Information on EC 4.2.1.33 - 3-isopropylmalate dehydratase

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

EC NUMBER
COMMENTARY hide
4.2.1.33
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RECOMMENDED NAME
GeneOntology No.
3-isopropylmalate dehydratase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(2R,3S)-3-isopropylmalate = (2S)-2-isopropylmalate
show the reaction diagram
overall reaction
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-
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(2R,3S)-3-isopropylmalate = 2-isopropylmaleate + H2O
show the reaction diagram
2-isopropylmaleate + H2O = (2S)-2-isopropylmalate
show the reaction diagram
(1b)
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-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
condensation
elimination
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of H2O, C-O bond cleavage, , trans elimination
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
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leucine metabolism
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Metabolic pathways
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Valine, leucine and isoleucine biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
(2R,3S)-3-isopropylmalate hydro-lyase (2-isopropylmaleate-forming)
Forms part of the leucine biosynthesis pathway. The enzyme brings about the interconversion of the two isomers of isopropylmalate. It contains an iron-sulfur cluster.
CAS REGISTRY NUMBER
COMMENTARY hide
37290-72-5
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
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Manually annotated by BRENDA team
strain B-6
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Manually annotated by BRENDA team
strain B-6
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
large subunit of 3-isopropylmalate dehydratase
SwissProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
wild type and Leu- -mutants
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Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
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isopropylmalate isomerase large subunit and small subunits form heterodimers to catalyze the isomerization of 2-isopropylmalate to 3-isopropylmalate in leucine biosynthesis in bacteria and archaea. Reverse genetics and metabolite profiling show that AtLeuD1 and AtLeuD2 function redundantly in aliphatic glucosinolate biosynthesis, but AtLeuD3 is not likely to be involved in this pathway
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
(2R,3S)-3-isopropylmalate
(2S)-2-isopropylmalate
show the reaction diagram
the isopropylmalate isomerase small subunit of the hyperthermophilic archaea Pyrococcus horikoshii (PhIPMI-s) functions as isopropylmalate isomerase in the leucine biosynthesis pathway, and as homoaconitase (HACN) in the lysine biosynthesis pathway via alpha-aminoadipic acid
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-
?
(2R,3S)-3-isopropylmalate
2-isopropylmaleate + H2O
show the reaction diagram
(2S)-2-isopropylmalate + H2O
(2R,3S)-3-isopropylmalate
show the reaction diagram
2-isopropylmaleate + H2O
3-isopropylmalate
show the reaction diagram
2-isopropylmaleate + H2O
?
show the reaction diagram
3-isopropylmalate
2-isopropylmaleate + H2O
show the reaction diagram
additional information
?
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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
(2R,3S)-3-isopropylmalate
(2S)-2-isopropylmalate
show the reaction diagram
O59393
the isopropylmalate isomerase small subunit of the hyperthermophilic archaea Pyrococcus horikoshii (PhIPMI-s) functions as isopropylmalate isomerase in the leucine biosynthesis pathway, and as homoaconitase (HACN) in the lysine biosynthesis pathway via alpha-aminoadipic acid
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-
?
(2R,3S)-3-isopropylmalate
2-isopropylmaleate + H2O
show the reaction diagram
(2S)-2-isopropylmalate + H2O
(2R,3S)-3-isopropylmalate
show the reaction diagram
2-isopropylmaleate + H2O
?
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Iron-sulfur cluster
the structure of the protein has unbound Fe-S clusters and contains a fourth cysteine in the active site
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(NH4)2SO4
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recovery after restoration of lower ionic strength shows hysteresis effect
1,10-phenanthroline
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16% inhibition at 0.1 mM
8-hydroxyquinolinesulfonate
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16% inhibition at 0.1 mM
beta-carboxy-beta-hydroxyisocaproate
Cd2+
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complete inhibition at 1 mM
Co2+
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3% inhibition at 1 mM
cysteine
dimethylmesaconate
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Fe2+
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13% inhibition at 1 mM
glutathione
isopropylmalate
KF
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most potent inhibitor
mercaptoethanol
Mg2+
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26% inhibition at 1 mM
Mn2+
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45% inhibition at 1 mM
N-ethylmaleimide
sulfhydryl compounds
Zn2+
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complete inhibition at 1 mM
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.078 - 4.57
alpha-hydroxy-beta-carboxyisocaproate
0.75 - 0.95
beta-carboxy-beta-hydroxyisocaproate
0.083 - 1.79
citraconate
0.0216 - 0.18
dimethylcitraconate
additional information
additional information
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Ks for standard substrates
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
12.3
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additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
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enzyme assay at
7 - 8
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8.5
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enzyme assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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enzyme assay at
34
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enzyme assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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expression in all floral tissues including sepal, stamen, pistil as well as pollen grains, but it is only present at both ends of developing siliques
Manually annotated by BRENDA team
additional information
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semi-quantitative RT-PCR analysis of temporal and spatial expression of AtLeuC and AtLeuD genes, overview. The tissue-specifis expression analysis reveals that the patterns of small subunits AtLeuD1 and AtLeuD2 expression are similar, but distinct from that of AtLeuD3
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
presence of different heterodimeric IPMIs in chloroplasts with distinct substrate specificities for Leu or glucosinolate metabolism; presence of different heterodimeric IPMIs in chloroplasts with distinct substrate specificities for Leu or glucosinolate metabolism; presence of different heterodimeric IPMIs in chloroplasts with distinct substrate specificities for Leu or glucosinolate metabolism; presence of different heterodimeric IPMIs in chloroplasts with distinct substrate specificities for Leu or glucosinolate metabolism. Import of the IPMI small subunit 2:GFP fusion protein into chloroplasts
Manually annotated by BRENDA team
additional information
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analysis of subcellular localization of AtLeuC and AtLeuDs within Arabidopsis chloroplast compartments, overview
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Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Campylobacter jejuni subsp. jejuni (strain IA3902)
Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440)
Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Streptococcus mutans serotype c (strain ATCC 700610 / UA159)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70000 - 90000
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sucrose density gradient centrifugation
88500
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sucrose density gradient centrifugation, gel filtration
additional information
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complementation pair enzymes have higher molecular weights of 169000-185000
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 46000 + x * 18000, SDS-PAGE
heterodimer
monomer
multimer
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active as monomer, can aggregate to dimers or multimers in cell
tetramer
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trimer or tetramer of 2 different polypeptide subunits
trimer
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trimer or tetramer of 2 different polypeptide subunits
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting drop vapor diffusion method, using 25% (w/v) polyethylene glycol monomethyl ether 2000 and 0.1 MTris-HCl (pH 7.9)
sitting-drop vapour-diffusion methodand hanging-drop vapour diffusion at 20C, structures of oxidized and reduced forms of the large subunit of isopropylmalate isomerase (ox-MJ0499 and red-MJ0499, respectively) are reported at 1.8 and 2.7 A resolution, respectively. Significant large conformational changes are observed in the active site of red-MJ0499 when compared with ox-MJ0499
small subunit LeuD variants, X-ray diffraction structure determination and analysis at resolutions of 2.0 A for LeuD_1-156, 1.2 A for LeuD_1-168, and 2.5 A for LeuD_1-186, respectively
variants LeuD-1-156 and LeuD-1-168, by sitting-drop vapour-diffusion method, crystals of LeuD-1-156 belong to the hexagonal system (space group P6122 or P6522) with up to four subunits in the asymmetric unit, whereas the crystals of LeuD-1-168 belong to the monoclinic system (space group P21) with two subunits in the asymmetric unit. Both crystals diffract to beyond 2.0 A resolution
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crystal structure isopropylmalate isomerase small subunit. Four molecules create an interlocked assembly with intermolecular disulfide linkages having a skewed 222 point-group symmetry. The structure reveals the formation of intermolecular disulfide linkages, and it provides insight into the dual substrate specificity of the enzyme
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 6.5
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unstable at pH 7.0
5658
6
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unstable at pH 7.0 or above
5659
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
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half-life: 4.5 h, in stabilizing ammonium sulfate buffer
62
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half-life: 1 min, in stabilizing ammonium sulfate buffer
additional information
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very heat sensitive, high ionic strenght stabilizes enzyme against heat inactivation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
10% inactivation at 0C in phosphate buffer at pH 7 or above
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half-life 2-3 h in potassium phosphate buffer, ph 6.8., 0-5C, beta-isopropylmalate at 1 mM increases half-life to 15 h
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loss of activity after repeated freezing and thawing
stabilized by high concentrations of glycerol and ammonium sulfate which also inhibit activity, half-life with 50% glycerol, 2.07 M ammonium sulfate, and 30% glycerol plus 830 mM ammonium sulfate: 50 h, 57 h, and 90 h, respectively, instead of 2-3 h without, stabilization probably due to changes in intramolecular interactions
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substrate stabilizes
unstable in phosphate buffer at pH 7.0, 10% loss of activity per h at 0C
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, sodium phosphate buffer, pH 7.2, 6 M urea, several days
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
nickel-affinity column chromatography and Superdex 200 gel filtration
variants LeuD-1-156 and LeuD-1-168, by centrifugation, on Ni-NTA column and by gel filtration, more than 95% pure
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
expression in Escherichia coli BL21(DE3) Star cells
expression of FLAG-tagged LeuC and of the three LeuD isoforms in transgenic Arabidosis thaliana plants, interaction analysis, overview
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expression of His6-tagged LeuD in Escherichia coli strain BL21(DE3)
full length reading frame of IPMI small subunit 2 fused in frame to the 5' end of the gene encoding the green fluorescent protein in the vector psmGFP4. Construct transiently expressed in tobacco protoplasts and stably transformed into Arabidopsis plants
variants LeuD-1-156 and LeuD-1-168, ligated into the pETM-11 expression vector, expressed in Escherichia coli BL21(DE3) RP
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
in ipmi ssu2-1 plants, absence of mature mRNAs in homozygous plants and the knockout of these genes by T-DNAs; in ipmi ssu3-1 plants, absence of mature mRNAs in homozygous plants and the knockout of these genes by T-DNAs; in the ipmilsu1-2mutant mRNA levels are reduced to 65 and 50% of the wild-type standard in seedlings and rosette leaves, respectively, while IPMI large subunit 1 transcript levels are reduced to 40 and 27% of wild-type in these tissues of ipmi lsu1-1 plants. Strongest reduction in ipmi lsu1-3 seedlings (21% of wild-type), while the IPMI large subunit 1 mRNA level in ipmi lsu1-3 reaches about 29% of the wild-type level in leaves
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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