Literature summary for 1.1.3.12 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.

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

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

KM Value [mM]

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Mesorhizobium loti	Q5NT46	gene mll6785	-

Purification (Commentary)

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

Substrates and Products (Substrate)

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

Subunits

Subunits	Comment	Organism
monomer	-	Mesorhizobium loti

Synonyms

Synonyms	Comment	Organism
PNOX	-	Mesorhizobium loti

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

Cofactor

Cofactor	Comment	Organism	Structure
FAD	dependent on	Mesorhizobium loti

General Information

General Information	Comment	Organism
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
metabolism	first enzyme in pathway I for the degradation of pyridoxine	Mesorhizobium loti
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]

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