Cloned (Comment) | Organism |
---|---|
gene MdPPO1, cloned from apple leaves, recombinant expression of GST-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 | Malus domestica |
Crystallization (Comment) | Organism |
---|---|
purified recombinant wild-type enzyme and mutants A239T and F259A, hanging drop vapour diffusion technique, mixing 0f 0.001 ml of 5-10 mg/ml protein solution with 0.002 ml reservoir solution containing 50 mM Tris-HCl, pH 7.0, 19-21% PEG 3350, at 20°C, 10-15 days, method optimization, X-ray diffraction structure determination and analysis at 1.35, 1.55 and 1.70 A resolution, respectively. Soaking of crystals with a monophenolic (tyramine) and a diphenolic (dopamine) substrate in 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 20% PEG 3350, 20-25% PEG 1500, using SDS as an activator in order to perform in crystallo activity tests | Malus domestica |
Protein Variants | Comment | Organism |
---|---|---|
A239T | site-directed mutagenesis, mutation of an activity controller residue | Malus domestica |
E234A | site-directed mutagenesis, mutation of the water-keeper residue | Malus domestica |
F259A | site-directed mutagenesis, mutation of the gatekeeper residue | Malus domestica |
L243R | site-directed mutagenesis, mutation of an activity controller residue | Malus domestica |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
chloroplast | - |
Malus domestica | 9507 | - |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Cu2+ | a copper-containing enzyme, two Cu2+ ions CuA and CuB, copper domain structure with the conserved histidines coordinating CuA (His86, His107 and His116) and CuB (His238, His242 and His272) and the positions of the mutated residues, namely the two activity controllers alanine (Ala239) and leucine (Leu243), the water keeper glutamic acid (Glu234) and the gatekeeper phenylalanine (Phe259), overview | Malus domestica |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | Malus domestica | tyrosinases are able to catalyze the ortho-hydroxylation of monophenols to o-diphenols (monophenolase activity, EC 1.14.18.1) coupled with the subsequent two-electron oxidation of o-diphenols to the corresponding o-quinones (diphenolase activity, EC 1.10.3.1). The o-diphenols formed in the hydroxylation step remain in the active centre and are oxidized to the quinonic state. During the TYR mediated hydroxylation and oxidation of one molecule of monophenol, one molecule of dioxygen is reduced to water. Catechol oxidases, EC 1.10.3.1, lack the monophenolase activity and are thus only capable of oxidizing o-diphenols | ? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Malus domestica | P43309 | cv. Golden Delicious | - |
Purification (Comment) | Organism |
---|---|
recombinant GST-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 by glutathione affinity chromatography, and by GST-tag cleavage through HRV3C protease, followed by another step of glutathione affinity chromatography | Malus domestica |
Source Tissue | Comment | Organism | Textmining |
---|---|---|---|
leaf | - |
Malus domestica | - |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2 L-dopa + O2 | - |
Malus domestica | 2 dopaquinone + 2 H2O | - |
? | |
additional information | tyrosinases are able to catalyze the ortho-hydroxylation of monophenols to o-diphenols (monophenolase activity, EC 1.14.18.1) coupled with the subsequent two-electron oxidation of o-diphenols to the corresponding o-quinones (diphenolase activity, EC 1.10.3.1). The o-diphenols formed in the hydroxylation step remain in the active centre and are oxidized to the quinonic state. During the TYR mediated hydroxylation and oxidation of one molecule of monophenol, one molecule of dioxygen is reduced to water. Catechol oxidases, EC 1.10.3.1, lack the monophenolase activity and are thus only capable of oxidizing o-diphenols | Malus domestica | ? | - |
? | |
additional information | soaking of crystals with a monophenolic (tyramine) and a diphenolic (dopamine) substrate in 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 20% PEG 3350, 20-25% PEG 1500, using SDS as an activator in order to perform in crystallo activity tests | Malus domestica | ? | - |
? | |
tyramine + O2 | - |
Malus domestica | 4-(2-aminoethyl)cyclohexa-3,5-diene-1,2-dione + H2O | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 56400, latent enzyme form, SDS-PAGE | Malus domestica |
Synonyms | Comment | Organism |
---|---|---|
MdPPO1 | - |
Malus domestica |
polyphenol oxidase | - |
Malus domestica |
PPO | - |
Malus domestica |
tyr | - |
Malus domestica |
General Information | Comment | Organism |
---|---|---|
additional information | enzyme active domain structure, modeling, overview | Malus domestica |