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

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The expected taxonomic range for this enzyme is: Paenarthrobacter nicotinovorans

EC NUMBER
COMMENTARY hide
1.5.3.6
-
RECOMMENDED NAME
GeneOntology No.
(R)-6-hydroxynicotine oxidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(R)-6-hydroxynicotine + H2O + O2 = 1-(6-hydroxypyridin-3-yl)-4-(methylamino)butan-1-one + H2O2
show the reaction diagram
overall reaction
-
-
-
(R)-6-hydroxynicotine + O2 = 5-(N-methyl-4,5-dihydro-1H-pyrrol-2-yl)pyridin-2-ol + H2O2
show the reaction diagram
(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
show the reaction diagram
(1b), spontaneous
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-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Microbial metabolism in diverse environments
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-
Nicotinate and nicotinamide metabolism
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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).
CAS REGISTRY NUMBER
COMMENTARY hide
37233-46-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Arthrobacter nicotinovorans
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
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
Arthrobacter nicotinovorans
-
-
-
-
?
(R)-6-hydroxynicotine + H2O + electron acceptor
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + electron donor
show the reaction diagram
Arthrobacter nicotinovorans
-
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
6-hydroxy-nornicotine + H2O + O2
4-amino-1-(6-aminopyridin-3-yl)-butan-1-one + H2O2
show the reaction diagram
Arthrobacter nicotinovorans
-
-
-
-
?
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
(R)-6-hydroxynicotine + H2O + O2
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one + H2O2
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Arthrobacter nicotinovorans
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molybdate is not required for the induction of 6-hydroxy-D-nicotine
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one
Arthrobacter nicotinovorans
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-
3-(4-aminobutyl)-pyridine
Arthrobacter nicotinovorans
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-
6-hydroxy-L-nicotine
D,L-2-Hydroxynicotine
Arthrobacter nicotinovorans
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-
Dithionitrobenzoic acid
Arthrobacter nicotinovorans
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inhibits holoenzyme formation from apoenzyme and FAD, can be prevented by addition of 2-mercaptoethanol
Phenanthroline
Arthrobacter nicotinovorans
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inactivation above 1 mM
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,6-dichlorophenolindophenol
Arthrobacter nicotinovorans
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the rate of the overall process is higher than with oxygen
glycerate-3-phosphate
Arthrobacter nicotinovorans
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activation of autocatalytical flavinylation
glycerol
Arthrobacter nicotinovorans
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activation of autocatalytical flavinylation of the enzyme at 45% v/v
glycerol-3-phosphate
methylene blue
Arthrobacter nicotinovorans
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the rate of the overall process is higher than with oxygen
phosphoenolpyruvate
Arthrobacter nicotinovorans
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activation of autocatalytical flavinylation, together with FAD protects the enzyme of proteolytic cleavage
Sucrose
Arthrobacter nicotinovorans
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activation of autocatalytical flavinylation of the enzyme at 20%
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.2
6-amino-D-nicotine
Arthrobacter nicotinovorans
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-
0.05 - 0.1
6-hydroxy-D-nicotine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
19.8
6-hydroxy-D-nicotine
Arthrobacter nicotinovorans
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30°C pH 9.2
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.05
1-(6-hydroxypyrid-3-yl)-4-(methylamino)-butan-1-one
Arthrobacter nicotinovorans
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-
0.7
3-(4-aminobutyl)-pyridine
Arthrobacter nicotinovorans
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-
0.02 - 1.5
6-hydroxy-L-nicotine
1.7
D,L-2-Hydroxynicotine
Arthrobacter nicotinovorans
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-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.06
Arthrobacter nicotinovorans
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expressed in Escherichia coli HB101 cells
0.08
Arthrobacter nicotinovorans
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wild type enzyme
22.5
Arthrobacter nicotinovorans
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-
28
Arthrobacter nicotinovorans
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32
Arthrobacter nicotinovorans
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-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
Arthrobacter nicotinovorans
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in 0.1 M Tris-HCl buffer
8.5
Arthrobacter nicotinovorans
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0.1 M glycine-NaOH buffer
9.2
Arthrobacter nicotinovorans
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assay at
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
Arthrobacter nicotinovorans
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-
Manually annotated by BRENDA team
Arthrobacter nicotinovorans
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-
-
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Arthrobacter nicotinovorans
Arthrobacter nicotinovorans
Arthrobacter nicotinovorans
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
48000
Arthrobacter nicotinovorans
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SDS-PAGE
48980
Arthrobacter nicotinovorans
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with FAD, amino acid analysis
50000
Arthrobacter nicotinovorans
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SDS-PAGE
52000
Arthrobacter nicotinovorans
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expression in Escherichia coli maxicells
53000
Arthrobacter nicotinovorans
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sedimentation equilibrium and SDS-PAGE
56000
Arthrobacter nicotinovorans
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fusion protein between 6-hydroxy-D-nicotine oxidase and the mitochondrial targeting sequence of Neurospora crassa F0-ATPase subunit 9
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
Arthrobacter nicotinovorans
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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
Arthrobacter nicotinovorans
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fusion protein with glutathione S-transferase
Arthrobacter nicotinovorans
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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
Arthrobacter nicotinovorans
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
Arthrobacter nicotinovorans
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below this pH inactivation
392461
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
Arthrobacter nicotinovorans
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the ability of building the holoenzyme is abolished in absence of glycerol
45
Arthrobacter nicotinovorans
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inactivation above this temperature
56
Arthrobacter nicotinovorans
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denaturation of the holoenzyme
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, fairly stable at neutral or alkaline pH in presence of 10 mM mercaptoethanol
Arthrobacter nicotinovorans
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
-
Arthrobacter nicotinovorans
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partially
Arthrobacter nicotinovorans
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli HB101
Arthrobacter nicotinovorans
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expressed in Escherichia coli HB101 as a 6-hydroxy-D-nicotine oxidase/glutathione S-transferase fusion protein
Arthrobacter nicotinovorans
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expressed in Escherichia coli JM101
Arthrobacter nicotinovorans
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expressed in Escherichia coli JM109
Arthrobacter nicotinovorans
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expressed in Escherichia coli JM109 as a beta-galactosidase/6-hydroxynicotine oxidase fusion protein
Arthrobacter nicotinovorans
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expressed in Escherichia coli JM109 as a fusion protein with dimethylglycine dehydrogenase and translated into rabbit reticulocyte lysate
Arthrobacter nicotinovorans
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expressed in Escherichia coli JM109; expression is temperature dependent, higher specific activities are found at 30°C than at 37°C
Arthrobacter nicotinovorans
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expressed in Escherichia coli K12 strain HB101, maxicells
Arthrobacter nicotinovorans
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expressed in Escherichia coli S30 wild type and mutants
Arthrobacter nicotinovorans
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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
Arthrobacter nicotinovorans
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expression in Escherichia coli
Arthrobacter nicotinovorans
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C136S
Arthrobacter nicotinovorans
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5% of wild-type activity, slight activation with glycerol-3-phosphate and FAD
C136S/C260S
Arthrobacter nicotinovorans
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no activity, no activation with glycerol-3-phosphate and FAD
C260S
Arthrobacter nicotinovorans
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15% of wild-type activity, no activation with glycerol-3-phosphate and FAD
C433S
Arthrobacter nicotinovorans
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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
Arthrobacter nicotinovorans
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40% of wild-type activity, no activation with glycerol-3-phosphate and FAD
P73A
Arthrobacter nicotinovorans
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unable to bind FAD
R67A
Arthrobacter nicotinovorans
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no enzyme activity, no flavinylation
R67K
Arthrobacter nicotinovorans
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3% activity of wild-type, better flavinylation rate than wild type enzyme
S68A
Arthrobacter nicotinovorans
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80% activity of wild-type, flavinylation