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Enzyme Nomenclature
Information on EC 1.14.13.242 - 3-hydroxy-2-methylpyridine-5-carboxylate monooxygenase
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1.14.13.242 3-hydroxy-2-methylpyridine-5-carboxylate monooxygenaseIUBMB Comments
Contains FAD. The enzyme, characterized from the bacteria Pseudomonas sp. MA-1 and Mesorhizobium loti, participates in the degradation of pyridoxine (vitamin B6). Although the enzyme was initially thought to be a dioxygenase, oxygen-tracer experiments have shown that it is a monooxygenase, incorporating only one oxygen atom from molecular oxygen. The second oxygen atom that is incorporated into the product originates from a water molecule, which is regenerated during the reaction and thus does not show up in the reaction equation.
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Word Map
- 1.14.13.242
- flavin
- flavoproteins
- fad
- hydroxylases
-
mesorhizobium
-
flavoenzyme
-
electrophilic
- loti
-
acyclic
-
flavin-dependent
-
ring-cleavage
-
hydride
- isoalloxazine
The enzyme appears in viruses and cellular organisms
Reaction Schemes
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
2-methyl-3-hydroxypyridine 5-carboxylic acid dioxygenase
-
-
-
-
2-methyl-3-hydroxypyridine 5-carboxylic acid oxygenase
2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase
3-hydroxy-2-methylpyridine carboxylate dioxygenase
-
-
-
-
3-hydroxy-2-methylpyridinecarboxylate dioxygenase
-
-
misleading
-
EC 1.14.12.4
-
-
formerly
-
methylhydroxypyridine carboxylate dioxygenase
-
-
-
-
methylhydroxypyridinecarboxylate oxidase
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-
-
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MHPCO
mlr6788
2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2 = 2-(acetamidomethylidene)succinate + NAD(P)+
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2 = 2-(acetamidomethylidene)succinate + NAD(P)+
reaction proceeds in a concerted fashion via a ternary complex of oxygenase, NADH and 3-hydroxy-2-methylpyridine-5-carboxylate
-
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2 = 2-(acetamidomethylidene)succinate + NAD(P)+
ordered mechanism in which 3-hydroxy-2-methylpyridine-5-carboxylate binds first, followed by NADH. The first product NAD+ is then released, followed by oxygen binding and finally release of the oxygenated and reduced cleavage product 2-(acetamidomethylene)succinate
-
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2 = 2-(acetamidomethylidene)succinate + NAD(P)+
the enzyme catalyzes both a classical hydroxylation and a subsequent unique hydrolysis of the hydroxylated substrate to yield the acyclic product
-
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2 = 2-(acetamidomethylidene)succinate + NAD(P)+
the binding proceeds in two steps: an enzyme-substrate complex initially formed is followed by a ligand-induced isomerization
-
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2 = 2-(acetamidomethylidene)succinate + NAD(P)+
oxygenation reaction occurs via an electrophilic aromatic substitution mechanism
-
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2 = 2-(acetamidomethylidene)succinate + NAD(P)+
-
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
-
-
-
-
redox reaction
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-
-
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reduction
-
-
-
-
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PATHWAY SOURCE
PATHWAYS
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SYSTEMATIC NAME
IUBMB Comments
3-hydroxy-2-methylpyridine-5-carboxylate,NAD(P)H:oxygen oxidoreductase (ring-opening)
Contains FAD. The enzyme, characterized from the bacteria Pseudomonas sp. MA-1 and Mesorhizobium loti, participates in the degradation of pyridoxine (vitamin B6). Although the enzyme was initially thought to be a dioxygenase, oxygen-tracer experiments have shown that it is a monooxygenase, incorporating only one oxygen atom from molecular oxygen. The second oxygen atom that is incorporated into the product originates from a water molecule, which is regenerated during the reaction and thus does not show up in the reaction equation.
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CAS REGISTRY NUMBER
COMMENTARY
37256-69-2
-
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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
2-methyl-3-hydroxypyridine-5-carboxylic acid + NADH + O2 + H+
2-(acetamidomethylene)succinate + NAD+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2
2-(acetamidomethylene)succinate + NAD(P)+
-
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2
2-(acetamidomethylidene)succinate + NAD(P)+
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + H+ + O2
2-(acetamidomethylene)succinate + NAD+
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + H+ + O2
2-(acetamidomethylene)succinate + NAD+ + H2O
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2
2-(acetamidomethylene)succinate + NAD(P)+
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2
2-(acetamidomethylene)succinate + NAD+
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2 + H+
2-(acetamidomethylene)succinate + NAD+
3-hydroxy-2-methylpyridine-5-carboxylate + NADPH + H+ + O2
2-(acetamidomethylene)succinate + NADP+
-
-
-
-
?
5-hydroxynicotinic acid + NADH + H+ + O2
alpha-(N-formylaminomethylene)succinic acid + NAD+ + H2O
5-pyridoxic acid + NADH + H+ + O2
alpha-(N-acetylaminomethylene)-beta-hydroxymethyl succinic acid + NAD+ + H2O
5-pyridoxic acid + NADH + O2 + H+
2-(acetamidomethylene)-beta-hydroxymethyl succinate + NAD+
5% of the activity with 2-methyl-3-hydroxypyridine-5-carboxylic acid
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2
2-(acetamidomethylidene)succinate + NAD(P)+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2
2-(acetamidomethylidene)succinate + NAD(P)+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2
2-(acetamidomethylidene)succinate + NAD(P)+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + H+ + O2
2-(acetamidomethylene)succinate + NAD+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + H+ + O2
2-(acetamidomethylene)succinate + NAD+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + H+ + O2
2-(acetamidomethylene)succinate + NAD+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + H+ + O2
2-(acetamidomethylene)succinate + NAD+ + H2O
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + H+ + O2
2-(acetamidomethylene)succinate + NAD+ + H2O
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + H+ + O2
2-(acetamidomethylene)succinate + NAD+ + H2O
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2
2-(acetamidomethylene)succinate + NAD(P)+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2
2-(acetamidomethylene)succinate + NAD(P)+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2
2-(acetamidomethylene)succinate + NAD(P)+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2
2-(acetamidomethylene)succinate + NAD(P)+
-
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2
2-(acetamidomethylene)succinate + NAD(P)+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2
2-(acetamidomethylene)succinate + NAD(P)+
-
the nitrogen atom of the 3-hydroxy-2-methylpyridine-5-carboxylate is invariably protonated during the catalytic reaction
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2
2-(acetamidomethylene)succinate + NAD+
-
inducible enzyme opens the pyridine ring during the metabolic degradation of vitamin B6
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2
2-(acetamidomethylene)succinate + NAD+
-
enzyme is involved in degradation of vitamin B6
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2 + H+
2-(acetamidomethylene)succinate + NAD+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2 + H+
2-(acetamidomethylene)succinate + NAD+
-
-
-
?
5-hydroxynicotinic acid + NADH + H+ + O2
alpha-(N-formylaminomethylene)succinic acid + NAD+ + H2O
-
-
-
?
5-hydroxynicotinic acid + NADH + H+ + O2
alpha-(N-formylaminomethylene)succinic acid + NAD+ + H2O
-
-
-
?
5-hydroxynicotinic acid + NADH + H+ + O2
alpha-(N-formylaminomethylene)succinic acid + NAD+ + H2O
-
-
-
?
5-hydroxynicotinic acid + NADH + O2 + H+
2-(formylamidomethylene)succinate + NAD+
-
-
-
?
5-hydroxynicotinic acid + NADH + O2 + H+
2-(formylamidomethylene)succinate + NAD+
about 100% of the activity with 2-methyl-3-hydroxypyridine-5-carboxylic acid
-
-
?
5-hydroxynicotinic acid + NADH + O2 + H+
2-(formylamidomethylene)succinate + NAD+
-
-
-
?
5-pyridoxic acid + NADH + H+ + O2
alpha-(N-acetylaminomethylene)-beta-hydroxymethyl succinic acid + NAD+ + H2O
5% activity compared to 3-hydroxy-2-methylpyridine-5-carboxylate
-
-
?
5-pyridoxic acid + NADH + H+ + O2
alpha-(N-acetylaminomethylene)-beta-hydroxymethyl succinic acid + NAD+ + H2O
5% activity compared to 3-hydroxy-2-methylpyridine-5-carboxylate
-
-
?
5-pyridoxic acid + NADH + H+ + O2
alpha-(N-acetylaminomethylene)-beta-hydroxymethyl succinic acid + NAD+ + H2O
5% activity compared to 3-hydroxy-2-methylpyridine-5-carboxylate
-
-
?
additional information
?
-
comparisons of substrate binding structure analysis mechanism
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-
-
additional information
?
-
-
comparisons of substrate binding structure analysis mechanism
-
-
-
additional information
?
-
comparisons of substrate binding structure analysis mechanism
-
-
-
additional information
?
-
comparisons of substrate binding structure analysis mechanism
-
-
-
additional information
?
-
also catalyzes the NADH oxidation reaction uncoupled with ring opening in the absence of 2-methyl-3-hydroxypyridine-5-carboxylic acid
-
-
?
additional information
?
-
-
also catalyzes the NADH oxidation reaction uncoupled with ring opening in the absence of 2-methyl-3-hydroxypyridine-5-carboxylic acid
-
-
?
additional information
?
-
also catalyzes the NADH oxidation reaction uncoupled with ring opening in the absence of 2-methyl-3-hydroxypyridine-5-carboxylic acid
-
-
?
additional information
?
-
also catalyzes the NADH oxidation reaction uncoupled with ring opening in the absence of 2-methyl-3-hydroxypyridine-5-carboxylic acid
-
-
?
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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
2-methyl-3-hydroxypyridine-5-carboxylic acid + NADH + O2 + H+
2-(acetamidomethylene)succinate + NAD+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2
2-(acetamidomethylidene)succinate + NAD(P)+
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + H+ + O2
2-(acetamidomethylene)succinate + NAD+
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2
2-(acetamidomethylene)succinate + NAD+
3-hydroxy-2-methylpyridine-5-carboxylate + NADPH + H+ + O2
2-(acetamidomethylene)succinate + NADP+
-
-
-
-
?
5-hydroxynicotinic acid + NADH + O2 + H+
2-(formylamidomethylene)succinate + NAD+
about 100% of the activity with 2-methyl-3-hydroxypyridine-5-carboxylic acid
-
-
?
5-pyridoxic acid + NADH + O2 + H+
2-(acetamidomethylene)-beta-hydroxymethyl succinate + NAD+
5% of the activity with 2-methyl-3-hydroxypyridine-5-carboxylic acid
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2
2-(acetamidomethylidene)succinate + NAD(P)+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2
2-(acetamidomethylidene)succinate + NAD(P)+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NAD(P)H + H+ + O2
2-(acetamidomethylidene)succinate + NAD(P)+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + H+ + O2
2-(acetamidomethylene)succinate + NAD+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + H+ + O2
2-(acetamidomethylene)succinate + NAD+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + H+ + O2
2-(acetamidomethylene)succinate + NAD+
-
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2
2-(acetamidomethylene)succinate + NAD+
-
inducible enzyme opens the pyridine ring during the metabolic degradation of vitamin B6
-
-
?
3-hydroxy-2-methylpyridine-5-carboxylate + NADH + O2
2-(acetamidomethylene)succinate + NAD+
-
enzyme is involved in degradation of vitamin B6
-
-
?
additional information
?
-
also catalyzes the NADH oxidation reaction uncoupled with ring opening in the absence of 2-methyl-3-hydroxypyridine-5-carboxylic acid
-
-
?
additional information
?
-
-
also catalyzes the NADH oxidation reaction uncoupled with ring opening in the absence of 2-methyl-3-hydroxypyridine-5-carboxylic acid
-
-
?
additional information
?
-
also catalyzes the NADH oxidation reaction uncoupled with ring opening in the absence of 2-methyl-3-hydroxypyridine-5-carboxylic acid
-
-
?
additional information
?
-
also catalyzes the NADH oxidation reaction uncoupled with ring opening in the absence of 2-methyl-3-hydroxypyridine-5-carboxylic acid
-
-
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
FAD
Km value 624 nM, one mol of the enzyme contains 1.93 mol of FAD
NADH
-
interacts with the holoenzyme in a slow catalytically irrelevant manner
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
6-methylnicotinic acid
-
competitive with 3-hydroxy-2-methylpyridine-5-carboxylate
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DISEASE
TITLE OF PUBLICATION
LINK TO PUBMED
Infections
Molecular cloning, identification and characterization of 2-methyl-3-hydroxypyridine-5-carboxylic-acid-dioxygenase-coding gene from the nitrogen-fixing symbiotic bacterium Mesorhizobium loti.
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.045
5-hydroxynicotinic acid
mutant enzyme Y270A, at pH 8.0 and 25°C
0.21
5-hydroxynicotinic acid
mutant enzyme Y270F, at pH 8.0 and 25°C
0.00056
N-methyl-5-hydroxynicotinic acid
-
calculation from stopped-flow data
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.05
5-hydroxynicotinic acid
mutant enzyme Y270A, at pH 8.0 and 25°C
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.1
5-hydroxynicotinic acid
mutant enzyme Y270A, at pH 8.0 and 25°C
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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IC50 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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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
additional information
physiological function
conversion of 2-methyl-3-hydroxypyridine-5-carboxylic acid to alpha-(N-acetylaminomethylene)succinic acid is the essential ring-opening step in the bacterial degradation of vitamin B6. The rearomatisation of the hydroxylated intermediate occurs spontaneously in aqueous solution. This implies that the ring-opening process occurs inside the enzyme's active site. Proposal of two pathways with reasonable energy barriers
physiological function
MHPCO is essential for the assimilation of pyridoxine, but not for its growth in a nutrient-rich medium. MHPCO is dispensable for at least nodule formation on roots of seedlings in symbiosis
physiological function
2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO) is a flavoenzyme that catalyzes oxidative ring opening of 2-methyl-3-hydroxypyridine-5-carboxylic acid
physiological function
-
2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO) is a flavoenzyme that catalyzes oxidative ring opening of 2-methyl-3-hydroxypyridine-5-carboxylic acid
-
physiological function
-
2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO) is a flavoenzyme that catalyzes oxidative ring opening of 2-methyl-3-hydroxypyridine-5-carboxylic acid
-
physiological function
-
conversion of 2-methyl-3-hydroxypyridine-5-carboxylic acid to alpha-(N-acetylaminomethylene)succinic acid is the essential ring-opening step in the bacterial degradation of vitamin B6. The rearomatisation of the hydroxylated intermediate occurs spontaneously in aqueous solution. This implies that the ring-opening process occurs inside the enzyme's active site. Proposal of two pathways with reasonable energy barriers
-
physiological function
-
MHPCO is essential for the assimilation of pyridoxine, but not for its growth in a nutrient-rich medium. MHPCO is dispensable for at least nodule formation on roots of seedlings in symbiosis
-
additional information
sequence comparisons, three-dimensional enzyme structure analysis, and structure comparisons with 2-hydroxybiphenyl 3-monooxygenase (HbpA) from Pseudomonas nitroreducens and 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO, PDB ID 5hxi) from Mesorhizobium japonicum, overview. Despite having only 14% similarity in their primary sequences, pairwise structure alignments of PobA from Pseudomonas putida with HbpA from Pseudomonas nitroreducens and MHPCO from Mesorhizobium japonicum reveal local similarities between these structures. Key residues in the FAD-binding and substrate-binding sites of PobA are highly conserved spatially across the proteins from all three species. The PobA from Pseudomonas putida is structurally very similar to PobA from Pseudomonas fluorescens and from Pseudomonas aeruginosa. Key secondary-structure elements important for catalysis, such as the betaalphabeta fold, beta-sheet wall and alpha12 helix, are conserved across this expanded class of oxygenases
additional information
-
sequence comparisons, three-dimensional enzyme structure analysis, and structure comparisons with 2-hydroxybiphenyl 3-monooxygenase (HbpA) from Pseudomonas nitroreducens and 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO, PDB ID 5hxi) from Mesorhizobium japonicum, overview. Despite having only 14% similarity in their primary sequences, pairwise structure alignments of PobA from Pseudomonas putida with HbpA from Pseudomonas nitroreducens and MHPCO from Mesorhizobium japonicum reveal local similarities between these structures. Key residues in the FAD-binding and substrate-binding sites of PobA are highly conserved spatially across the proteins from all three species. The PobA from Pseudomonas putida is structurally very similar to PobA from Pseudomonas fluorescens and from Pseudomonas aeruginosa. Key secondary-structure elements important for catalysis, such as the betaalphabeta fold, beta-sheet wall and alpha12 helix, are conserved across this expanded class of oxygenases
additional information
-
sequence comparisons, three-dimensional enzyme structure analysis, and structure comparisons with 2-hydroxybiphenyl 3-monooxygenase (HbpA) from Pseudomonas nitroreducens and 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO, PDB ID 5hxi) from Mesorhizobium japonicum, overview. Despite having only 14% similarity in their primary sequences, pairwise structure alignments of PobA from Pseudomonas putida with HbpA from Pseudomonas nitroreducens and MHPCO from Mesorhizobium japonicum reveal local similarities between these structures. Key residues in the FAD-binding and substrate-binding sites of PobA are highly conserved spatially across the proteins from all three species. The PobA from Pseudomonas putida is structurally very similar to PobA from Pseudomonas fluorescens and from Pseudomonas aeruginosa. Key secondary-structure elements important for catalysis, such as the betaalphabeta fold, beta-sheet wall and alpha12 helix, are conserved across this expanded class of oxygenases
-
additional information
-
sequence comparisons, three-dimensional enzyme structure analysis, and structure comparisons with 2-hydroxybiphenyl 3-monooxygenase (HbpA) from Pseudomonas nitroreducens and 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO, PDB ID 5hxi) from Mesorhizobium japonicum, overview. Despite having only 14% similarity in their primary sequences, pairwise structure alignments of PobA from Pseudomonas putida with HbpA from Pseudomonas nitroreducens and MHPCO from Mesorhizobium japonicum reveal local similarities between these structures. Key residues in the FAD-binding and substrate-binding sites of PobA are highly conserved spatially across the proteins from all three species. The PobA from Pseudomonas putida is structurally very similar to PobA from Pseudomonas fluorescens and from Pseudomonas aeruginosa. Key secondary-structure elements important for catalysis, such as the betaalphabeta fold, beta-sheet wall and alpha12 helix, are conserved across this expanded class of oxygenases
-
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). A0A852TMD2_9ACTN
384
0
42029
TrEMBL
-
Q988D3_RHILO
Mesorhizobium japonicum (strain LMG 29417 / CECT 9101 / MAFF 303099)
379
0
41748
TrEMBL
other Location (Reliability: 5)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
tetramer
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structure analysis, PDB ID 5hxi, comparison to the structure of HbpA from Pseudomonas nitroreducens strain HBP1 (PDB ID 4cy8)
structure in complex with FAD. Structural comparison of p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas putida, EC 1.14.13.2, 2-hydroxybiphenyl 3-monooxygenase (HbpA) from Pseudomonas nitroreducens, EC 1.14.13.44, and 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO) from Mesorhizobium japonicum. The portions of the residues required for substrate and FAD binding are identical, including the betaalphabeta fold and beta-sheet wall
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
-
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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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Y270A
Y270F
Y270A
Y270F
Y270A
Y270F
Y223E
-
the mutant shows reduced activity compared to the wild type enzyme
Y223F
-
the mutant shows reduced activity compared to the wild type enzyme
Y223H
-
the mutant shows reduced activity compared to the wild type enzyme
Y223T
-
the mutant shows reduced activity compared to the wild type enzyme
Y82F
-
the mutant shows reduced activity compared to the wild type enzyme
Y82H
-
the mutant shows reduced activity compared to the wild type enzyme
Y82R
-
the mutant shows reduced activity compared to the wild type enzyme
Y270A
the mutant shows reduced ring opening activity but increased NADH oxidation activity compared to the wild type enzyme
Y270A
less than 0.1% of the pyridine ring cleavage capacity of wild-type
Y270F
the mutant shows reduced ring opening activity but increased NADH oxidation activity compared to the wild type enzyme
Y270F
about 1.5% of the pyridine ring cleavage capacity of wild-type
Y270A
-
the mutant shows reduced ring opening activity but increased NADH oxidation activity compared to the wild type enzyme
-
Y270A
-
less than 0.1% of the pyridine ring cleavage capacity of wild-type
-
Y270F
-
the mutant shows reduced ring opening activity but increased NADH oxidation activity compared to the wild type enzyme
-
Y270F
-
about 1.5% of the pyridine ring cleavage capacity of wild-type
-
Y270A
-
the mutant shows reduced ring opening activity but increased NADH oxidation activity compared to the wild type enzyme
-
Y270A
-
less than 0.1% of the pyridine ring cleavage capacity of wild-type
-
Y270F
-
the mutant shows reduced ring opening activity but increased NADH oxidation activity compared to the wild type enzyme
-
Y270F
-
about 1.5% of the pyridine ring cleavage capacity of wild-type
-
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
the enzyme is very sensitive to oxidation, it loses activity rapidly in absence of mercaptoethanol even at 4°C, it is further stabilized in presence of high concentrations of glycerol or by serum albumin
-
439056
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
expression in Escherichia coli
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Sparrow, L.G.; Ho, P.P.K.; Sundaram, T.K.; Zach, D.; Nyns, E.J.; Snell, E.E.
The bacterial oxidation of vitamin B6. VII. Purification, properties, and mechanism of action of an oxygenase which cleaves the 3-hydroxypyridine ring
J. Biol. Chem.
244
2590-2600
1969
Pseudomonas sp.
brenda
Kishore, G.M.; Snell, E.E.
Interaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase with FAD, substrates, and analogues. Spectral and fluorescence investigations
J. Biol. Chem.
256
4234-4240
1981
Pseudomonas sp.
brenda
Kishore, G.M.; Snell, E.E.
Kinetic investigations on a flavoprotein oxygenase, 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase
J. Biol. Chem.
256
4228-4233
1981
Pseudomonas sp.
brenda
Chaiyen, P.; Ballou, D.P.; Massey, V.
Gene cloning, sequence analysis, and expression of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase
Proc. Natl. Acad. Sci. USA
94
7233-7238
1997
Pseudomonas sp. (O08453), Pseudomonas sp.
brenda
Chaiyen, P.; Brissette, P.; Ballou, D.P.; Massey, V.
Reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase with N-methyl-5-hydroxynicotinic acid: studies on the mode of binding, and Protonation Status of the Substrate
Biochemistry
36
13856-13864
1997
Pseudomonas sp.
brenda
Chaiyen, P.; Brissette, P.; Ballou, D.P.; Massey, V.
Unusual mechanism of oxygen atom transfer and product rearrangement in the catalytic reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase
Biochemistry
36
8060-8070
1997
Pseudomonas sp.
brenda
Chaiyen, P.; Brissette, P.; Ballou, D.P.; Massey, V.
Thermodynamics and reduction kinetics properties of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase
Biochemistry
36
2612-2621
1997
Pseudomonas sp.
brenda
Oonanant, W.; Sucharitakul, J.; Yuvaniyama, J.; Chaiyen, P.
Crystallization and preliminary x-ray crystallographic analysis of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase from Pseudomonas sp. MA-1
Acta Crystallogr. Sect. F
61
312-314
2005
Pseudomonas sp.
brenda
Chaiyen, P.; Sucharitakul, J.; Svasti, J.; Entsch, B.; Massey, V.; Ballou, D.P.
Use of 8-substituted-FAD analogues to investigate the hydroxylation mechanism of the flavoprotein 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase
Biochemistry
43
3933-3943
2004
Pseudomonas sp.
brenda
McCulloch, K.; Mukherjee, T.; Begley, T.; Ealick, S.
Structure of the PLP degradative enzyme 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase from Mesorhizobium loti MAFF303099 and its mechanistic implications
Biochemistry
48
4139-4149
2009
Mesorhizobium loti (Q988D3)
brenda
Tian, B.; Strid, A.; Eriksson, L.A.
Catalytic roles of active-site residues in 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase: an ONIOM/DFT study
J. Phys. Chem. B
115
1918-1926
2011
Mesorhizobium loti, Mesorhizobium loti MAFF303099
brenda
Luanloet, T.; Sucharitakul, J.; Chaiyen, P.
Selectivity of substrate binding and ionization of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase
FEBS J.
282
3107-3125
2015
Pseudomonas sp. MA-1
brenda
Kobayashi, J.; Yoshida, H.; Yagi, T.; Kamitori, S.; Hayashi, H.; Mizutani, K.; Takahashi, N.; Mikami, B.
Role of the Tyr270 residue in 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase from Mesorhizobium loti
J. Biosci. Bioeng.
123
154-162
2017
Mesorhizobium loti (Q988D3), Mesorhizobium loti, Mesorhizobium loti MAFF 303099 (Q988D3), Mesorhizobium loti MAFF303099 (Q988D3)
brenda
Tian, B.; Tu, Y.; Strid, A.; Eriksson, L.A.
Hydroxylation and ring-opening mechanism of an unusual flavoprotein monooxygenase, 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase a theoretical study
Chemistry
16
2557-2566
2010
Mesorhizobium loti (Q988D3), Mesorhizobium loti MAFF 303099 (Q988D3)
brenda
Yuan, B.; Yokochi, N.; Yoshikane, Y.; Ohnishi, K.; Yagi, T.
Molecular cloning, identification and characterization of 2-methyl-3-hydroxypyridine-5-carboxylic-acid-dioxygenase-coding gene from the nitrogen-fixing symbiotic bacterium Mesorhizobium loti
J. Biosci. Bioeng.
102
504-510
2006
Mesorhizobium loti (Q988D3), Mesorhizobium loti, Mesorhizobium loti MAFF 303099 (Q988D3)
brenda
Lazar, J.T.; Shuvalova, L.; Rosas-Lemus, M.; Kiryukhina, O.; Satchell, K.J.F.; Minasov, G.
Structural comparison of p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas putida with PobA from other Pseudomonas spp. and other monooxygenases
Acta Crystallogr. F Struct. Biol. Commun.
75
507-514
2019
Mesorhizobium japonicum (Q988D3), Mesorhizobium japonicum, Mesorhizobium japonicum CECT 9101 (Q988D3)
brenda
Lazar, J.T.; Shuvalova, L.; Rosas-Lemus, M.; Kiryukhina, O.; Satchell, K.J.F.; Minasov, G.
Structural comparison of p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas putida with PobA from other Pseudomonas spp. and other monooxygenases
Acta Crystallogr. Sect. F
75
507-514
2019
Mesorhizobium japonicum (Q988D3), Mesorhizobium japonicum, Mesorhizobium japonicum LMG 29417 (Q988D3), Mesorhizobium japonicum CECT 9101 (Q988D3)
brenda
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