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Information on EC 1.5.3.6 - (R)-6-hydroxynicotine oxidase
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EC Tree
1.5.3.6 (R)-6-hydroxynicotine oxidaseIUBMB Comments
A flavoprotein (FAD). The enzyme, which participates in nicotine degradation, is specific for (R) isomer of 6-hydroxynicotine, derived from the uncommon (R)-nicotine. The bacterium Arthrobacter nicotinovorans, in which this enzyme was originally discovered, has a different enzyme that catalyses a similar reaction with the (S)-isomer (cf. EC 1.5.3.5, (S)-6-hydroxynicotine oxidase).
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Word Map
- 1.5.3.6
- fad
- arthrobacter
- oxidans
-
flavinylation
-
holoenzyme
- flavin
- flavoproteins
- 6-hydroxy-l-nicotine
-
flavoenzymes
-
apoenzyme
- nicotinovorans
-
nicotine-degrading
- l-nicotine
- dimethylglycine
-
glycerol-3-p
-
cotranslational
-
fad-binding
-
l-enantiomer
The enzyme appears in viruses and cellular organisms
Reaction Schemes
Synonyms
6-hdno, 6-hydroxy-d-nicotine oxidase, d-6-hydroxynicotine oxidase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
6-HDNO
-
-
-
-
6-hydroxy-D-nicotine oxidase
D-6-hydroxynicotine oxidase
-
-
-
-
6-hydroxy-D-nicotine oxidase
-
-
-
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(R)-6-hydroxynicotine + H2O + O2 = 1-(6-hydroxypyridin-3-yl)-4-(methylamino)butan-1-one + H2O2
overall reaction
-
-
-
(R)-6-hydroxynicotine + O2 = 5-(N-methyl-4,5-dihydro-1H-pyrrol-2-yl)pyridin-2-ol + H2O2
(1a)
-
-
-
5-(N-methyl-4,5-dihydro-1H-pyrrol-2-yl)pyridin-2-ol + H2O = 1-(6-hydroxypyridin-3-yl)-4-(methylamino)butan-1-one
(1b), spontaneous
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
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PATHWAY SOURCE
PATHWAYS
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SYSTEMATIC NAME
IUBMB Comments
(R)-6-hydroxynicotine:oxygen oxidoreductase
A flavoprotein (FAD). The enzyme, which participates in nicotine degradation, is specific for (R) isomer of 6-hydroxynicotine, derived from the uncommon (R)-nicotine. The bacterium Arthrobacter nicotinovorans, in which this enzyme was originally discovered, has a different enzyme that catalyses a similar reaction with the (S)-isomer (cf. EC 1.5.3.5, (S)-6-hydroxynicotine oxidase).
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CAS REGISTRY NUMBER
COMMENTARY
37233-46-8
-
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(R)-4-(1-methylpyrrolidine-2-yl)phenol + H2O + O2
? + H2O2
Q8GAG1
-
-
-
?
(R)-6-aminonicotine + H2O + O2
1-(-aminopyridin-3-yl)-4-methylamino-butan-1-one + H2O2
-
-
-
-
?
(R)-6-chloronicotine + H2O + O2
4-amino-1-(6-chloropyridin-3-yl)-butan-1-one + H2O2
Q8GAG1
-
-
-
?
(R)-6-hydroxynicotine + H2O + electron acceptor
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + electron donor
-
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
(R)-6-hydroxynornicotine + H2O + O2
4-amino-1-(6-hydroxypyridin-3-yl)-butan-1-one + H2O2
Q8GAG1
-
-
-
?
(R)-nicotine + H2O + O2
1-(pyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
Q8GAG1
-
-
-
?
6-hydroxy-nornicotine + H2O + O2
4-amino-1-(6-hydroxypyridin-3-yl)-butan-1-one + H2O2
-
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
-
-
392431, 392434, 392435, 392436, 392438, 392439, 392440, 392441, 392443, 392445, 392446, 392447, 392449, 392450, 392451, 392452, 392453, 392454, 392456, 392457, 392460, 392461, 392462
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
Q8GAG1
-
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
-
-
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
-
-
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
-
-
transcient product is 6-hydroxy-N-methylmyosmine that hydrolyses spontaneously
?
additional information
?
-
-
enzyme production is induced by growing cells in D,L-nicotine as only source of carbon and nitrogen
-
-
?
additional information
?
-
-
enzyme production is induced by growing cells in D,L-nicotine as only source of carbon and nitrogen
-
-
?
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
-
-
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
-
-
392431, 392434, 392435, 392436, 392438, 392439, 392440, 392441, 392443, 392445, 392446, 392447, 392449, 392450, 392451, 392452, 392453, 392454, 392456, 392457, 392460, 392461, 392462
-
-
?
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
-
-
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
-
-
transcient product is 6-hydroxy-N-methylmyosmine that hydrolyses spontaneously
?
additional information
?
-
-
enzyme production is induced by growing cells in D,L-nicotine as only source of carbon and nitrogen
-
-
?
additional information
?
-
-
enzyme production is induced by growing cells in D,L-nicotine as only source of carbon and nitrogen
-
-
?
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
-
-
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
FAD
-
-
FAD
-
bound as N6-(2-aminoethyl)-FAD 50% activity of the wild type enzyme
FAD
-
FAD is bound via an (8alpha)-isoalloxazine-(N3)histidyl linkage
FAD
-
bound as 8-(N-acetylcysteinyl)FAD 80% activity of the wild type enzyme, H71C mutant
FAD
-
formation of the bond to FAD proceeds autocatalytically
FAD
-
bound as N6-(6-carboxyhexyl)-FAD 60% activity of the wild type enzyme
FAD
-
flavinylation can be performed with purified apoenzyme post-translationally with chaperonins ATP, GroEL and GroES
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
molybdate is not required for the induction of 6-hydroxy-D-nicotine
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Dithionitrobenzoic acid
-
inhibits holoenzyme formation from apoenzyme and FAD, can be prevented by addition of 2-mercaptoethanol
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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
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%
glycerol-3-phosphate
-
activation of autocatalytical flavinylation of the enzyme in rabbit reticulocyte lysate
glycerol-3-phosphate
-
activation of autocatalytical flavinylation
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.36
(R)-4-(1-methylpyrrolidine-2-yl)phenol
Q8GAG1
wild-type, pH 8, 25°C
-
0.83
(R)-6-hydroxynornicotine
Q8GAG1
mutant K348M, pH 8, 25°C
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.5
(R)-4-(1-methylpyrrolidine-2-yl)phenol
Q8GAG1
mutant E352Q, pH 8, 25°C
-
5.4
(R)-6-hydroxynornicotine
Q8GAG1
mutant K348M, pH 8, 25°C
-
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
4
(R)-4-(1-methylpyrrolidine-2-yl)phenol, (R)-6-chloronicotine
Q8GAG1
wild-type, pH 8, 25°C
-
6.5
(R)-6-hydroxynornicotine
Q8GAG1
mutant K348M, pH 8, 25°C
-
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
392431, 392435, 392436, 392438, 392439, 392440, 392443, 392445, 392446, 392447, 392449, 392460, 392461, 392462, 675353, 712752
-
-
brenda
formerly Arthrobacter oxidans
-
-
brenda
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
Q8GAG1
a network of hydrogen-bonded residues in the active site is involved in binding the neutral form of the amine substrate, followed by the transfer of a hydride from the amine to the flavin. There is no solvent isotope effect on the kcat/Kamine value with either (R)-6-hydroxynicotine or the slower substrate (R)-6-hydroxynornicotine. Only the neutral form of the substrate and the correctly protonated form of the enzyme bind. The active-site residues Lys348, Glu350, and Glu352 are all properly positioned for substrate binding
Show AA Sequence and Transmembrane Helices (285 entries)
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Please use the AA Sequence and Transmembrane Helices Search for a specific query.
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
56000
-
fusion protein between 6-hydroxy-D-nicotine oxidase and the mitochondrial targeting sequence of Neurospora crassa F0-ATPase subunit 9
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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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
-
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
-
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PROTEIN VARIANTS
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
C59S
-
40% of wild-type activity, no activation with glycerol-3-phosphate and FAD
E350A
Q8GAG1
substitution decreases the kcat/Kamine value by about 20fold
E350Q
Q8GAG1
substitution decreases the kcat/Kamine value by about 220fold
E352Q
Q8GAG1
substitution decreases the kcat/Kamine value about 3800fold
H71C
K348M
Q8GAG1
substitution has only a small effect on the kinetic parameters
R67K
-
3% activity of wild-type, better flavinylation rate than wild type enzyme
C433S
-
40% of wild-type activity, 50% activation with glycerol-3-phosphate and FAD
C433S
-
deletions F448 and R449, no activity, no activation with glycerol-3-phosphate and FAD
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
-
the ability of building the holoenzyme is abolished in absence of glycerol
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, fairly stable at neutral or alkaline pH in presence of 10 mM mercaptoethanol
-
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli HB101 as a 6-hydroxy-D-nicotine oxidase/glutathione S-transferase fusion protein
-
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 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 is temperature dependent, higher specific activities are found at 30°C than at 37°C
-
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Schenk, S.; Hoelz, A.; Krauss, B.; Decker, K.
Gene structures and properties of enzymes of the plasmid-encoded nicotine catabolism of Arthrobacter nicotinovorans
J. Mol. Biol.
284
1323-1339
1998
Paenarthrobacter nicotinovorans
brenda
Grether-Beck, S.; Igloi, G.L.; Pust, S.; Schilz, E.; Decker, K.; Brandsch, R.
Structural analysis and molybdenum-dependent expression of the pAO1-encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans
Mol. Microbiol.
13
929-936
1994
Paenarthrobacter nicotinovorans
brenda
Brandsch, R.; Hinkkanen, A.E.; Mauch, L.; Nagursky, H.; Decker, K.
6-Hydroxy-D-nicotine oxidase of Arthrobacter oxidans. Gene structure of the flavoenzyme and its relationship to 6-hydroxy-L-nicotine oxidase
Eur. J. Biochem.
167
315-320
1987
Paenarthrobacter nicotinovorans
brenda
Swafford, J.R.; Reeves, H.C.; Brandsch, R.
Localization of the enantiozymes of 6-hydroxy-nicotine oxidase in Arthrobacter oxidans by electron immunochemistry
J. Bacteriol.
163
792-795
1985
Paenarthrobacter nicotinovorans
brenda
Hinkkanen, A.; Lilius, E.M.; Nowack, J.; Maas, R.; Decker, K.
Purification of the flavoproteins 6-hydroxy-D- and 6-hydroxy-L-nicotine oxidase using hydrophobic affinity chromatography
Hoppe-Seyler's Z. Physiol. Chem.
364
801-806
1983
Paenarthrobacter nicotinovorans
brenda
Decker, K.; Dai, V.D.; Mhler, H.; Bruhmuller, M.
D- and L-6-hydroxynicotine oxidase, enantioenzymes of Arthrobacter oxidans
Z. Naturforsch. B
27
1072-1073
1972
Paenarthrobacter nicotinovorans
brenda
Dai, V.D.; Decker, K.; Sund, H.
Purification and properties of L-6-hydroxynicotine oxidase
Eur. J. Biochem.
4
95-102
1968
Paenarthrobacter nicotinovorans
brenda