Information on EC 1.13.11.87 - endo-cleaving rubber dioxygenase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.13.11.87
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RECOMMENDED NAME
GeneOntology No.
endo-cleaving rubber dioxygenase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Cleavage of cis-1,4-polyisoprene polymers into a mixture of compounds, including a C20 compound ((4Z,8Z,12Z,16Z,20Z,24Z)-4,8,12,16,20,24-hexamethyl-28-oxononacosa-4,8,12,16,20,24-hexaenal), a C25 compound ((4Z,8Z,12Z,16Z,20Z)-4,8,12,16,20-pentamethyl-24-oxopentacosa-4,8,12,16,20-pentaenal), a C30 compound ((4Z,8Z,12Z,16Z)-4,8,12,16-tetramethyl-20-oxohenicosa-4,8,12,16-tetraenal), and larger isoprenologes such as C35, C40, C45, and higher analogues
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
rubber degradation II
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SYSTEMATIC NAME
IUBMB Comments
cis-1,4-polyisoprene:oxygen dioxygenase (endo-cleaving)
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
cis-1,4-polyisoprene + O2
?
show the reaction diagram
poly(cis-1,4-isoprene) + O2
(4Z,8Z)-4,8-dimethyl-12-oxotrideca-4,8-dienal
show the reaction diagram
12-oxo-4,8-dimethyltrideca-4,8-diene-1-al is the main cleavage product in the absence of (18)O-compounds. Incorporation of one (18)O atom in 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al is found if the cleavage reaction is performed in the presence of (18)O2 and H2(16)O. Incubation of poly(cis-1,4-isoprene) (with RoxA) or of isolated unlabeled 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (without RoxA) with H2(18)O in the presence of (16)O2 indicates that the carbonyl oxygen atoms of 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al is significantly exchanged with oxygen atoms derived from water. The isotope exchange is avoided by simultaneous enzymatic reduction of both carbonyl functions of 12-Oxo-4,8-dimethyltrideca-4,8-diene-1-al to the corresponding dialcohol 12-hydroxy-4,8-dimethyltrideca-4,8-diene-1-ol during RoxA-mediated in vitro cleavage of poly(cis-1,4-isoprene)
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?
poly(cis-1,4-isoprene) + O2
(4Z,8Z,12Z,16Z,20Z,24Z)-4,8,12,16,20,24-hexamethyl-28-oxononacosa-4,8,12,16,20,24-hexaenal + (4Z,8Z,12Z,16Z,20Z)-4,8,12,16,20-pentamethyl-24-oxopentacosa-4,8,12,16,20-pentaenal + (4Z,8Z,12Z,16Z)-4,8,12,16-tetramethyl-20-oxohenicosa-4,8,12,16-tetraenal + ?
show the reaction diagram
the enzyme cleaves cis-1,4-polyisoprene polymers into a mixture of C20, C25, and higher oligo-isoprenoids with terminal aldehyde and keto groups were identified
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?
poly(cis-1,4-isoprene) + O2
?
show the reaction diagram
rubber + O2
?
show the reaction diagram
rubber latex + O2
(4Z,8Z)-4,8-dimethyl-12-oxotrideca-4,8-dienal
show the reaction diagram
RoxA cleaves rubber by a dioxygenase mechanism
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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
cis-1,4-polyisoprene + O2
?
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cytochrome
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b-type cytochrome. The haem group of LcpK30 is ligated to the polypeptide by a proximal histidine (His198) and by a lysine residue (Lys167) as the distal axial ligand
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additional information
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no indication for the presence of iron, other metals, or cofactors in Lcp
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe3+
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purified Lcp contains an oxidized heme-Fe3+
additional information
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no indication for the presence of iron, other metals, or cofactors in Lcp
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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1 mM, 20% inhibition
alpha-tocopherol
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1 mM, 40% inhibition
ascorbate
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1 mM, 20% inhibition
carotene
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1 mM, 40% inhibition
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CO
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oxygen consumption by RoxB is inhibited when carbon monoxide is added to an ongoing polyisoprene cleavage reaction
Diethyl dithiocarbamate
82 % inhibition. No effect on the UV(vis) spectrum of the enzyme and this excludes a direct effect of the inhibitor at the heme site
diethyldithiocarbamate
Diethylthiocarbamate
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1 mM, 56% inhibition
Dithionite
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1 mM, 15% inhibition
dithiothreitol
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10 mM, 90% inhibition
EDTA
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1 mM, 30% inhibition
ethanol
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5%, 10% inhibition
ethyl xanthogenate
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1 mM, 7% inhibition
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imidazole
Phenanthroline
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1 mM, 10% inhibition
Pyridine
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binding of this compounds to the active heme site inhibits the enzyme
squalene
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1 mM, 10% inhibition
additional information
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.5
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23°C. pH 7.0
4.5
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23°C, pH and temperature not specified in the publication
4.6
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37°C, pH 7.0
4.8
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23°C, pH not specified in the publication
5.7
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30°C, pH and temperature not specified in the publication
6.4
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37°C, pH and temperature not specified in the publication
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9
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pH 5.0: about 80% of maximal activity, pH 9.0: about 70% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40000
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SDS-PAGE
42200
mature protein, SDS-PAGE
45200
preprotein, SDS-PAGE
46800
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His-tagged enzyme, SDS-PAGE
48000
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SDS-PAGE
70000
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SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting-drop vapour diffusion method
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23
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Lcp activity decreases by incubation at 23°C by 70% within the 22 h of incubation time
37
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Lcp activity decreases by incubation at 37°C by 80% within the 22 h of incubation time
61.5
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melting point
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
almost stable during storage on ice
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in a DELTAroxA35Y/DELTAroxB35Y background of Steroidobacter cummioxidans 35Y
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expression in Escherichia coli
expression of Lcp of Streptomyces sp. K30 in a DELTAroxA background of Xanthomonas sp. strain 35Y
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extression in Streptomyces lividans TK23
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heterologous expression of the NVL3 lcp in Escherichia coli BL21(DE3)
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when Lcp from Streptomyces sp. strain K30 is heterologously expressed in strains TK23 and TK24 of Streptomyces lividans and a strain of Saccharopolyspora erythraea with plasmid pIJ6021::lcp, the recombinant strains acquire the ability to cleave synthetic poly(cis-1,4-isoprene)
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
basal expression of Lcp in glucose-grown cells. Transcription of lcp is induced in the presence of poly(cis-1,4-isoprene). Expression of lcp at a low level is required for sensing the polymer in the medium. Rubber degradation products may then induce the transcription of genes coding for enzymes catalyzing the later steps of poly(cis-1,4-isoprene) degradation and the transcription of lcp itself
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expression of the roxA gene is upregulated during growth on polyisoprene
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strong transcriptional induction of the lcp gene in NVL3 in the presence of synthetic poly(cis-1,4-isoprene)
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H198A
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mutant protein is stable, contains no heme group and is inactive
H203A
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mutant enzyme reveals wild-type properties
H232A
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mutant enzyme reveals wild-type properties
H259A
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mutant enzyme reveals wild-type properties
H266A
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mutant enzyme reveals wild-type properties
K167A
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specific activity of polyisoprene oxidation is reduced to 20%, the product spectrum is unchanged
K167H
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specific activity of polyisoprene oxidation is reduced to 12%, the product spectrum is unchanged
R195A
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mutant enzyme is colorless and instable
R202A
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mutant enzyme is colorless and instable
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis