Information on EC 1.14.13.242 - 3-hydroxy-2-methylpyridine-5-carboxylate monooxygenase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.14.13.242
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RECOMMENDED NAME
GeneOntology No.
3-hydroxy-2-methylpyridine-5-carboxylate monooxygenase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2 = 2-(acetamidomethylene)succinate + NAD(P)+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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-
-
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redox reaction
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-
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reduction
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
vitamin B6 degradation
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Vitamin B6 metabolism
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
3-hydroxy-2-methylpyridine-5-carboxylate,NAD(P)H:oxygen oxidoreductase (ring-opening)
A flavoprotein (FAD).
CAS REGISTRY NUMBER
COMMENTARY hide
37256-69-2
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-methyl-3-hydroxypyridine-5-carboxylic acid + NADH + O2 + H+
2-(acetamidomethylene)succinate + NAD+
show the reaction diagram
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2
2-(acetamidomethylene)succinate + NAD(P)+
show the reaction diagram
-
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + H+ + O2
2-(acetamidomethylene)succinate + NAD+
show the reaction diagram
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + H+ + O2
2-(acetamidomethylene)succinate + NAD+ + H2O
show the reaction diagram
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2
2-(acetamidomethylene)succinate + NAD(P)+
show the reaction diagram
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2
2-(acetamidomethylene)succinate + NAD+
show the reaction diagram
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2 + H+
2-(acetamidomethylene)succinate + NAD+
show the reaction diagram
3-hydroxy-2-methylpyridine-5-carboxylate + NADPH + H+ + O2
2-(acetamidomethylene)succinate + NADP+
show the reaction diagram
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-
-
-
?
5-hydroxynicotinate + NAD(P)H + H+ + O2
? + NAD(P)+
show the reaction diagram
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-
-
-
?
5-hydroxynicotinic acid + NADH + H+ + O2
alpha-(N-formylaminomethylene)succinic acid + NAD+ + H2O
show the reaction diagram
5-hydroxynicotinic acid + NADH + O2
?
show the reaction diagram
5-hydroxynicotinic acid + NADH + O2 + H+
2-(formylamidomethylene)succinate + NAD+
show the reaction diagram
5-pyridoxic acid + NADH + H+ + O2
alpha-(N-acetylaminomethylene)-beta-hydroxymethyl succinic acid + NAD+ + H2O
show the reaction diagram
5-pyridoxic acid + NADH + O2
?
show the reaction diagram
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-
-
-
?
5-pyridoxic acid + NADH + O2 + H+
2-(acetamidomethylene)-beta-hydroxymethyl succinate + NAD+
show the reaction diagram
5% of the activity with 2-methyl-3-hydroxypyridine-5-carboxylic acid
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-
?
N-methyl-5-hydroxynicotinic acid + NADH + O2
?
show the reaction diagram
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-
-
-
?
additional information
?
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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
2-methyl-3-hydroxypyridine-5-carboxylic acid + NADH + O2 + H+
2-(acetamidomethylene)succinate + NAD+
show the reaction diagram
Q988D3
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + H+ + O2
2-(acetamidomethylene)succinate + NAD+
show the reaction diagram
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2
2-(acetamidomethylene)succinate + NAD+
show the reaction diagram
3-hydroxy-2-methylpyridine-5-carboxylate + NADPH + H+ + O2
2-(acetamidomethylene)succinate + NADP+
show the reaction diagram
-
-
-
-
?
5-hydroxynicotinic acid + NADH + O2 + H+
2-(formylamidomethylene)succinate + NAD+
show the reaction diagram
Q988D3
about 100% of the activity with 2-methyl-3-hydroxypyridine-5-carboxylic acid
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-
?
5-pyridoxic acid + NADH + O2 + H+
2-(acetamidomethylene)-beta-hydroxymethyl succinate + NAD+
show the reaction diagram
Q988D3
5% of the activity with 2-methyl-3-hydroxypyridine-5-carboxylic acid
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-
?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NAD(P)H
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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no metal ion required
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-deaza-FAD
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5-hydroxynicotinic acid
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5-pyridoxic acid
6-Methylnicotinate
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6-methylnicotinic acid
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competitive with 3-hydroxy-2-methylpyridine-5-carboxylate
NAD+
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binds competitively with O2, but not with NADH
p-chloromercuribenzoate
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; 0.05 mM, quick and complete inhibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0254
3-Hydroxy-2-methylpyridine-5-carboxylate
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pH 8.0, 25C
0.045 - 0.21
5-hydroxynicotinic acid
0.00056 - 0.0011
N-methyl-5-hydroxynicotinic acid
0.0054 - 0.47
NADH
0.0059 - 0.148
O2
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
11.8
3-Hydroxy-2-methylpyridine-5-carboxylate
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pH 8.0, 25C
0.05 - 1300
5-hydroxynicotinic acid
additional information
additional information
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.1 - 22000
5-hydroxynicotinic acid
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00046
1-deaza-FAD
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0.023
5-pyridoxic acid
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.48
5-hydroxynicotinic acid
Mesorhizobium loti
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wild type enzyme, at pH 8.0 and 25C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.28
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pH 8.0, 25C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8
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7
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substrate 3-hydroxy-2-methylpyridine-5-carboxylate
8
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substrate 5-hydroxynicotinic acid
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
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79% of maximum activity
40
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82% of maximum activity
PDB
SCOP
CATH
ORGANISM
UNIPROT
Mesorhizobium japonicum (strain LMG 29417 / CECT 9101 / MAFF 303099)
Mesorhizobium japonicum (strain LMG 29417 / CECT 9101 / MAFF 303099)
Mesorhizobium japonicum (strain LMG 29417 / CECT 9101 / MAFF 303099)
Mesorhizobium japonicum (strain LMG 29417 / CECT 9101 / MAFF 303099)
Mesorhizobium japonicum (strain LMG 29417 / CECT 9101 / MAFF 303099)
Mesorhizobium japonicum (strain LMG 29417 / CECT 9101 / MAFF 303099)
Mesorhizobium japonicum (strain LMG 29417 / CECT 9101 / MAFF 303099)
Mesorhizobium japonicum (strain LMG 29417 / CECT 9101 / MAFF 303099)
Mesorhizobium japonicum (strain LMG 29417 / CECT 9101 / MAFF 303099)
Mesorhizobium japonicum (strain LMG 29417 / CECT 9101 / MAFF 303099)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
41700
4 * 41700, calculation from nucleotide sequence
43000
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4 * 43000, SDS-PAGE
160000
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equilibrium sedimentation
164000
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gel fitlration
166000
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equilibrium sedimentation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
crystal structure, the tetramer may have been disrupted
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
density functional theory/molecular mechanics calculations show that the active-site residues Arg211 and Tyr223 have a minor effect on the reaction, while the peptide bond of Pro295-Ala296, the side chain of Tyr82 and several crystal water molecules affect the reaction energy profile considerably. The ring-opening pathway, in which an epoxy transition state is formed, is more favored than the direct C2-C3 cleavage pathway. Both the reaction barriers for the hydroxylation and the ring-opening pathways are sensitive to the quantum mechanics/molecular mechanics partitioning
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hanging drop vapor diffusion method, using 8% (w/v) PEG 8000, 0.1 M Tris-HCl, pH 8.5; structure of enzyme with and without substrates 2-methyl-3-hydroxypyridine-5-carboxylic acid, 5-hydroxynicotinic acid and 5-pyridoxic acid, and of mutant Y270F. Residue Y270 is located in the active site
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
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stable for 10 min
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
the enzyme is very sensitive to oxidation, it loses activity rapidly in absence of mercaptoethanol even at 4C, it is further stabilized in presence of high concentrations of glycerol or by serum albumin
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439056
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 50% glycerol, 0.1% 2-mercaptoethanol, 1 month
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Q-Sepharose column chromatography and G-25 Sephadex gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli BL21(DE3) cells
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expression in Escherichia coli
His-tag, expressed in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Y270A
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less than 0.1% of the pyridine ring cleavage capacity of wild-type; the mutant shows reduced ring opening activity but increased NADH oxidation activity compared to the wild type enzyme
Y270F
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about 1.5% of the pyridine ring cleavage capacity of wild-type; the mutant shows reduced ring opening activity but increased NADH oxidation activity compared to the wild type enzyme
Y223E
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the mutant shows reduced activity compared to the wild type enzyme
Y223F
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the mutant shows reduced activity compared to the wild type enzyme
Y223H
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the mutant shows reduced activity compared to the wild type enzyme
Y223T
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the mutant shows reduced activity compared to the wild type enzyme
Y82F
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the mutant shows reduced activity compared to the wild type enzyme
Y82H
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the mutant shows reduced activity compared to the wild type enzyme
Y82R
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the mutant shows reduced activity compared to the wild type enzyme