the role of the substrate in terms of the enzyme activity and stability
-
to 1.45 A resolution. Space group P212121
-
ligand-free form, binary complexes with NADP+ or substrate shikimate, and ternary complex with both NADP+ and shikimate. Structures of the closed subunits with and without NADP(H) show no significant difference. Residues K64 and D100 are involved in hydride transfer
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 10
-
stable for 45 min
286398
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
purified recombinant AroE, stable for at least 1 h
40
-
at temperatures higher than 40C the enzyme is quickly inactivated
50
-
5 min, 85% loss of activity
55
purified recombinant AroE, 20% remaining activity after 1 h
63
-
melting temperature
100
-
half-life about 2.1 h, presence of 1 M NaCl; half-life about 30 min, no addition of salt
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
stable in crude extract
-
the purified recombinant enzyme is stable to freezing
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 50% glycerol, 1 mM benzamidine, 0.4 mM dithiothreitol
-
-20C, Tris-HCl, pH 7.5, 0.4 mM DTT, 50% glycerol, long-term
-
-70C, purified recombinant enzyme, stable for several months
-
1C, storage for 10 days, increase of enzyme activity
-
4C, 10 mM Tris-HCl buffer, pH 7.5, 500 mM NaCl, 5% glycerol
-
4C, purified recombinant enzyme, stable for 2 weeks
-
storage at 1C, SKDH activity increases in all strawberries
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by nickel-nitrilotriacetic acid affinity chromatography; by nickel-nitrilotriacetic acid affinity chromatography
Cultures with expressed SDH enzymes are incubated and then induced with 0.4 mM isopropyl-beta-D-thiogalactopyranoside. Harvested cells are disrupted and the insoluble cellular material is removed by centrifugation. The recombinants are purified from other contaminating proteins using nickel-nitrilotriacetic acid affinity chromatography. Protein samples for kinetic studies are dialyzed and stored at 4 C in 10 mM TrisHCl, 500 mM NaCl, and 5% glycerol.; Cultures with expressed SDH enzymes are incubated and then induced with 0.4 mM isopropyl-beta-D-thiogalactopyranoside. Harvested cells are disrupted and the insoluble cellular material is removed by centrifugation. The recombinants are purified from other contaminating proteins using nickel-nitrilotriacetic acid affinity chromatography. Protein samples for kinetic studies are dialyzed and stored at 4 C in 10 mM TrisHCl, 500 mM NaCl, and 5% glycerol.; Cultures with expressed SDH enzymes are incubated and then induced with 0.4 mM isopropyl-beta-D-thiogalactopyranoside. Harvested cells are disrupted and the insoluble cellular material is removed by centrifugation. The recombinants are purified from other contaminating proteins using nickel-nitrilotriacetic acid affinity chromatography. Protein samples for kinetic studies are dialyzed and stored at 4C in 10 mM Tris-HCl, 500 mM NaCl, and 5% glycerol.; Cultures with expressed SDH enzymes are incubated and then induced with 0.4 mM isopropyl-beta-D-thiogalactopyranoside. Harvested cells are disrupted and the insoluble cellular material is removed by centrifugation. The recombinants are purified from other contaminating proteins using nickel-nitrilotriacetic acid affinity chromatography. Protein samples for kinetic studies are dialyzed and stored at 4C in 10 mM TrisHCl, 500 mM NaCl, and 5% glycerol.; Cultures with expressed SDH enzymes are incubated and then induced with 0.4 mM isopropyl-beta-D-thiogalactopyranoside. Harvested cells are disrupted and the insoluble cellular material is removed by centrifugation. The recombinants are purified from other contaminating proteins using nickel-nitrilotriacetic acid affinity chromatography. Protein samples for kinetic studies are dialyzed and stored at 4C in 10 mM TrisHCl, 500 mM NaCl, and 5% glycerol.
enzyme complex of EC 4.2.1.10 and EC 1.1.1.25
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homogeneous recombinant mutant K69A and wild-type proteins are obtained by a three-step purification protocol, by gel filtration, 11.8fold
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multienzyme complex
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one-step purification by nickel-affinity chromatography
recombinant enzyme expressed in Escherichia coli, formation of insoluble aggregates, solubilization by repeated freezing and thawing
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recombinant enzyme from aroE auxotrophic mutant strain AB2834 by ammonium sulfate fractionation, anion exchange chromatography, ultrafiltration, and gel filtration
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recombinant GST-tagged SeMet-labeled enzyme from Escherichia coli strain BL21 by glutathione affinity and ion exchange chromatography, proteolytic removal of the GST-tag
recombinant His-tagged ARoE from Escherichia coli strain BL21(DE3) by nickel affinity chromatograohy
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recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3)
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recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant protein; recombinant protein
recombinant soluble enzyme 8.5fold from Escherichia coli strain BL21(DE3) by ion exchange and hydrophobic interaction chromatography, gel filtration, and again ion exchange chromatography, to homogeneity
separation of isoenzymes by regaining the protein from PA-gel slices
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis; DNA and amino acid sequence determination and analysis, expression of Hs6-tagged isozyme 1 in Escherichia coli, transfection of Nicotina plants; expression in Escherichia coli; expression in Escherichia coli
expression in Escherichia coli
expression in Escherichia coli in native and His-tagged form
expression in Escherichia coli; expression in Escherichia coli
expression in Escherichia coli; overexpression in Escherichia coli
expression of C-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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expression of wild-type and selenomethionine-labeled enzyme in Escherichia coli strain DL41
five putative SDH-encoding genes amplified and cloned into either a modified pET28a vector or the p15TVLIC vector, a ligation-independent vector due to incompatible cloning sites between the insert and pET28.3. Expressed in either the Escherichia coli strain BL21 gold (p15TV-LIC constructs) or the Escherichia coli BL21CodonPlus (pET28.3constructs); five putative SDH-encoding genes amplified and cloned into either a modified pET28a vector or the p15TVLIC vector, a ligation-independent vector due to incompatible cloning sites between the insert and pET28.3. Expressed in either the Escherichia coli strain BL21 gold (p15TV-LIC constructs) or the Escherichia coli BL21CodonPlus (pET28.3constructs)
gene aroE, DNA and amino acid sequence determination, functional expression as soluble enzyme in Escherichia coli strain BL21(DE3)
gene aroE, expression of GST-tagged SeMet-labeled enzyme in Escherichia coli strain BL21
gene aroE, expression of His-tagged AroE in Escherichia coli strain BL21(DE3)
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gene aroE, expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
gene aroE, overexpression in the aroE auxotrophic mutant strain AB2834
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overexpression in Escherichia coli K12
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overexpression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
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phylogenetic analysis of the three paralogues YdiB, AroE, and HI0607, DNA and amino acid sequence determination and analysis of paralogue HI0607, expression of wild-type paralogue HI0607 in Escherichia coli strain BL21(DE3) and of selenomethionine-labeled enzyme in strain B834(DE3)
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plasmid pET23a(+)::aroE (either wild-type or mutants) transformed into Escherichia coli C41(DE3)
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SDH-encoding gene is amplified by polymerase chain reaction from Pseudomonas putida KT2440 genomic DNA using primers with incorporated restriction sites. Resulting product is cloned into a vector and expressed in the Escherichia coli strain BL21 with appropriate antibiotics.; SDH-encoding gene is amplified by polymerase chain reaction from Pseudomonas putida KT2440 genomic DNA using primers with incorporated restriction sites. Resulting product is cloned into a vector and expressed in the Escherichia coli strain BL21 with appropriate antibiotics.; SDH-encoding gene is amplified by polymerase chain reaction from Pseudomonas putida KT2440 genomic DNA using primers with incorporated restriction sites. Resulting product is cloned into vector and expressed in the Escherichia coli strain BL21 with appropriate antibiotics.; SDH-encoding gene is amplified by polymerase chain reaction from Pseudomonas putida KT2440 genomic DNA using primers with incorporated restriction sites. Resulting product is cloned into vector and expressed in the Escherichia coli strain BL21 with appropriate antibiotics.; SDH-encoding gene is amplified by polymerase chain reaction from Pseudomonas putida KT2440 genomic DNA using primers with incorporated restriction sites. Resulting product is cloned into vector and expressed in the Escherichia coli strain BL21 with appropriate antibiotics.
wild-type and mutant cloned into vector pET22b and transformed into Escherichia coli strain BL21(DE3). Selenomethionine-substituted SDH generated in the methionine auxotrophic Escherichia coli strain B834(DE3)
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
isoform qsuD mRNA expression in shikimate-grown cells is upregulated relative to that in the glucose-grown cells
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D423A
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site-directed mutagenesis of enzyme variant DELTA88DHQ-SDH, inactive mutant
D423N
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site-directed mutagenesis of enzyme variant DELTA88DHQ-SDH, highly reduced activity compared to the wild-type enzyme
H335A
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site-directed mutagenesis, tenfold decrease in kcat value
K385A
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site-directed mutagenesis of enzyme variant DELTA88DHQ-SDH, highly reduced activity compared to the wild-type enzyme
K385N
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site-directed mutagenesis of enzyme variant DELTA88DHQ-SDH, highly reduced activity compared to the wild-type enzyme
K385N/D423N
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site-directed mutagenesis of enzyme variant DELTA88DHQ-SDH, highly reduced activity compared to the wild-type enzyme
N406A
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site-directed mutagenesis, very strong decrease in kcat value
Q578L
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site-directed mutagenesis, sixtyfold decrease in kcat value
Q582L
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site-directed mutagenesis, very strong decrease in kcat value
S336A
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site-directed mutagenesis of enzyme variant DELTA88DHQ-SDH, reduced activity compared to the wild-type enzyme
S338A
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site-directed mutagenesis of enzyme variant DELTA88DHQ-SDH, reduced activity compared to the wild-type enzyme
T381A
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site-directed mutagenesis, more than twentyfold decrease in kcat value
T381S
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site-directed mutagenesis, fourfold decrease in kcat value, slight decrease in Km value
T407A
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site-directed mutagenesis, 6.5fold decrease in kcat value, increase in Km-value
T422S
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site-directed mutagenesis, tenfold decrease in kcat value
Y550A
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site-directed mutagenesis of enzyme variant DELTA88DHQ-SDH, reduced activity compared to the wild-type enzyme
Y550F
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site-directed mutagenesis of enzyme variant DELTA88DHQ-SDH, reduced activity compared to the wild-type enzyme
D103X
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site-directed mutagenesis of paralogue HI0607, inactive mutant
K67H
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site-directed mutagenesis of paralogue HI0607, inactive mutant
D105A
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the freeze-thaw method is able to yield the mutant protein in soluble form, after growth at 37C for 24 h with IPTG induction of Escherichia coli C41 (DE3) cells harboring the recombinant plasmid
D105N
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the freeze-thaw method is able to yield the mutant protein in soluble form, after growth at 37C for 24 h with IPTG induction of Escherichia coli C41 (DE3) cells harboring the recombinant plasmid
K69H
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the freeze-thaw method is able to yield the mutant protein in soluble form, after growth at 37C for 24 h with IPTG induction of Escherichia coli C41 (DE3) cells harboring the recombinant plasmid
K69I
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the freeze-thaw method is able to yield the mutant protein in soluble form, after growth at 37C for 24 h with IPTG induction of Escherichia coli C41 (DE3) cells harboring the recombinant plasmid
K69Q
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the freeze-thaw method is able to yield the mutant protein in soluble form, after growth at 37C for 24 h with IPTG induction of Escherichia coli C41 (DE3) cells harboring the recombinant plasmid
Y211F
results in a remarkable reduction in enzyme activity, leads to a significant decrease in kcat (345fold) and a minor increase in the Km (3fold) for shikimate. Tyr211 may play a major role in the catalytic process and a minor role in the initial substrate binding
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme is stable up to 50C presenting a cooperative unfolding profile. Unfolding at 63C is irreversible
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agriculture
drug development
the enzyme can be a target for rational design of specific inhibitors, aiming at the development of antitubercular drugs
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