Information on EC 1.5.3.6 - (R)-6-hydroxynicotine oxidase

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The expected taxonomic range for this enzyme is: Arthrobacter nicotinovorans

EC NUMBER
COMMENTARY
1.5.3.6
-
RECOMMENDED NAME
GeneOntology No.
(R)-6-hydroxynicotine oxidase
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(R)-6-hydroxynicotine + H2O + O2 = 1-(6-hydroxypyridin-3-yl)-4-(methylamino)butan-1-one + H2O2
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Nicotinate and nicotinamide metabolism
-
-
Microbial metabolism in diverse environments
-
-
SYSTEMATIC NAME
IUBMB Comments
(R)-6-hydroxynicotine:oxygen oxidoreductase
A flavoprotein (FAD).
SYNONYMS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6-HDNO
-
-
-
-
6-hydroxy-D-nicotine oxidase
-
-
-
-
6-hydroxy-D-nicotine oxidase
-
-
D-6-hydroxynicotine oxidase
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
37233-46-8
-
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
formerly Arthrobacter oxidans; strain DSM 420
-
-
Manually annotated by BRENDA team
strain DSM 420
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(R)-6-aminonicotine + H2O + O2
1-(-aminopyridin-3-yl)-4-methylamino-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + electron acceptor
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + electron donor
show the reaction diagram
-
as electron acceptors methylene blue and 2,6-dichlorophenolindophenol aerobically and anaerobically
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
transcient product is 6-hydroxy-N-methylmyosmine that hydrolyses spontaneously
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
transcient product is 6-hydroxy-N-methylmyosmine that hydrolyses spontaneously
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
transcient product is 6-hydroxy-N-methylmyosmine that hydrolyses spontaneously
?
6-hydroxy-nornicotine + H2O + O2
4-amino-1-(6-aminopyridin-3-yl)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
additional information
?
-
-
-
-
-
-
additional information
?
-
-
enzyme production is induced by growing cells in D,L-nicotine as only source of carbon and nitrogen
-
-
-
additional information
?
-
-
it is synthesized only during the late logarithmic or early stationary phases of growth
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
transcient product is 6-hydroxy-N-methylmyosmine that hydrolyses spontaneously
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
-
-
transcient product is 6-hydroxy-N-methylmyosmine that hydrolyses spontaneously
?
additional information
?
-
-
-
-
-
-
additional information
?
-
-
enzyme production is induced by growing cells in D,L-nicotine as only source of carbon and nitrogen
-
-
-
additional information
?
-
-
it is synthesized only during the late logarithmic or early stationary phases of growth
-
-
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
FAD
-
an adenylate-binding domain is absent
FAD
-
covalently bound to a histidine
FAD
-
bound as N6-(2-aminoethyl)-FAD 50% activity of the wild type enzyme; bound as N6-(6-carboxyhexyl)-FAD 60% activity of the wild type enzyme; FAD is bound via an (8alpha)-isoalloxazine-(N3)histidyl linkage; formation of the bond to FAD proceeds autocatalytically
FAD
-
bound as 8-(N-acetylcysteinyl)FAD 80% activity of the wild type enzyme, H71C mutant; formation of the bond to FAD proceeds autocatalytically
FAD
-
flavinylation can be performed with purified apoenzyme post-translationally with chaperonins ATP, GroEL and GroES
FAD
-
covalently bound to a histidine; formation of the bond to FAD proceeds autocatalytically
FAD
-
FAD is bound via an (8alpha)-isoalloxazine-(N3)histidyl linkage
FAD
-
covalently bound to a histidine; one mol FAD per mol enzyme
FAD
-
covalently bound to a histidine; FAD is bound via an (8alpha)-isoalloxazine-(N3)histidyl linkage
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
molybdate is not required for the induction of 6-hydroxy-D-nicotine
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one
-
-
3-(4-aminobutyl)-pyridine
-
-
6-hydroxy-L-nicotine
-
competitive inhibitor
6-hydroxy-L-nicotine
-
at 0.12 M 50% inhibition
6-hydroxy-L-nicotine
-
-
D,L-2-Hydroxynicotine
-
-
Dithionitrobenzoic acid
-
inhibits holoenzyme formation from apoenzyme and FAD, can be prevented by addition of 2-mercaptoethanol
Phenanthroline
-
inactivation above 1 mM
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2,6-dichlorophenolindophenol
-
the rate of the overall process is higher than with oxygen
glycerate-3-phosphate
-
activation of autocatalytical flavinylation
glycerol
-
activation of autocatalytical flavinylation of the enzyme at 45% v/v
glycerol-3-phosphate
-
activation of autocatalytical flavinylation of the enzyme in rabbit reticulocyte lysate
glycerol-3-phosphate
-
activation of autocatalytical flavinylation
methylene blue
-
the rate of the overall process is higher than with oxygen
phosphoenolpyruvate
-
activation of autocatalytical flavinylation, together with FAD protects the enzyme of proteolytic cleavage
Sucrose
-
activation of autocatalytical flavinylation of the enzyme at 20%
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.2
6-amino-D-nicotine
-
-
0.05
6-Hydroxy-D-nicotine
-
-
0.1
6-Hydroxy-D-nicotine
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
19.8
6-Hydroxy-D-nicotine
Arthrobacter nicotinovorans
-
30C pH 9.2
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.05
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one
-
-
0.7
3-(4-aminobutyl)-pyridine
-
-
0.02
6-hydroxy-L-nicotine
-
-
1.5
6-hydroxy-L-nicotine
-
-
1.7
D,L-2-Hydroxynicotine
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.06
-
expressed in Escherichia coli HB101 cells
0.08
-
wild type enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
8
-
in 0.1 M Tris-HCl buffer
8.5
-
0.1 M glycine-NaOH buffer
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
48000
-
SDS-PAGE
392450
48980
-
with FAD, amino acid analysis
392430
50000
-
SDS-PAGE
392435
52000
-
expression in Escherichia coli maxicells
392456, 392457
53000
-
sedimentation equilibrium and SDS-PAGE
392461
56000
-
fusion protein between 6-hydroxy-D-nicotine oxidase and the mitochondrial targeting sequence of Neurospora crassa F0-ATPase subunit 9
392445
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a comparison of the substrate-binding modes of 6-hydroxy-D-nicotine oxidase and 6-hydroxy-L-nicotine oxidase, EC 1.5.3.5, based on models of complexes with the D-substrate, suggests that the two enzymes orient the enantiomeric substrates in mirror symmetry with respect to the plane of the flavin
-
fusion protein with glutathione S-transferase
-
hanging dropp method, the crystal structure of 6-hydroxy-D-nicotine oxidase is solved by X-ray diffraction analysis in three crystal forms at resolutions up to 1.9 A
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6
-
below this pH inactivation
392461
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
-
the ability of building the holoenzyme is abolished in absence of glycerol
392451
45
-
inactivation above this temperature
392461
56
-
denaturation of the holoenzyme
392453
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, fairly stable at neutral or alkaline pH in presence of 10 mM mercaptoethanol
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli HB101
-
expressed in Escherichia coli HB101 as a 6-hydroxy-D-nicotine oxidase/glutathione S-transferase fusion protein
-
expressed in Escherichia coli JM101
-
expressed in Escherichia coli JM109
-
expressed in Escherichia coli JM109 as a beta-galactosidase/6-hydroxynicotine oxidase fusion protein
-
expressed in Escherichia coli JM109 as a fusion protein with dimethylglycine dehydrogenase and translated into rabbit reticulocyte lysate
-
expressed in Escherichia coli JM109; expression is temperature dependent, higher specific activities are found at 30C than at 37C
-
expressed in Escherichia coli K12 strain HB101, maxicells
-
expressed in Escherichia coli S30 wild type and mutants
-
expressed in Saccharomyces cerevisiae as a fusion protein between 6-hydroxy-D-nicotine oxidase and the mitochondrial targeting sequence of Neurospora crassa F0-ATPase subunit 9 (Su9-6-HDNO) and translated into rabbit reticulocyte lysate
-
expression in Escherichia coli
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C136S
-
5% of wild-type activity, slight activation with glycerol-3-phosphate and FAD
C136S/C260S
-
no activity, no activation with glycerol-3-phosphate and FAD
C260S
-
15% of wild-type activity, no activation with glycerol-3-phosphate and FAD
C433S
-
40% of wild-type activity, 50% activation with glycerol-3-phosphate and FAD; deletions F448 and R449, no activity, no activation with glycerol-3-phosphate and FAD
C59S
-
40% of wild-type activity, no activation with glycerol-3-phosphate and FAD
H71C
-
only residual enzyme activity
H71C
-
80% activity of wild-type
P73A
-
unable to bind FAD
R67A
-
no enzyme activity, no flavinylation
R67K
-
3% activity of wild-type, better flavinylation rate than wild type enzyme
S68A
-
80% activity of wild-type, flavinylation