Information on EC 1.14.13.101 - senecionine N-oxygenase

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The expected taxonomic range for this enzyme is: Eukaryota

EC NUMBER
COMMENTARY hide
1.14.13.101
-
RECOMMENDED NAME
GeneOntology No.
senecionine N-oxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
senecionine + NADPH + H+ + O2 = senecionine N-oxide + NADP+ + H2O
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
N-oxidation
oxidation
redox reaction
reduction
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
senecionine N-oxide biosynthesis
-
-
Tropane, piperidine and pyridine alkaloid biosynthesis
-
-
SYSTEMATIC NAME
IUBMB Comments
senecionine,NADPH:oxygen oxidoreductase (N-oxide-forming)
A flavoprotein. NADH cannot replace NADPH. While pyrrolizidine alkaloids of the senecionine and monocrotaline types are generally good substrates (e.g. senecionine, retrorsine and monocrotaline), the enzyme does not use ester alkaloids lacking an hydroxy group at C-7 (e.g. supinine and phalaenopsine), 1,2-dihydro-alkaloids (e.g. sarracine) or unesterified necine bases (e.g. senkirkine) as substrates [1]. Senecionine N-oxide is used by insects as a chemical defense: senecionine N-oxide is non-toxic, but it is bioactivated to a toxic form by the action of cytochrome P-450 oxidase when absorbed by insectivores.
CAS REGISTRY NUMBER
COMMENTARY hide
220581-68-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Amerila phaedra
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Arctia intercalaris
-
-
-
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
no activity in Spodoptera littoralis
-
-
-
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
atropine + NADPH + H+ + O2
atropine N-oxide + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
atropine + NADPH + O2
atropine N-oxide + NADP+ + H2O
show the reaction diagram
21% of the activity with monocrotaline
-
-
?
axillaridine + NADPH + H+ + O2
axillaridine N-oxide + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
axillaridine + NADPH + O2
axillaridine N-oxide + NADP+ + H2O
show the reaction diagram
axillarine + NADPH + H+ + O2
axillarine N-oxide + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
axillarine + NADPH + O2
axillarine N-oxide + NADP+ + H2O
show the reaction diagram
cysteamine + NADPH + H+ + O2
cysteamine N-oxide + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
heliotrine + NADPH + H+ + O2
heliotrine N-oxide + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
heliotrine + NADPH + O2
heliotrine N-oxide + NADP+ + H2O
show the reaction diagram
indicine + NADPH + H+ + O2
indicine N-oxide + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
indicine + NADPH + O2
indicine N-oxide + NADP+ + H2O
show the reaction diagram
lycopsamine + NADPH + O2
lycopsamine N-oxide + NADP+ + H2O
show the reaction diagram
monocrotaline + NADPH + H+ + O2
monocrotaline N-oxide + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
monocrotaline + NADPH + O2
monocrotaline N-oxide + NADP+ + H2O
show the reaction diagram
nicotine + NADPH + H+ + O2
nicotine N-oxide + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
phalaenopsine + NADPH + H+ + O2
phalaenopsine N-oxide + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
phalaenopsine + NADPH + O2
phalaenopsine N-oxide + NADP+ + H2O
show the reaction diagram
19% of the activity with monocrotaline
-
-
?
retronecine + NADPH + H+ + O2
retronecine N-oxide + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
retrorsine + NADPH + H+ + O2
retrosine N-oxide + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
retrorsine + NADPH + O2
retrorsine N-oxide + NADP+ + H2O
show the reaction diagram
rinderine + NADPH + H+ + O2
rinderine N-oxide + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
rinderine + NADPH + O2
rinderine N-oxide + NADP+ + H2O
show the reaction diagram
sarracine + NADPH + H+ + O2
sarracine N-oxide + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
senecionine + NADPH + H+ + O2
senecionine N-oxide + NADP+ + H2O
show the reaction diagram
senecionine + NADPH + O2
senecionine N-oxide + NADP+ + H2O
show the reaction diagram
seneciphylline + NADPH + H+ + O2
seneciphylline N-oxide + NADP+ + H2O
show the reaction diagram
-
second best substrate
-
-
?
seneciphylline + NADPH + O2
seneciphylline N-oxide + NADP+ + H2O
show the reaction diagram
senecivernine + NADPH + H+ + O2
senecivernine N-oxide + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
senecivernine + NADPH + O2
senecivernine N-oxide + NADP+ + H2O
show the reaction diagram
triangularine + NADPH + O2
triangularine N-oxide + NADP+ + H2O
show the reaction diagram
-
60% of the activity with senecionine
-
?
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
senecionine + NADPH + H+ + O2
senecionine N-oxide + NADP+ + H2O
show the reaction diagram
-
most efficient substrate
-
-
?
senecionine + NADPH + O2
senecionine N-oxide + NADP+ + H2O
show the reaction diagram
Q8MP06
-
-
-
?
seneciphylline + NADPH + H+ + O2
seneciphylline N-oxide + NADP+ + H2O
show the reaction diagram
-
second best substrate
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cytochrome c
-
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
guanethidine
-
enhances activity
Metyrapone
-
enhances activity
n-octylamine
-
activity of solubilized enzyme is 1.6fold increased by 0.5 mM n-octylamine
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00983
atropine
-
in 100 mM glycine/NaOH buffer pH 9.0, at 37C
0.0186 - 0.284
heliotrine
0.0029 - 0.0125
monocrotaline
0.0013
NADPH
-
pH 7.0, pH 37C
0.8376
phalaenopsine
-
in 100 mM glycine/NaOH buffer pH 9.0, at 37C
0.00108 - 0.0048
senecionine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.37
atropine
Zonocerus variegatus
-
in 100 mM glycine/NaOH buffer pH 9.0, at 37C
0.12
heliotrine
Zonocerus variegatus
-
in 100 mM glycine/NaOH buffer pH 9.0, at 37C
0.96
monocrotaline
Zonocerus variegatus
-
in 100 mM glycine/NaOH buffer pH 9.0, at 37C
0.13
phalaenopsine
Zonocerus variegatus
-
in 100 mM glycine/NaOH buffer pH 9.0, at 37C
0.99
senecionine
Zonocerus variegatus
-
in 100 mM glycine/NaOH buffer pH 9.0, at 37C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
37.64
atropine
Zonocerus variegatus
-
in 100 mM glycine/NaOH buffer pH 9.0, at 37C
4789
0.454
heliotrine
Zonocerus variegatus
-
in 100 mM glycine/NaOH buffer pH 9.0, at 37C
22428
80.5
monocrotaline
Zonocerus variegatus
-
in 100 mM glycine/NaOH buffer pH 9.0, at 37C
13488
0.155
phalaenopsine
Zonocerus variegatus
-
in 100 mM glycine/NaOH buffer pH 9.0, at 37C
117306
916.7
senecionine
Zonocerus variegatus
-
in 100 mM glycine/NaOH buffer pH 9.0, at 37C
9685
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.3
purified enzyme with senecionine as substrate
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
-
solubilized partially purified enzyme
9
-
in potassium phosphate buffer (10 mM), Tris/HCl buffer (10 mM), and in glycine/NaOH buffer (100 mM)
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40 - 45
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.9
-
determination on an Ampholine PAGplate pH 3.5-9.5 using a horizantal electrophoresis system
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
Amerila phaedra
-
-
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
-
not a microsomal enzyme
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
91200
-
gel filtration
200000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
-
2 * 47793, calculated from amino acid sequence
tetramer
-
4 * 51000, SDS-PAGE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
20 min, solubilized enzyme is completely inactivated in absence of NADPH, preincubation the presence of NADPH has no effect
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni2+-NTA agarose column chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
-
expressing GgPNO without the signal peptide in at least a partially soluble and active form. Betaine is added to the medium and promotes its uptake by osmotic stress by sorbitol. Further improvements are achieved by coexpression with the Escherichia coli chaperones DnaK/DnaJ/GrpE by using the plasmid pRDKJG and by reducing the expression temperature to 4C
expression in active form in Escherichia coli BL21
the heterologous expression results in the formation of an insoluble and inactive protein. Despite the finding that protein solubilization and subsequent renaturation results in an active protein, the specific activity remains low. Active SNO is detected by using an an Sf9 insect cell expression system, but the yield of protein is too low for further biochemical characterization. Using an alignment of senecionine N-oxygenase of Tyria jacobaeae and of flavin-dependent monooxygenase-like sequences of the dipteran species Drosophila melanogaster and Anopheles gambiae, several new flavin-dependent monooxygenase sequences of arctiids and other Lepidoptera are idenified