Information on EC 1.1.1.87 - homoisocitrate dehydrogenase

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

EC NUMBER
COMMENTARY hide
1.1.1.87
-
RECOMMENDED NAME
GeneOntology No.
homoisocitrate dehydrogenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate + NAD+ = 2-oxoadipate + CO2 + NADH + H+
show the reaction diagram
retinol + NAD(P)+ = retinal + NAD(P)H + H+
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
oxidative decarboxylation
-
-
-
-
reduction
-
-
-
-
reductive carboxylation
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of antibiotics
-
-
coenzyme B biosynthesis
-
-
L-lysine biosynthesis IV
-
-
L-lysine biosynthesis V
-
-
Lysine biosynthesis
-
-
lysine metabolism
-
-
Metabolic pathways
-
-
Microbial metabolism in diverse environments
-
-
SYSTEMATIC NAME
IUBMB Comments
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate:NAD+ oxidoreductase (decarboxylating)
Forms part of the lysine biosynthesis pathway in fungi [3].
CAS REGISTRY NUMBER
COMMENTARY hide
37250-23-0
-
9067-90-7
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
UniProt
Manually annotated by BRENDA team
Candida albicans ATCC 10231
gene LYS12
-
-
Manually annotated by BRENDA team
gene PH1722 or hdh
SwissProt
Manually annotated by BRENDA team
Saccharomycopsis lipolytica
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
homoisocitrate dehydrogenase is involved in the alpha-aminoadipate pathway of lysine biosynthesis
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
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate + NAD+
2-oxoadipate + CO2 + NADH + H+
show the reaction diagram
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate + NAD+
2-oxoadipate + NADH + H+ + CO2
show the reaction diagram
(2R,3S)-3-(2-hydroxyethyl)malate + NAD+
?
show the reaction diagram
(2R,3S)-3-(3-aminopropyl)malate + NAD+
?
show the reaction diagram
(2R,3S)-3-(3-hydroxypropyl)malate + NAD+
?
show the reaction diagram
(2R,3S)-3-(4-hydroxybutyl)malate + NAD+
?
show the reaction diagram
(2R,3S)-3-(4-pentenyl)malic acid + NAD+
?
show the reaction diagram
(2R,3S)-3-allylmalic acid + NAD+
?
show the reaction diagram
(2R,3S)-3-propylmalic acid + NAD+
?
show the reaction diagram
1-hydroxy-1,2,3-propanetricarboxylate + NAD+
?
show the reaction diagram
-
more effective substrate than 1-hydroxy-1,2,4-butanetricarboxylate
-
-
?
1-hydroxy-1,2,4-butanetricarboxylate + NAD+
2-oxoadipate + CO2 + NADH
show the reaction diagram
11-cis-retinol + NAD+
11-cis-retinal + NADH + H+
show the reaction diagram
11-cis-retinol + NADP+
11-cis-retinal + NADPH
show the reaction diagram
-
-
-
-
?
11-cis-retinol + NADP+
11-cis-retinal + NADPH + H+
show the reaction diagram
2(R),3(S)-homoisocitrate + NAD+
alpha-ketoadipate + NADH + CO2 + H+
show the reaction diagram
-
with homoisocitrate as the substrate, no primary deuterium isotope effect is observed, and a small 13C kinetic isotope effect indicates that the decarboxylation step contributes only slightly to rate limitation
-
-
?
3-carboxy-2-hydroxyadipate + NAD+
2-oxoadipate + CO2 + NADH
show the reaction diagram
3-isopropylmalate + NAD+
?
show the reaction diagram
3-vinylmalate + NAD+
?
show the reaction diagram
9-cis-retinol + NAD+
9-cis-retinal + NADH + H+
show the reaction diagram
-
microsomal preparations of RDH10 are not active in presence of NADP+
-
-
r
9-cis-retinol + NADP+
9-cis-retinal + NADPH
show the reaction diagram
-
-
-
-
?
all-trans retinol + NAD+
all-trans-retinal + NADH + H+
show the reaction diagram
-
no significant difference in the binding constants of NADP+ and NADPH versus NAD+ and NADH
-
-
r
all-trans retinol + NADP+
all-trans-retinal + NADPH + H+
show the reaction diagram
-
no significant difference in the binding constants of NADP+ and NADPH versus NAD+ and NADH
-
-
r
all-trans-retinal + NADPH + H+
all-tans-retinol + NADP+
show the reaction diagram
-
-
-
-
r
all-trans-retinol + NAD+
all-trans-retinal + NADH + H+
show the reaction diagram
all-trans-retinol + NADP+
all-trans-retinal + NADPH + H+
show the reaction diagram
all-trans-retinol + NADPH
all-trans-retinal + NADP+
show the reaction diagram
-
-
-
-
r
ethylmalate + NAD+
?
show the reaction diagram
homoisocitrate + NAD+
?
show the reaction diagram
homoisocitrate + NAD+
alpha-ketoadipate + NADH + CO2
show the reaction diagram
-
-
-
-
r
homoisocitrate + NAD+
alpha-ketoadipate + NADH + CO2 + H+
show the reaction diagram
-
-
-
-
r
isocitrate + NAD+
?
show the reaction diagram
isocitrate + NAD+
? + NADH + H+
show the reaction diagram
-
low activity
-
-
?
isopropylmalate + NAD+
?
show the reaction diagram
-
-
-
-
?
threo-D-isocitric acid + NAD+
?
show the reaction diagram
-
with isocitrate as the substrate, primary deuterium and 13C isotope effects indicate that hydride transfer and decarboxylation steps contribute to rate limitation, and that the decarboxylation step is the more rate-limiting of the two. The multiple-substrate deuterium/13C isotope effects suggest a stepwise mechanism with hydride transfer preceding decarboxylation
-
-
?
trisodium (2S,3R)-2-(carboxylatomethoxy)-3-hydroxybutanedioate + NAD+
? + NADH + CO2
show the reaction diagram
-
-
-
-
?
trisodium (2S,3R)-2-[(carboxylatomethyl)amino]-3-hydroxybutanedioate + NAD+
? + NADH + CO2
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
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate + NAD+
2-oxoadipate + CO2 + NADH + H+
show the reaction diagram
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate + NAD+
2-oxoadipate + NADH + H+ + CO2
show the reaction diagram
-
-
-
-
?
1-hydroxy-1,2,4-butanetricarboxylate + NAD+
2-oxoadipate + CO2 + NADH
show the reaction diagram
11-cis-retinol + NAD+
11-cis-retinal + NADH + H+
show the reaction diagram
-
RDH10 can utilize both NAD+ and NADP+ as cofactors for 11-cis-retinol dehydrogenase activity. NAD+ cofactor confers more robust activity. RDH10 may function in the RPE retinoid visual cycle as an 11-cis-retinol dehydrogenase, and thereby partially compensate for the loss of RDH5 function in human patients with fundus albipunctatus
-
-
?
11-cis-retinol + NADP+
11-cis-retinal + NADPH + H+
show the reaction diagram
-
RDH10 can utilize both NAD+ and NADP+ as cofactors for 11-cis-retinol dehydrogenase activity. NAD+ cofactor confers more robust activity. RDH10 may function in the RPE retinoid visual cycle as an 11-cis-retinol dehydrogenase, and thereby partially compensate for the loss of RDH5 function in human patients with fundus albipunctatus
-
-
?
3-carboxy-2-hydroxyadipate + NAD+
2-oxoadipate + CO2 + NADH
show the reaction diagram
-
intermediate in lysine biosynthesis
-
-
?
3-isopropylmalate + NAD+
?
show the reaction diagram
A4CYJ9
-
-
-
?
homoisocitrate + NAD+
?
show the reaction diagram
A4CYJ9
-
-
-
?
isocitrate + NAD+
?
show the reaction diagram
A4CYJ9
-
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
NH4+
-
activates
additional information
-
selectivity of the activator site for monovalent ions, K+ is the best activator, and NH4+ and Rb+ are also activators of the reaction, while Cs+, Li+, and Na+ are not, overview. Substitution of potassium acetate for KCl changes the kinetic mechanism of HIcDH from a steady state random to a fully ordered mechanism with the binding of Mg-HIc followed by K+ and NAD+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(2R,3S)-3-(p-carboxybenzyl)malate
-
-
(2S)-thiahomocitrate
-
-
(2S,3S)-(-)-3-methylmercaptomalic acid
-
(2S,3S)-thiahomoisocitrate
interacts through hydrogen bonding to Arg 118, Tyr 125 and Lys 171 in the active site, enzyme binding structure, overview
(R,E)-(3-carboxypropylidene)malic acid
(R,E)-(3-hydroxypropylidene)malic acid
(R,Z)-(3-carboxypropylidene)malic acid
(R,Z)-(3-hydroxypropylidene)malic acid
11-cis-retinol
-
substrate inhibition above 0.005 mM
2-oxoadipate
Saccharomycopsis lipolytica
-
-
3-carboxypropylidenemalate
-
-
3-vinylmalate
-
-
3-[(carboxymethyl)sulfanyl]-2-oxopropanoic acid
-
-
9-cis-retinol
-
substrate inhibition above 0.001 mM
acetate
-
slight inhibition
all-trans-retinol
-
substrate inhibition
homoisocitrate
Mg-homoisocitrate
-
-
NAD+
-
substrate inhibition at high concentrations and in absence of K+, kinetics, overview
Oxaloglutarate
Saccharomycopsis lipolytica
-
-
trisodium (2S,3R)-2-(carboxylatomethoxy)-3-hydroxybutanedioate
-
-
trisodium (2S,3R)-2-[(carboxylatomethyl)amino]-3-hydroxybutanedioate
-
-
trisodium (2S,3S)-2-[(carboxylatomethyl)sulfanyl]-3-hydroxybutanedioate
-
-
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-oxoadipate
-
required for reductive carboxylation
cellular retinaldehyde-binding protein
-
RDH10 oxidizes 11-cis-retinol to generate 11-cis-retinaldehyde in vitro in the presence of cellular retinaldehyde-binding protein
-
NaHCO3-CO2
-
required for reductive carboxylation
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.074 - 7.5
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate
0.59 - 2.1
(2R,3S)-3-(2-hydroxyethyl)malate
0.51 - 0.58
(2R,3S)-3-(3-hydroxypropyl)malate
0.44 - 0.47
(2R,3S)-3-(4-hydroxybutyl)malate
2.7 - 9.4
(2R,3S)-3-(4-pentenyl)malic acid
0.41 - 2.8
(2R,3S)-3-allylmalic acid
0.29 - 0.78
(2R,3S)-3-propylmalic acid
0.4
1-hydroxy-1,2,3-propanetricarboxylate
-
purified enzyme, pH 8.0, 60C
7.5
1-hydroxy-1,2,4-butanetricarboxylate
-
purified enzyme, pH 8.0, 60C
0.06
11-cis-retinol
-
microsomal preparations of RDH10
1.4
3-carboxy-2-hydroxyadipate
-
-
1.33
3-isopropylmalate
-
pH 7.8, 25C, recombinant wild-type enzyme
0.083 - 3.4
3-vinylmalate
0.04 - 6
9-cis-retinol
0.57
all-trans retinal
-
pH 7.4, 37C, wild-type enzyme
0.00018 - 0.0041
all-trans retinol
0.4
all-trans-retinal
-
; microsomal preparations of RDH10
0.035
all-trans-retinol
-
; microsomal preparations of RDH10, cofactor: NAD+
1.5
alpha-ketoadipate
-
-
16.3
CO2
-
reductive carboxylation of alpha-ketoadipate
0.6 - 1
ethylmalate
0.01 - 1.5
homoisocitrate
1.4
homoisocitric acid
-
-
0.0164 - 0.521
isocitrate
1.33
isopropylmalate
-
pH 7.8, 28C, recombinant enzyme
0.0042 - 3.2
Mg-homoisocitrate
0.036 - 9
NAD+
0.065 - 0.11
NADH
0.027
NADP+
-
pH 7.4, 37C, wild-type enzyme
0.015
NADPH
-
pH 7.4, 37C, wild-type enzyme
0.02
trisodium (2S,3R)-2-(carboxylatomethoxy)-3-hydroxybutanedioate
-
-
0.15
trisodium (2S,3R)-2-[(carboxylatomethyl)amino]-3-hydroxybutanedioate
-
-
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.38 - 0.4
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate
0.78 - 25
(2R,3S)-3-(2-hydroxyethyl)malate
4.8 - 29
(2R,3S)-3-(3-hydroxypropyl)malate
2 - 8
(2R,3S)-3-(4-hydroxybutyl)malate
0.14 - 3
(2R,3S)-3-(4-pentenyl)malic acid
0.34 - 37
(2R,3S)-3-allylmalic acid
0.7 - 53
(2R,3S)-3-propylmalic acid
171
1-hydroxy-1,2,3-propanetricarboxylate
0.371
3-isopropylmalate
Deinococcus radiodurans
-
pH 7.8, 25C, recombinant wild-type enzyme
0.5 - 0.77
3-vinylmalate
0.61 - 43
ethylmalate
5.46 - 46.2
homoisocitrate
10.9 - 438
isocitrate
0.37
isopropylmalate
Deinococcus radiodurans
-
pH 7.8, 28C, recombinant enzyme
0.38 - 0.4
NAD+
0.59
trisodium (2S,3R)-2-(carboxylatomethoxy)-3-hydroxybutanedioate
Saccharomyces cerevisiae
-
-
2
trisodium (2S,3R)-2-[(carboxylatomethyl)amino]-3-hydroxybutanedioate
Saccharomyces cerevisiae
-
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.16 - 8.75
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate
0.35 - 0.44
NAD+
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.013
(2S,3S)-thiahomoisocitrate
pH 7.8, 60C
0.79 - 3.1
(R,E)-(3-carboxypropylidene)malic acid
1.4 - 5.2
(R,E)-(3-hydroxypropylidene)malic acid
0.072 - 0.26
(R,Z)-(3-carboxypropylidene)malic acid
0.51 - 15.3
(R,Z)-(3-hydroxypropylidene)malic acid
0.088
3-vinylmalate
-
pH 7.8, 28C, recombinant enzyme
12
3-[(carboxymethyl)sulfanyl]-2-oxopropanoic acid
-
-
0.002
Mg-homoisocitrate
-
oxidate decarboxylation of homoisocitrate
77
NAD+
-
pH 8.0, 25C, in absence of KOAc
0.01
trisodium (2S,3R)-2-(carboxylatomethoxy)-3-hydroxybutanedioate
-
-
0.000097
trisodium (2S,3S)-2-[(carboxylatomethyl)sulfanyl]-3-hydroxybutanedioate
-
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.97 - 3.78
(2R,3S)-3-(p-carboxybenzyl)malate
0.000097
(2S)-thiahomocitrate
Candida albicans
-
recombinant His-tagged enzyme, pH 7.8, 20C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.304
Saccharomycopsis lipolytica
-
-
564
-
purified enzyme, pH 8.0, 60C, with 1-hydroxy-1,2,4-butanetricarboxylate as substrate
8579
-
purified enzyme, pH 8.0, 60C, with 1-hydroxy-1,2,3-propanetricarboxylate as substrate
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
-
reductive carboxylation
8.3 - 8.8
-
oxidative decarboxylation
additional information
-
mutant enzymes kinetics and pH-dependencies, overview
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.1 - 7.7
-
pH 6.1: about 60% of activity maximum, pH 7.7: about 50% of activity maximum, reductive carboxylation
6.5 - 8.5
-
activity drops sharply below pH 6.5 and above pH 8.5
7.4 - 9.3
-
pH 7.4: about 50% of activity maximum, pH 9.3: about 90% of activity maximum, oxidative decarboxylation
additional information
-
pH-rate profile in the absence of K+, overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
-
reductive carboxylation
7.5
-
oxidative decarboxylation
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4
-
calculated from sequence
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
mRNA level of isoform Rdh10 declines during development, with strong and lasting expression in the meninges and choroid plexuses. Expression is also present in the striatum
Manually annotated by BRENDA team
-
mRNA level of isoform Rdh10 declines during development, with strong and lasting expression in the meninges and choroid plexuses. Expression is also present in the striatum
Manually annotated by BRENDA team
-
inner ear, specific expression of isoform Rdh10 in the endolymphatic system and stria vascularis
Manually annotated by BRENDA team
-
expression of Rdh10 gene correlates with many sites of retinoid signalling during embryogenesis and organ differentiation
Manually annotated by BRENDA team
-
mRNA expression of isoform Rdh10 both in the prospective pigmented epithelium and neural retina
Manually annotated by BRENDA team
-
the 3 kb isoform is the most abundant one
Manually annotated by BRENDA team
-
mRNA level of isoform Rdh10 declines during development, with strong and lasting expression in the meninges and choroid plexuses. Expression is also present in the striatum
Manually annotated by BRENDA team
-
and vibrissae follicles, expression of isoform Rdh10 from early stages in regions where sensory receptors appear and mesenchymal/epithelial interactions take place
Manually annotated by BRENDA team
low activity
Manually annotated by BRENDA team
-
strong expression, the 3 kb isoform is the most abundant one
Manually annotated by BRENDA team
-
mRNA expression of isoform Rdh10 both in the prospective pigmented epithelium and neural retina
Manually annotated by BRENDA team
-
the 3 kb isoform is the most abundant one
Manually annotated by BRENDA team
additional information
-
no expression in H-460 cells (non-small-cell lung cancer). Very low level of expression is detected in cell lines SKMES (squamous lung cancer) and SCLC (small-cell lung carcinoma)
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Thermus thermophilus (strain HB27 / ATCC BAA-163 / DSM 7039)
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
38090
-
calculated from sequence
154000
-
recombinant enzyme, gel filtration
158000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
Saccharomycopsis lipolytica
-
2 * 48000, at high protein concentration or in presence of substrate the enzyme exists as dimer
monomer
Saccharomycopsis lipolytica
-
1 * 48000, native or denaturing PAGE
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme in binary complex with inhibitor (2S,3S)-thiahomoisocitrate, sitting drop vapour diffusion method, 0.00 ml of 8.7 mg/ml protein in 5 mM Tris-HCl, pH 7.8. 0.8 mM inhibitor, and 1.7 mM NAD, is mixed with 0.002 ml of reservoir solution containing 40% 2-methyl-2,4-pentandiol and 100 mM citrate pH 4.85, eqilibration against 1 ml resetvoir solution, X-ray diffrcation structure determination and analysis at 2.6 A resolution, molecular replacement
purified recombinant enzyme, hanging drop vapour diffusion method, 10 mg/ml protein with reservoir solution containing 24% PEG 400, and 0.1 M citrate, pH 4.8, addition of 0.003 ml drops of 0.1 M MgCl2, 5 mM isocitrate or homoisocitrate, and of a 0.001 ml drop 50 mM CdCl2, equilibration against 0.5 ml reservoir solution, 5 days at 20C, X-ray diffraction structure determination and analysis at 1.85 A resolution, molecular replacement
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
enzyme is stable when incubated for at least 15 min over the pH range of 5.0-10.0
685232
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
90
-
half life: 16.7 h
93.6
-
melting point
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
Mg2+ stabilizes
Saccharomycopsis lipolytica
-
Mn2+ stabilizes
Saccharomycopsis lipolytica
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, ammonium sulfate precipitate, partially purified, stable
-
-20C, some loss of activity after repeated freeze-thawing cycles
-
-20C, stable as ammonium sulfate precipitate
Saccharomycopsis lipolytica
-
0C, pH 7.2, rapid loss of activity
Saccharomycopsis lipolytica
-
0C, potassium phosphate 20 mM, pH 7.2, 4-5 hours
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purification of His-tagged enzyme using nickel affinity chromatography results in an inactive enzyme
-
recombinant enzyme from Escherichia coli by anion exchange chromatography and gel filtration
-
recombinant enzyme from Escherichia coli strain OM17 by anion exchange and hydrophobic interaction chromatography, followed by another step of anion exchange chromatography
-
recombinant His-tagged enzyme from Escherichia coli strain TOP 10F by nickel affinity chromatography'
-
recombinnat His-tagged enzyme from Escherichia coli by metal affinity chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, expression of His-tagged enzyme in Escherichia coli
DNA and amino acid sequence determination and analysis, overexpression in Escherichia coli strain OM17
-
expressed in COS1 cells
-
expressed in Escherichia coli BL21(DE3)
-
expression in Cos-1 cells
-
expression in Escherichia coli
-
expression in Sf9 cell; RDH10 is expressed the enzyme in insect Sf9 cells using the Baculovirus expression system. Purification of RDH10-His6 from Sf9 cells using nickel affinity chromatography produces an inactive enzyme. Therefore, microsomal preparations of RDH10 are used for its kinetic characterization
-
expression of His-tagged enzyme in Escherichia coli strain TOP 10F'
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LYS1 gene codes for the alpha-aminoadipate pathway enzymes in fungi, can complement LYS1-deficient mutants of Candida albicans and Saccharomyces cerevisiae, amplification in Escherichia coli
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RDH10 is expressed in COS cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A80del
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site-directed mutagenesis, the mutant shows altered substrate specificity preferring isocitrate to homoisocitrate, it is unable to oxidize 3-isopropylmalate, the specificity is similar to the enzyme from Thermus thermophilus
R87T
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site-directed mutagenesis, the mutant oxidizes homoisocitrate, but not isocitrate and 3-isopropylmalate
R87V
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site-directed mutagenesis, the mutant oxidizes homoisocitrate, but not isocitrate and 3-isopropylmalate
D169A
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mutant enzyme completely loses enzymatic activity
D169N
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mutant enzyme completely loses enzymatic activity
G43A/G47A/G49A
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mutant enzyme completely loses enzymatic activity
K214A
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mutant enzyme completely loses enzymatic activity
K214R
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mutant enzyme completely loses enzymatic activity
S197A
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mutation does not abolish activity
S197C
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mutant enzyme completely loses enzymatic activity
S197G
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mutation does not abolish activity
S197T
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mutant enzyme completely loses enzymatic activity
S197V
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mutant enzyme completely loses enzymatic activity
Y210A
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mutant enzyme completely loses enzymatic activity
Y210F
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mutant enzyme completely loses enzymatic activity
K206M
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site-directed mutagenesis, the active site mutant shows about 2400fold reduced activity compared to the wild-type enzyme, the Km for HIc does not change significantly
Y150F
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site-directed mutagenesis, the active site mutant shows about 680fold reduced activity compared to the wild-type enzyme, the Km for HIc does not change significantly
R85V
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complete loss of activity with isocitrate, significant activity with 3-isopropylmalate, no effect on activity with homoisocitrate
V135M
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site-directed mutagenesis, tetramer-to-dimer structural transition enhances the activity with isocitrate 1.6fold
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine