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Literature summary extracted from

  • Mugo, A.; Kobayashi, J.; Yamasaki, T.; Mikami, B.; Ohnishi, K.; Yoshikane, Y.; Yagi, T.
    Crystal structure of pyridoxine 4-oxidase from Mesorhizobium loti (2013), Biochim. Biophys. Acta, 1834, 953-963.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.3.12 gene mll6785, expression of C-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain JM109 Mesorhizobium loti

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.1.3.12 purified recombinant C-terminally His6-tagged PNOX and PNOX-pyridoxamine complex, sitting drop vapour diffusion mthod and micro-seeding method, mixing of 0.002 ml of 10 mg/ml PNOX with 0.002 ml of a precipitant solution comtaining 0.1 M HEPES-NaOH, pH 7.5, 10% v/v 2-propanol, and 20% w/v PEG 4000, equilibration against 01 ml of precipitant solution, 4°C, primary crystals are crushed and suspended in 0.5 ml of a solution consisting of equal volumes of a crystallization buffer containing 50 mM Tris-HCl, pH 8.0, 10% w/v glycerol, 0.005 mM FAD, 0.1% v/v 2-mercaptoethanol, and 0.01% v/v Tween 20, and precipitant solution as seeding solution, or seeding crystals are prepared by vapor diffusion in a mixture consisting of 0.002 ml of 13 mg/ml PNOX, and 0.002 ml of the precipitant solution comprising 0.1 M HEPES-NaOH, pH 7.5, 25% v/v 2-propanol, 20% w/v PEG 4000, and 10 mM pyridoxamine, 3 weeks, X-ray diffraction structuure determination and analysis at 2.2 A and 2.1 A resolutions, respectively, molecular replacement Mesorhizobium loti

Protein Variants

EC Number Protein Variants Comment Organism
1.1.3.12 H460A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Mesorhizobium loti
1.1.3.12 H460A/H462A site-directed mutagenesis, inactive mutant Mesorhizobium loti
1.1.3.12 H462A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Mesorhizobium loti

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.3.12 0.0153
-
pyridoxine pH 8.0, 30°C, recombinant His6-tagged wild-type enzyme Mesorhizobium loti
1.1.3.12 0.0687
-
pyridoxine pH 8.0, 30°C, recombinant His6-tagged mutant H460A Mesorhizobium loti
1.1.3.12 0.0876
-
pyridoxine pH 8.0, 30°C, recombinant His6-tagged mutant H462A Mesorhizobium loti

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.3.12 pyridoxine + O2 Mesorhizobium loti
-
pyridoxal + H2O2
-
?

Organism

EC Number Organism UniProt Comment Textmining
1.1.3.12 Mesorhizobium loti Q5NT46 gene mll6785
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.3.12 recombinant C-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain JM109 Mesorhizobium loti

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.3.12 pyridoxine + O2
-
Mesorhizobium loti pyridoxal + H2O2
-
?

Subunits

EC Number Subunits Comment Organism
1.1.3.12 monomer
-
Mesorhizobium loti

Synonyms

EC Number Synonyms Comment Organism
1.1.3.12 PNOX
-
Mesorhizobium loti

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.3.12 30
-
assay at Mesorhizobium loti

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.3.12 0.4
-
pyridoxine pH 8.0, 30°C, recombinant His6-tagged mutant H460A Mesorhizobium loti
1.1.3.12 1.3
-
pyridoxine pH 8.0, 30°C, recombinant His6-tagged mutant H462A Mesorhizobium loti
1.1.3.12 19.9
-
pyridoxine pH 8.0, 30°C, recombinant His6-tagged wild-type enzyme Mesorhizobium loti

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.3.12 8
-
assay at Mesorhizobium loti

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.3.12 FAD dependent on Mesorhizobium loti

General Information

EC Number General Information Comment Organism
1.1.3.12 evolution the enzyme belongs to the glucose methanol choline (GMC) oxidoreductase family of enzymes. Active site Pro504 in PNOX corresponds to Asn or His of the conserved His-Asn or His-His pair in other GMC oxidoreductase active sites Mesorhizobium loti
1.1.3.12 metabolism first enzyme in pathway I for the degradation of pyridoxine Mesorhizobium loti
1.1.3.12 additional information in the active site, His460, His462, and Pro504 are located on the re-face of the isoalloxazine ring of FAD, pyridoxamine binds to the active site through several hydrogen bonds, mode, overview. His462 may act as a general base for the abstraction of a proton from the 4'-hydroxyl of pyridoxine. His460 may play a role in the binding and positioning of pyridoxine Mesorhizobium loti

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.3.12 6
-
pyridoxine pH 8.0, 30°C, recombinant His6-tagged mutant H460A Mesorhizobium loti
1.1.3.12 15
-
pyridoxine pH 8.0, 30°C, recombinant His6-tagged mutant H462A Mesorhizobium loti
1.1.3.12 1300
-
pyridoxine pH 8.0, 30°C, recombinant His6-tagged wild-type enzyme Mesorhizobium loti