Information on EC 1.1.1.85 - 3-isopropylmalate dehydrogenase

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

EC NUMBER
COMMENTARY hide
1.1.1.85
-
RECOMMENDED NAME
GeneOntology No.
3-isopropylmalate dehydrogenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(2R,3S)-3-isopropylmalate + NAD+ = (2S)-2-isopropyl-3-oxosuccinate + NADH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
butanol and isobutanol biosynthesis (engineered)
-
-
isoleucine metabolism
-
-
leucine metabolism
-
-
Valine, leucine and isoleucine biosynthesis
-
-
C5-Branched dibasic acid metabolism
-
-
Metabolic pathways
-
-
Biosynthesis of secondary metabolites
-
-
SYSTEMATIC NAME
IUBMB Comments
(2R,3S)-3-isopropylmalate:NAD+ oxidoreductase
The product decarboxylates spontaneously to yield 4-methyl-2-oxopentanoate.
CAS REGISTRY NUMBER
COMMENTARY hide
9030-97-1
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
No 1023
-
-
Manually annotated by BRENDA team
No 1023
-
-
Manually annotated by BRENDA team
gene leuB
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
gene leu2A
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
no 38-2
-
-
Manually annotated by BRENDA team
Bacillus sp. no 38-2
no 38-2
-
-
Manually annotated by BRENDA team
gene leuB
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
gene leu2
SwissProt
Manually annotated by BRENDA team
strain C175/CBS 8195, gene LEU2
SwissProt
Manually annotated by BRENDA team
strain C175/CBS 8195, gene LEU2
SwissProt
Manually annotated by BRENDA team
gene leu2
SwissProt
Manually annotated by BRENDA team
TIT 01010
-
-
Manually annotated by BRENDA team
M588
-
-
Manually annotated by BRENDA team
i.e. Eremothecium gossypii, gene leu2
SwissProt
Manually annotated by BRENDA team
gene leuB
SwissProt
Manually annotated by BRENDA team
gene leuB
SwissProt
Manually annotated by BRENDA team
gene leu2
SwissProt
Manually annotated by BRENDA team
strain SK103
-
-
Manually annotated by BRENDA team
gene leu2
SwissProt
Manually annotated by BRENDA team
gene leuB
SwissProt
Manually annotated by BRENDA team
strain HB-8
-
-
Manually annotated by BRENDA team
strain I5
-
-
Manually annotated by BRENDA team
strain I5
-
-
Manually annotated by BRENDA team
i.e. Septoria tritici, gene leuC
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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 + NAD+
(2S)-2-isopropyl-3-oxosuccinate + NADH + H+
show the reaction diagram
(2R,3S)-3-isopropylmalate + NAD+
2-ketoisocaproate + NADH + CO2
show the reaction diagram
-
-
-
-
?
(2R,3S)-3-isopropylmalate + NAD+
2-oxoisocaproate + NADH + CO2
show the reaction diagram
(2R,3S)-3-isopropylmalate + NAD+
2-oxoisocaproate + NADH + H+ + CO2
show the reaction diagram
-
-
-
-
?
(2R,3S)-3-isopropylmalate + NAD+
?
show the reaction diagram
(2R,3S)-3-isopropylmalate + NAD+
D-3-isopropyl-2-oxosuccinate + NADH + H+
show the reaction diagram
(2R,3S)-3-isopropylmalate + NAD+
isopropyl-3-oxosuccinate + NADH + H+
show the reaction diagram
-
-
-
r
(2R,3S)-3-isopropylmalate + thionicotinamide NAD+
2-oxoisohexanoate + thionicotinamideNADH + CO2
show the reaction diagram
(S)-malate + NAD+
pyruvate + NADH + CO2
show the reaction diagram
-
12% of the activity with 3-isopropylmalate
-
-
?
3-(2'-methylthio)ethylmalate + NAD+
? + NADH + H+
show the reaction diagram
-
-
-
?
3-ethylmalate + NAD+
3-ethylpyruvate + NADH + CO2
show the reaction diagram
3-isoamylmalate + NAD+
3-isoamylpyruvate + NADH + CO2
show the reaction diagram
-
-
-
-
?
3-isopropylmalate + NAD+
2-ketoisocaproate + CO2 + NADH + H+
show the reaction diagram
3-isopropylmalate + NAD+
2-ketoisocaproate + NADH + CO2
show the reaction diagram
3-isopropylmalate + NAD+
?
show the reaction diagram
-
-
-
-
?
3-methylmalate + NAD+
3-methylpyruvate + NADH + CO2
show the reaction diagram
-
-
-
-
?
3-tert-butylmalate + NAD+
3-tert-butylpyruvate + NADH + CO2
show the reaction diagram
-
-
-
-
?
beta-methylmalate + NAD+
2-oxobutyrate + NADH + CO2
show the reaction diagram
-
enzyme catalyzes the final step in the pyruvate pathway to 2-oxobutyrate
-
-
?
D-3-ethylmalate + NAD+
D-3-ethyl-2-oxosuccinate + NADH + H+
show the reaction diagram
D-3-isobutylmalate + NAD+
D-3-isobutyl-2-oxosuccinate + NADH + H+
show the reaction diagram
D-3-isopentylmalate + NAD+
D-3-isopentyl-2-oxosuccinate + NADH + H+
show the reaction diagram
D-3-methylmalate + NAD+
D-3-methyl-2-oxosuccinate + NADH + H+
show the reaction diagram
-
-
-
?
D-3-t-butylmalate + NAD+
D-3-t-butyl-2-oxosuccinate + NADH + H+
show the reaction diagram
-
-
-
?
D-malate + NAD+
pyruvate + NADH + CO2
show the reaction diagram
DL-3-isopropylmalate + NAD+
DL-isopropyl-3-oxosuccinate + NADH + H+
show the reaction diagram
-
-
-
-
?
ethylmalate + NAD+
ethylpyruvate + NADH + CO2
show the reaction diagram
-
-
-
-
?
isopropylmalate + NAD+
?
show the reaction diagram
-
-
-
-
?
malate + NAD+
3-oxosuccinate + NADH + H+
show the reaction diagram
-
-
-
?
malate + NAD+
pyruvate + NADH + CO2
show the reaction diagram
malate + NADP+
pyruvate + NADPH + CO2
show the reaction diagram
-
-
-
-
?
tartrate + NAD+
?
show the reaction diagram
-
-
-
-
?
tartrate + NADP+
?
show the reaction diagram
-
-
-
-
?
tert-butylmalate + NAD+
tert-butylpyruvate + NADH + CO2
show the reaction diagram
-
-
-
-
?
threo-D-3-isopropylmalate + NAD+
?
show the reaction diagram
threo-DL-isopropylmalate + NAD+
2-isopropyl-3-oxosuccinate + NADH
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
(2R,3S)-3-isopropylmalate + NAD+
2-oxoisocaproate + NADH + CO2
show the reaction diagram
(2R,3S)-3-isopropylmalate + NAD+
?
show the reaction diagram
P50455
the enzyme is enzyme involved in leucine biosynthesis
-
-
?
3-isopropylmalate + NAD+
2-ketoisocaproate + CO2 + NADH + H+
show the reaction diagram
-
the enzyme catalyzes a step in leucine biosynthesis
-
-
?
additional information
?
-
-
the enzyme catalyzes a step in leucine biosynthesis
-
-
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADP+
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
0.5 mM, very minor stimulatory effect
Fe2+
-
0.5 mM, very minor stimulatory effect
Rb+
-
RbCl enhances activity
Zn2+
-
0.5 mM, very minor stimulatory effect
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(2S,3S)-3-methylmercaptomalate
-
strong competitive inhibitor
-
1,2-Cyclohexanediamine-N,N,N',N'-tetraacetate
-
-
Fe2+
-
0.5 mM, 41% inhibition
iodoacetamide
iodoacetate
isocitrate
-
-
NaCl
-
250 mM, 48% inhibition
NADH
-
NADH always produces competitive inhibition patterns with respect to NAD and noncompetitive inhibition patterns with respect to the substrates
O-isobutenyloxalylhydroxamate
O-isobutenyloxalylhydroxamate binds to the active site of enzyme in a mode similar to the substrate isopropylmalate
p-hydroxymercuribenzoate
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0087 - 2.6
(2R,3S)-3-isopropylmalate
0.045 - 0.502
3-(2'-methylthio)ethylmalate
0.013
3-ethylmalate
-
-
0.11
3-isoamylmalate
-
-
0.0039 - 0.026
3-isopropylmalate
0.05
3-methylmalate
-
-
0.074
3-tert-butylmalate
-
-
0.0011
D-3-ethylmalate
pH 8.0
0.0015
D-3-isobutylmalate
pH 8.0
0.0058
D-3-isopentylmalate
pH 8.0
0.0012 - 0.0267
D-3-isopropylmalate
0.0037
D-3-methylmalate
pH 8.0
0.0023
D-3-t-butylmalate
pH 8.0
0.41 - 4
D-malate
0.002 - 0.0133
DL-3-isopropylmalate
0.019
dl-erythro-beta-ethylmalate
-
pH 9.0, 22C
0.0092
erythro-Lg-beta-isopropylmalate
-
pH 9.0, 22C
-
0.09
ethylmalate
-
pH 7.6
0.000017 - 0.316
isopropylmalate
12
L-Malate
-
pH 9.0
0.0326 - 35.59
malate
0.00321 - 3.56
NAD+
0.014 - 6.579
NADP+
0.105
propylmalate
-
pH 7.6
0.0407 - 1.21
Tartrate
0.214
tert-butylmalate
-
pH 7.6
0.051
thionicotinamide-NAD+
-
pH 9.0, 22C
0.0061 - 0.0161
threo-D,L-isopropylmalate
0.08
threo-Ds-3-Isopropylmalate
-
60C
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.08 - 17
(2R,3S)-3-isopropylmalate
0.016 - 0.85
3-(2'-methylthio)ethylmalate
38
3-ethylmalate
Acidithiobacillus ferrooxidans
-
-
20
3-isoamylmalate
Acidithiobacillus ferrooxidans
-
-
3.8 - 28
3-isopropylmalate
28
3-methylmalate
Acidithiobacillus ferrooxidans
-
-
2.7
3-tert-butylmalate
Acidithiobacillus ferrooxidans
-
-
3.1
D-3-ethylmalate
2.7
D-3-isopentylmalate
Sulfolobus tokodaii
P50455
pH 8.0
2.36 - 117
D-3-isopropylmalate
3.5
D-3-methylmalate
Sulfolobus tokodaii
P50455
pH 8.0
0.96
D-3-t-butylmalate
Sulfolobus tokodaii
P50455
pH 8.0
70
ethylmalate
Escherichia coli
-
pH 7.6
0.09 - 69
isopropylmalate
0.19 - 10.6
malate
0.15 - 117
NAD+
0.09 - 4.69
NADP+
0.1 - 0.26
Tartrate
6
tert-butylmalate
Escherichia coli
-
pH 7.6
additional information
additional information
Thermus thermophilus
-
the cold adaption results from the destabilization of the ternary complex caused by increase in the volume of the residue at position 126
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.16 - 983.7
(2R,3S)-3-isopropylmalate
1131
0.038 - 18.77
3-(2'-methylthio)ethylmalate
5879
0.12 - 4.2
NAD+
7
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000062
(2S,3S)-3-methylmercaptomalate
-
pH 7.8, 60C
-
additional information
additional information
-
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.54
-
slow electrophoretic form
4.7
-
mutant E253A
5.2
-
mutant E253W
5.9
-
mutant E253G
7
-
mutant E253Q
7.4
-
mutant E253I
7.6
-
mutant E253F; mutant E253L
9
-
mutant E253V
13.9
-
wild-type
19.4
-
55C; pH 7.0, 55C
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 11.5
-
about 75% of maximal activity at pH 7.5 and 11.5
8 - 10.1
-
pH 8.0: 75% of maximal activity, pH 8.9-10.1: optimum
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
-
assay at
55
-
assay at
96
Tm-value of wild-type enzyme is 96C. Tm-value of mutant enzyme K152R is 95.6C. Tm-value of mutant enzyme (M91L/I95L/K152R/G154A/A259S/F261P/Y282L) is 96.4C
97
Tm-value of mutant enzyme A259S/F261P is 97.1C; Tm-value of mutant enzyme K152R/G154A is 97.2C
98
Tm-value of mutant enzyme G154A is 97.9C
99
t1/2: 3.67 min (wild-type enzyme), 5.52 min (mutant enzyme M91L/I95L), 6.56 min (K152R/G154A mutant enzyme), 5.72 min (K152R mutant enzyme), 7.13 min (G154A mutant enzyme), 4.79 min (A259S/F261P mutant enzyme), 3.79 min (Y282L mutant enzyme), 6.5 min (M91L/I95L/K152R/G154A/A259S/F261P/Y282L mutant enzyme); Tm-value of mutant enzyme M91L/I95L is 99.2C
additional information
-
reaction temperature dependence, overview
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 40
-
the van t Hoff plot of Km IPM shows sigmoid-like transition within this range
25 - 95
2% of maximal activity at 25C, maximal activity at 95C
40 - 80
-
40C: about 45% of maximal activity, 80C: about 85% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.9
-
isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
high expression level of IMD3 and IMD2
Manually annotated by BRENDA team
-
very low expression level of IMD1
Manually annotated by BRENDA team
-
high expression level of IMD1 and IMD2
Manually annotated by BRENDA team
-
high expression level of IMD3 and IMD2
Manually annotated by BRENDA team
additional information
-
tissue specific expression of genes IMD1, IMD2, and IMD3, overview
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
the enzyme contains a transit peptide
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Burkholderia thailandensis (strain ATCC 700388 / DSM 13276 / CIP 106301 / E264)
Burkholderia thailandensis (strain ATCC 700388 / DSM 13276 / CIP 106301 / E264)
Campylobacter jejuni subsp. jejuni serotype O:2 (strain ATCC 700819 / NCTC 11168)
Campylobacter jejuni subsp. jejuni serotype O:2 (strain ATCC 700819 / NCTC 11168)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Shewanella oneidensis (strain MR-1)
Shewanella oneidensis (strain MR-1)
Shewanella oneidensis (strain MR-1)
Shewanella oneidensis (strain MR-1)
Shewanella oneidensis (strain MR-1)
Shewanella oneidensis (strain MR-1)
Shewanella oneidensis (strain MR-1)
Shewanella oneidensis (strain MR-1)
Shewanella oneidensis (strain MR-1)
Shewanella oneidensis (strain MR-1)
Shewanella oneidensis (strain MR-1)
Sulfolobus acidocaldarius (strain ATCC 33909 / DSM 639 / JCM 8929 / NBRC 15157 / NCIMB 11770)
Sulfolobus tokodaii (strain DSM 16993 / JCM 10545 / NBRC 100140 / 7)
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)
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)
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)
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)
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)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50000
-
gel filtration
58000
-
gel filtration
68000
-
monomer-dimer equilibrium in aqueous solution, gel filtration
72000
-
gel filtration
73000
-
equilibrium sedimentation
73300
-
SDS-PAGE
90000
-
gel electrophoresis
110000
-
gel filtration
136000
sedimentation equilibrium analysis
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
monomer
tetramer
trimer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme in complex with 3-isopropylmalate
-
to 2.25 A resolution. Modeling of 3-isopropylmalate binding in the isoform IPMDH2 active site. Results suggest that substitution of one active site residue may lead to altered substrate specificity and metabolic function
hanging drop vapor diffusion method, chimeric enzyme constructed by fusing the gene of Bacillus subtilis and Thermus thermophilus coding for 3-isopropylmalate dehydrogenase, expression in Escherichia coli
-
crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus
-
The crystal structure is determined at 1.65 A resolution. The crystals contain two functional dimers in the asymmetric unit in an arrangement close to a tetramer of D2 symmetry.
crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus
-
to 1.5 A resolution. Comparison of enzyme structures from the obligate piezophile Shewanella benthica DB21MT-2 and the nonpiezophile Shewanella oneidensis MR-1. The Shewanella benthica enzyme is in a more open form and has a larger internal cavity volume than Shewanella oneidensis IPMDH at atmospheric pressure. The loosely packed structure of Shewanella benthica IPMDH may help it to avoid pressure-induced distortion of the native structure and to remain active at higher pressures than Shewanella oneidensis IPMDH
-
structures of 3-isopropylmalate dehydrogenase at about 2 A resolution under pressures ranging from 0.1 to 650 MPa. Most of the protein cavities are monotonically compressed as the pressure increases. The volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity occurs at pressures over 410 MPa.The generation of a new cleft on the molecular surface accompanied by water penetration can be observed at pressures over 580 MPa
-
to 1.5 A resolution. Comparison of enzyme structures from the obligate piezophile Shewanella benthica DB21MT-2 and the nonpiezophile Shewanella oneidensis MR-1. The Shewanella benthica enzyme is in a more open form and has a larger internal cavity volume than Shewanella oneidensis IPMDH at atmospheric pressure. The loosely packed structure of Shewanella benthica IPMDH may help it to avoid pressure-induced distortion of the native structure and to remain active at higher pressures than Shewanella oneidensis IPMDH
-
crystallized by the vapor-diffusion method, space group P2221. The crystallization requires 2-methyl-2,4-pentanediol to avoid twinning of the crystals
-
ammonium sulfate precipitation, crystals diffract beyond 2.5 A resolution and are quite stable against X-rays, recombinant enzyme expressed in Escherichia coli
-
apo-form without substrate and in complex with the divalent metalion, in complexes with both Mn2+ and 3-isopropylmalate, as well as with both Mn2+ and NADH, at resolutions ranging from 1.8 to 2.5 A. Identification of two hinges at the interdomain region, hinge 1 between alphad and betaF as well as hinge 2 between alphah and betaE with a possible operational mechanism upon the action of the substrates. The interactions of the protein with Mn2+ and isopropylmalate are mainly responsible for the domain closure. Upon binding into the cleft of the interdomain region, the substrate isopropylmalate induces a relative movement of the secondary structural elements betaE, betaF, betaG, alphad and alphah. A movement of the loop bearing the amino acid Tyr139 precedes the interacting arm of the subunit. The tyrosyl ring rotates and moves by at least 5 A upon substrate binding. Thereby, new hydrophobic interactions are formed above the buried isopropyl-group of isopropylmalate. Domain closure is then completed only through subunit interactions. A loop of one subunit that is inserted into the interdomain cavity of the other subunit extends the area with the hydrophobic interactions, providing an example of the cooperativity between interdomain and intersubunit interactions
-
crystal structure of mutant H15Y/E57V/S72I/R85M/Y86A/M208T/F217Y/V238M/R310M is determined at 2.4 A
crystal structure of Thermus thermophilus IPMDH in a ternary complex with NAD+ and the inhibitor ((2S,3S)-3-methylmercaptomalate) is determined at 2.8 A resolution. The inhibitor exists as a decarboxylated product with an enol/enolate form in the active site. The product interacts with Arg94, Asn102, Ser259, Glu270, and a water molecule hydrogen-bonding with Arg132. All interactions between the product and the enzyme are observed in the position associated with keto-enol tautomerization
-
enzyme in complex with NAD+
-
hanging drop vapor diffusion method, chimeric enzyme constructed by fusing the gene of Bacillus subtilis and Thermus thermophilus coding for 3-isopropylmalate dehydrogenase, expression in Escherichia coli
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mutant enzymes A172V, A172G and A172F. As in the case of A172L enzyme, the A172F mutant can not be crystallized by the salting-out technique with ammonium sulfate. The crystal is obtained at pH 4.8 using polyethylene glycol 4000 as a precipitant. Crystals of mutant enzyme A172E are obtained from a drop equilibrated with reservoir solution consisting of 0.8 M ammonium sulfate pH 6.0 at either 15C or 20C K. Two types of crystals: one hexagonal bipyramidal and the other is tetragonal
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recombinant enzyme expressed in Escherichia coli, ammonium sulfate precipitation
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study of the mutant enzymes G240A and L246E/V249M by X-ray crystallography
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using the hanging-drop vapour diffusion method crystals of Tt-IPMDH are grown in the following combinations: Apo Tt-IPMDH (crystal diffracted to a resolution of about 1.8 A), Tt-IPMDH in complex with Mn2+ (crystal diffracted to a resolution of about 2.5 A), Tt-IPMDH in complex with Mn2+ and beta-3-isopropylmalate (crystal diffracted to a resolution of 2.2 A), Tt-IPMDH in complex with Mn2+ and NADH (crystal diffracted to a resolution of 2.5 A), and Tt-IPMDH in complex with Mn2+, beta-3-isopropylmalate and NADH (crystal diffracted to a resolution of 2.75 A)
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 7.5
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stable if stored concentrated
639148
8.3
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5 min, half-life at 0C and at 23C is 5 min
639147
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0
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pH 8.3, half-life: 5 min
23
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pH 8.3, half-life: 5 min, essentially full protection against inactivation is provided by either 20% v/v glycerol or by simultaneous presence of Mn2+ and dithiothreitol, Mn2+ and beta-isopropylmalate or Mn2+ and alpha-ketoisocaproate
50
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thermal stability curves of wild-type and mutant enzymes at pH 7.6 in presence of 0.5 mM EDTA, overview
60
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5 min, 92% loss of activity in absence of NaCl, 6% loss of activity in presence of 1.5 M NaCl
61
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midpoint of thermal unfolding curve, mutant enzyme A172D
63
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midpoint of irreversible denaturation
65
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midpoint of thermal unfolding curve, mutant enzyme A172V
66
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midpoint of thermal unfolding curve, mutant enzyme A172I and A172E
67
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midpoint of thermal unfolding curve, mutant enzyme A172L and A172F
70
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10 min, 40% loss of activity
79
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midpoint of irreversible denaturation
82
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10 min, 50% inactivation, mutant enzyme A31G/G43A/A709G
83
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10 min, 50% inactivation
85 - 95
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thermal degeneration curves of wild-type and mutant enzymes at pH 7.6, overview
85
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10 min, 50% inactivation, mutant enzyme G43A
97
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melting temperature; pH 7.0, 50% inactivation; Tm-value
additional information
GENERAL STABILITY