Information on EC 2.5.1.20 - rubber cis-polyprenylcistransferase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.5.1.20
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RECOMMENDED NAME
GeneOntology No.
rubber cis-polyprenylcistransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
polycis-polyprenyl diphosphate + isopentenyl diphosphate = diphosphate + a polycis-polyprenyl diphosphate longer by one C5 unit
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
alkenyl group transfer
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
rubber biosynthesis
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Terpenoid backbone biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
poly-cis-polyprenyl-diphosphate:isopentenyl-diphosphate polyprenylcistransferase
Rubber particles act as acceptor.
CAS REGISTRY NUMBER
COMMENTARY hide
62213-41-6
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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
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-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
Ficus decora
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-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
cultivar Salinas
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Manually annotated by BRENDA team
prickly lettuce
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Manually annotated by BRENDA team
isozymes TbCPT1, TbCPT2, and TbCPT3
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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CPT-RNAi plants indicate a significant reduction in rubber biosynthesis and a corresponding 50% increase in the levels of triterpenes and inulin, the primary storage carbohydrate compared to wild-type plants. The laticifers in CPT-RNAi plants contained fewer and smaller rubber particles than wild-type laticifers. And a decrease is noted in the activity of hydroxymethylglutaryl-CoA reductase, a key enzyme in the mevalonate pathway, reflecting homeostatic control of the isopentenyl diphosphate pool, CPT RNAi silencing phenotypes, overview
metabolism
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
dimethylallyl diphosphate + isopentenyl diphosphate
(E,E)-farnesyl diphosphate + (E)-geranyl diphosphate
show the reaction diagram
isopentenyl diphosphate + (E,E)-farnesyl diphosphate + cis-1,4-polyisoprene
diphosphate + a poly-cis-polyprenyl diphosphate longer by one C5 unit
show the reaction diagram
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-
-
-
?
isopentenyl diphosphate + cis-1,4-polyisoprene
diphosphate + a poly-cis-polyprenyl diphosphate longer by one C5 unit
show the reaction diagram
isopentenyl diphosphate + cis-1,4-polyisoprene
diphosphate + cis-1,4-polyisoprene + a poly-cis-polyprenyl diphosphate longer by one C5 unit
show the reaction diagram
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50 mM phosphate buffer, pH 7.5, 40 mM 2-mercaptoethanol, 40 mM KF, 8 mM MgCl2 + substrates, 30C, 70% incorporation small rubber particles, 50% incorporation with FL-latex, 13% with washed small particles, 4% with highly purified washed small particles, with farnesyl diphosphate or dimethylallyl diphosphate as allylic initiators 70% incorporation, similar as for small rubber particles without allylic initiators
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-
?
isopentenyl diphosphate + farnesyl diphosphate
diphosphate + ?
show the reaction diagram
isopentenyl diphosphate + farnesyl diphosphate + cis-1,4-polyisoprene
diphosphate + a poly-cis-polyprenyl diphosphate longer by one C5 unit
show the reaction diagram
polycis-polyprenyl diphosphate + isopentenyl diphosphate
diphosphate + a polycis-polyprenyl diphosphate longer by one C5 unit
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
isopentenyl diphosphate + cis-1,4-polyisoprene
diphosphate + a poly-cis-polyprenyl diphosphate longer by one C5 unit
show the reaction diagram
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incorporation of isopentenyl diphosphate into rubber
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isopentenyl diphosphate + cis-1,4-polyisoprene
diphosphate + cis-1,4-polyisoprene + a poly-cis-polyprenyl diphosphate longer by one C5 unit
show the reaction diagram
-
50 mM phosphate buffer, pH 7.5, 40 mM 2-mercaptoethanol, 40 mM KF, 8 mM MgCl2 + substrates, 30C, 70% incorporation small rubber particles, 50% incorporation with FL-latex, 13% with washed small particles, 4% with highly purified washed small particles, with farnesyl diphosphate or dimethylallyl diphosphate as allylic initiators 70% incorporation, similar as for small rubber particles without allylic initiators
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-
?
polycis-polyprenyl diphosphate + isopentenyl diphosphate
diphosphate + a polycis-polyprenyl diphosphate longer by one C5 unit
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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maximal stimulation at 5 mM
Pb2+
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maximal stimulation at 5 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(Z)-2-farnesyl-3-methylbutenedioic acid dilithium salt
(Z)-2-octyl-3-methylbutenedioic acid dilithium salt
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i.e. SG-296, 40% inhibition at 0.18 mM
alpha-hydroxyfarnesylphosphonic acid
chaetomellic acid
Co2+
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0.1-1 mM, complete inhibition
diphosphate
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farnesyl diphosphate
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iodoacetamide
iodoacetic acid
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N-ethylmaleimide
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p-chloromercuribenzoate
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50 mM, strong inhibition
Sodium diphosphate
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Sodium phosphate
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Thiol alkylating agents
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-
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Zn2+
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0.1-1 mM, complete inhibition
additional information
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not inhibitory: (Z)-2-octyl-3-methylbutenedioic acid dilithium salt
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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10 mM, not as effective as dithiothreitol
dithiothreitol
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10 mM, required for maximal stability
EDTA
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approx. 3.5fold activation at 20 mM
glutathione
neryl diphosphate
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accelerates activity
Rubber elongation factor
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rubber particle
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sulfhydryl compound
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essential for full activity
additional information
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recombinant HRT2, expressed in Escherichia coli, is significantly activated in the presence of a centrifuged latex fraction, resulting in the formation of polyisoprenes corresponding to natural rubber, but the recombinant HRT2 does not exhibit significant activity independently. HRT1/HRT2 require additional cofactors from the eukaryotic cells to produce distinct cPT activity, which seem to be contained in latex species
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.096
dimethylallyl diphosphate
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0.0003 - 2.2
farnesyl diphosphate
0.083 - 0.5
isopentenyl diphosphate
additional information
additional information
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.025 - 0.14
(Z)-2-farnesyl-3-methylbutenedioic acid dilithium salt
0.064 - 0.42
alpha-hydroxyfarnesylphosphonic acid
0.0088 - 0.042
chaetomellic acid
200
diphosphate
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-
5
Sodium diphosphate
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-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0000026
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2-methyl-1,3-butadiene incorporation assay with rubber particles, 100 mM Tris-HCl, pH 7.5, 30C, 2 h
0.000003
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2-methyl-1,3-butadiene incorporation assay with rubber particles, 100 mM Tris-HCl, pH 7.5, 30C, 2 h
0.000695
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partially purified enzyme
additional information
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2.3 microM isopentenyl diphosphate incorporation/g dry rubber/4 h for small rubber particles, 0.7 microM isopentenyl diphosphate incorporation/g dry rubber/4 h for purified and washed small rubber particles, almost no isopentenyl diphosphate incorporation/g dry rubber/4 h for washed and highly purified small rubber particles, in vitro, 50 mM phosphate buffer, pH 7.5, 40 mM 2-mercaptoethanol, 40 mM KF, 8 mM MgCl2 + substrates, 30C, small rubber particle activity decreases with increasing dimethylallyl diphosphate concentration, dimethylallyl diphosphate slightly increases activity in washed small particles not in a dose-dependent manner
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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in young branches, enzyme activity is induced by cool night temperatures of 20C
Manually annotated by BRENDA team
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in seedlings younger than 200 days, enzyme activity is not induced by cool night temperatures of 20C
Manually annotated by BRENDA team
additional information
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only minimally expressed in leaves, pedicels, and roots
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
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localization of the enzyme on small rubber particle and lutoid particles, which are considered to be polydispersed lysosomal vacuole
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
32000
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SDS-PAGE, TkCPT1; SDS-PAGE, TkCPT2
34000
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SDS-PAGE, TkCPT3
60000
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gel filtration
70000
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gel filtration
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 41000, recombinant C-terminally His6-tagged HRT1 or HRT2, SDS-PAGE
dimer
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2 * 38000, SDS-PAGE
additional information
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 9
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maximal stability at pH 7
638735
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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enzyme activity in rubber particles purified from line 593 decreases below limits of detection in 1 h at 25C, whereas activity is stable for much longer in particles from line 11591
40
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10 min, partial denaturation
60
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10 min, complete inactivation
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
thiols stabilize
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638726
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80C, 25 mM Tris-HCl, pH 8, 10 mM dithiothreitol, 1 mM MgSO4, 30% glycerol, 0.1% sodium azide, at least 6 months, no loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate, DE52, Sephacryl S-200, hexyl-Sepharose, Mono Q
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ammonium sulfate, Sephadex G-25, DEAE-cellulose, partial purification
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centrifugation to separate small rubber particles from the lower layer, dilution with 0.01 M phosphate buffer, pH 7.0 to 30% dry rubber content, washed particles are produced by washing with 0.25 M Tris-HCl, pH 8.0, with 1% Tween-20 and 5 mM 2-mercaptoethanol, applied to a Ultrogel AcA-44 column, centrifuged and diluted to 15% dry rubber content, highly purified small particles are produced by further centrifugation with 0.5% Triton X-100, exchange by buffer and centrifugation, cream phase diluted to 15% dry rubber content
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Parthenium argentatum makes rubber in parenchyma cells, which must be homogenized to release the rubber particles, the method for purification of enzymatically active rubber particles from the plant requires homogenization that also releases copious amounts of proteases and other degradative enzymes, with a resultant decrease in enzyme stability and half-life, overview
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pH 5.4, ammonium sulfate, Sephadex G-100, DEAE-Sephadex, partial purification
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purification of rubber particles containing the enzyme by harvesting latex from petioles of roots, separating latex fractions by centrifugation with extraction buffer (100 mM Tris-HCl, pH 7.8, 350 mM sorbitol, 10 mM NaCl, 5 mM MgCl2, 5 mM DTT), rubber phase is further centrifuged, washed with buffer and used for enzyme assays
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Saccharomyces cerevisiae SNH23-D7 (temperature sensitive mutant strain for dehydrodoichyl diphosphate synthase) or Nicotiana tabacum protoplasts, enzyme remains fully functional, and expression in Escherichai coli BL21 for sequencing and Western blot analysis with antibodies
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expression of isozymes TbCPT1, TbCPT2, and TbCPT3 associated with rubber particles in transgenic Taraxacum brevicorniculatum plants
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recombinant expression of C-terminally His6-tagged HRT2 in Escherichia coli, expression of C-terminally His6-tagged HRT1 amd HRT2 in Saccharomyces cerevisiae and Arabidopsis thaliana T87 cultured cells, predominantly in the membrane fraction in yeast cells
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
CPT expression is reduced by laticifer-specific RNA interference, CPT-RNAi
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recombinant expression of HRT2 in Escherichia coli is significantly activated in the presence of a centrifuged latex fraction, resulting in the formation of polyisoprenes corresponding to natural rubber
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
molecular biology
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model for rubber biosynthesis
synthesis
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metal ion concentration regulates rubber initiation rate, biosythetic rate and molecular weight of product