Information on EC 4.2.3.27 - isoprene synthase

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

EC NUMBER
COMMENTARY hide
4.2.3.27
-
RECOMMENDED NAME
GeneOntology No.
isoprene synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
dimethylallyl diphosphate = isoprene + diphosphate
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
-
-
isoprene biosynthesis I
-
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isoprene biosynthesis II (engineered)
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Terpenoid backbone biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
dimethylallyl-diphosphate diphosphate-lyase (isoprene-forming)
Requires Mg2+ or Mn2+ for activity. This enzyme is located in the chloroplast of isoprene-emitting plants, such as poplar and aspen, and may be activitated by light-dependent changes in chloroplast pH and Mg2+ concentration [2,8].
CAS REGISTRY NUMBER
COMMENTARY hide
139172-14-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
transgenic, transformend with Grey poplar ISPS
-
-
Manually annotated by BRENDA team
strain 6051
-
-
Manually annotated by BRENDA team
strain 6051
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Eucalyptus sp.
-
-
-
Manually annotated by BRENDA team
var. utilis
-
-
Manually annotated by BRENDA team
Mucuna sp.
velvet bean
-
-
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
hybrid, Populus tremula x Populus tremuloides
-
-
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
soluble and membrane-bound isozymes
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
dimethylallyl diphosphate
isoprene + diphosphate
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
dimethylallyl diphosphate
isoprene + diphosphate
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
Mucuna sp.
-
stimulates at up to 2 mM, inhibits at 2-30 mM
Co2+
activates, 23% of the activity with Mg2+
Fe2+
2% of the activity with Mg2+
Ni2+
2% of the activity with Mg2+
Zn2+
3% of the activity with Mg2+
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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weak, 20% inhibition at 10 mM
Co2+
-
inhibits at 1 mM
diethyldicarbonate
-
34-59% inhibition at 1.1 mM, dimethylally diphosphate and Mg2+ protect the soluble enzyme, but not the solubilized thylakoid membrane isozyme
dimethylallyl diphosphate
-
substrate inhibition above 10 mM
diphosphate
-
69-100% inhibition
EDTA
-
40% inhibition at 1 mM, 70% inhibition at 10 mM
Fe2+
-
inhibits at 1 mM
Mg2+
-
activates at low concentration below 0.1 mM, inhibits at higher concentrations
Mn2+
-
activates at low concentration below 0.1 mM, inhibits at higher concentrations
NEM
-
complete inactivation at 1.1 mM, dimethylally diphosphate and Mg2+ protect the isozyme partially
Phenylglyoxal
-
50 mM, complete inactivation, dimethylally diphosphate and Mg2+ protect the isozyme partially
Zn2+
-
inhibits 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
0.0038 - 8
dimethylallyl diphosphate
additional information
additional information
-
soluble and membrane-bound isozymes
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.7 - 1.9
dimethylallyl diphosphate
additional information
additional information
Populus tremula x Populus alba
Q7XAS7
turnover values ranging from 3.7 to 5.1 mmol isoprene mol-1 ISPS under darkness, long-day conditions, i.e. 16 h light, 8 h dark, and continuous light conditions
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.000006
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PcISPS activity detected from 2-week-old plant material, Arabidopsis thaliana line 5
0.000012
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PcISPS activity detected from 2-week-old plant material, Arabidopsis thaliana line 10
0.000024
0.000027
continuous dark condition, time curve over 72h
0.000036
0.000042
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PcISPS activity detected from 2-week-old plant material, Arabidopsis thaliana line 3
0.000066
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fraction of transpirable soil water, FTSW, 30%
0.000072
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PcISPS activity detected from 2-week-old plant material, Arabidopsis thaliana line 9
0.000096
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14 days after recovery, DAR
0.00012
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2 days after recovery, DAR
0.000138
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fraction of transpirable soil water, FTSW, 100%
0.00014
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PcISPS activity detected from 2-week-old plant material, Arabidopsis thaliana line 8
0.00039
leaf cell culture
0.001
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purified thylakoid membrane-bound isozyme
0.011
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purified soluble isozyme
0.012 - 0.156
purified recombinant enzyme with or without leader sequence
0.333
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partially purified enzyme
0.521
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purified enzyme
1500
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cell free exract from leaf transformants
4967
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native level in Populus alba leaves
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.3
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first optimum
7.7
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second optimum
7.8 - 8.5
9
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recombinant N-terminally His-tagged enzyme
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.4 - 11.2
Mucuna sp.
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-
6.3 - 9.2
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sharp decrease in activity below pH 6.3
6.5 - 9.5
activity range
7.7 - 8.2
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broad pH-optimum
additional information
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pH profile
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
28
-
assay at
32
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assay at
37
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assay at
40 - 45
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optimal temperature for isoprene emission in vivo in warm and cooler regions, respectively
40 - 50
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recombinant C-terminally His-tagged enzyme
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15 - 50
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exponential increase of activity from 15C to 40C, optimum activity at 50C, inactivation at 60C
25 - 50
about half-maximal activity at 25C and 50C, temperature profile
26 - 45
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no activity in vivo below 20C and above 45C, temperature dependence of activity, it is not closely related to electron flux or CO2 assimilation arte, overview
40 - 55
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recombinant N-terminally His-tagged enzyme, recombinant untagged enzyme, and native enzyme
40 - 60
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recombinant C-terminally His-tagged enzyme
additional information
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the in vivo rate constant of IspS obeys the Arrhenius law, with an activation energy of 42.8 kJ per mol
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.7
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isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
from leaves
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
soluble isozyme
Manually annotated by BRENDA team
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soluble enzyme form
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Manually annotated by BRENDA team
additional information
PDB
SCOP
CATH
ORGANISM
UNIPROT
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
52000
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native enzyme, gel filtration
53000
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recombinant untagged enzyme, gel filtration
62000
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recombinant untagged enzyme, native PAGE
90000 - 100000
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gel filtration
98000 - 137000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
-
1 * 58000 + 1 * 63000, SDS-PAGE
monomer
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1 * 68000, C-terminally His-tagged enzyme, SDS-PAGE, 1 * 78000, N-terminally His-tagged enzyme, SDS-PAGE
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
X-ray crystal structure of recombinant ISPS at 2.7 A resolution, and the structure of its complex with three Mg2+ and theunreactive substrate analogue dimethylallyl-S-thiolodiphosphate determined at 2.8 A resolution. Analysis suggests that the generation of isoprene from substrate dimethylallyl diphosphate occurs via a syn-periplanar elimination mechanism in which the diphosphate-leaving group serves as a general base
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 45
-
stable within
additional information
thermotolerance of photosynthesis against transient high-temperature episodes can only be observed in lines emitting high levels of isoprene
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
endogenous isoprene emission of plants plays a protective role against three abiotic stresses (heat, combined heat/light and ozone-induced oxidative stress)
glycerol stabilizes the enzyme in the early stages of purification, PEG in the late stages
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isoprene biosynthesis by ISPS is relatively unaffected by drought
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the enzyme activity is very labile and only reproducibly detected when suitable protectants are used in the extraction buffer, especially PMSF is highly stabilizing
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
isoprene-emitting plants are highly resistant to ozone-induced oxidative damage compared with their non-emitting azygous controls, endogenously produced isoprene provides protection from oxidative damage
694648
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70C, partially purified enzyme, 40-55% ammonium sulfate fraction, in phosphate buffered saline, pH 7.4, 1mM DTT, stable for at least 1 month
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-80C, 30-60C ammonium sulfate fraction, resuspended in 50 mM Tris-HCl, pH 8.5, 20 mM MgCl2, 5% glycerol, 2 mM DTT, stable
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native enzyme 4000fold from leaves to homogeneity, ammonium sulfate fractionation of cell-free supernatant, dialysis, ion exchange chromatography, ultrafiltration, and gel filtration
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native enzyme from leaves
-
native enzyme partially by ammonium sulfate fractionation and anion exchange chromatography
Mucuna sp.
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native enzyme partially by anion exchange chromatography
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native enzyme partially from leaves by ammonium sulfate fractionation
native enzyme partially from leaves, recombinant His-tagged enzyme without plastidial targeting sequence from Escherichia coli strain TG1 by nickel affinity chromatography
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recombinant enzyme from Escherichia coli
recombinant His-tagged enzyme from Escherichia coli strain TG1
recombinant His-tagged ISPS from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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separation of soluble and membrane-bound isozymes, by ammonium sulfate fractionation, chloroplast preparation, PEG precipitation, hydrophobic interaction and ion exchange chromatography, and gel filtration, the thylakoid membrane isozyme is irreversibly solubilized by pH 10.0 treatment, overview
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli XL1-Blue; expressed in Escherichia coli XL1-Blue
expression analysis under different atmospheric conditions, overview, expression of the His-tagged ISPS in Escherichia coli strain BL21(DE3)
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expression in Escherichia coli
gene ispS, DNA and amino acid sequence determination and analysis, expression in Escherichia coli, expression as GFP-fusion protein in onion peels and tobacco leaves, transformations by particle bombardement
gene ispS, DNA and amino acid sequence determination and analysis, sequence comparisons, expression of His-tagged enzyme with or without leader sequence in Escherichia coli strain TG1
gene ispS, expression of N-terminally or C-terminally His-tagged enzyme without plastidial targeting sequence in Escherichia coli strain TG1
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His-tagged version expressed in Escherichia coli, after codon-use optimization expressed in Synechocystis sp. PCC6803
into the pET28a vector for production of a polyclonal primary antibody
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into the pQE50 vector for expression in Escherichia coli, into the binary vector pBinAR for transformation of Agrobacterium tumefaciens and generating transgenic Arabidopsis thaliana plants
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the plasmid pGWB2-PaIspS is introduced via Agrobacterium tumefaciens into Arabidopsis plants
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transformation of tobacco with an isoprene synthase gene extracted from poplar, obtained plants emitting isoprene at levels comparable to a naturally emitting species, full-length and truncated isoprene synthase cDNAs are amplified
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
environmental protection
synthesis