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Literature summary extracted from

  • Kampatsikas, I.; Bijelic, A.; Pretzler, M.; Rompel, A.
    In crystallo activity tests with latent apple tyrosinase and two mutants reveal the importance of the mutated sites for polyphenol oxidase activity (2017), Acta Crystallogr. Sect. F, 73, 491-499 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.18.1 gene MdPPO1, cloned from apple leaves, recombinant expression of GST-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 Malus domestica

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.14.18.1 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

EC Number Protein Variants Comment Organism
1.14.18.1 A239T site-directed mutagenesis, mutation of an activity controller residue Malus domestica
1.14.18.1 E234A site-directed mutagenesis, mutation of the water-keeper residue Malus domestica
1.14.18.1 F259A site-directed mutagenesis, mutation of the gatekeeper residue Malus domestica
1.14.18.1 L243R site-directed mutagenesis, mutation of an activity controller residue Malus domestica

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
1.14.18.1 chloroplast
-
 Malus domestica 9507
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.14.18.1 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/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.18.1 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

EC Number Organism UniProt Comment Textmining
1.14.18.1 Malus domestica P43309 cv. Golden Delicious
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.18.1 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

EC Number Source Tissue Comment Organism Textmining
1.14.18.1 leaf
-
 Malus domestica
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.18.1 2 L-dopa + O2
-
 Malus domestica 2 dopaquinone + 2 H2O
-
 ?
1.14.18.1 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 ?
-
 ?
1.14.18.1 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 ?
-
 ?
1.14.18.1 tyramine + O2
-
 Malus domestica 4-(2-aminoethyl)cyclohexa-3,5-diene-1,2-dione + H2O
-
 ?

Subunits

EC Number Subunits Comment Organism
1.14.18.1 ? x * 56400, latent enzyme form, SDS-PAGE Malus domestica

Synonyms

EC Number Synonyms Comment Organism
1.14.18.1 MdPPO1
-
 Malus domestica
1.14.18.1 polyphenol oxidase
-
 Malus domestica
1.14.18.1 PPO
-
 Malus domestica
1.14.18.1 tyr
-
 Malus domestica

General Information

EC Number General Information Comment Organism
1.14.18.1 additional information enzyme active domain structure, modeling, overview Malus domestica