The seed coat serves as the primary protective barrier, offering mechanical and chemical defense for the embryo. It contains various metabolites, including phenolic compounds, which can be oxidized by polyphenol oxidase (PPO) to form oligomers. In this study, we heterologously expressed a 515 amino acid protein derived from wild pea (Pisum elatius), omitting its N-terminal signal sequence, and analyzed its biochemical properties. The recombinant PeaPPO required sodium dodecyl sulfate (SDS) for activation and exhibited activity between pHs 5.2 and 7.0, peaking at pH 6.0 with 0.25 mM SDS. Tropolone and its isomer thujaplicin were the most effective inhibitors. PeaPPO catalyzed reactions with seed coat-derived substrates, displaying activity toward phenols, catechols, and pyrogallols, with the highest affinity for catechols. Principal component analysis of LC-MS/MS-derived phenolic profiles demonstrated that PPO+ and ppo- genotypes differ significantly in their accumulation of PPO substrates and inhibitors. These findings confirm that PeaPPO possesses both monophenolase and catechol oxidase activities, identifying it as a tyrosinase.

Czech Advanced Technology and Research Institute Palacký University Olomouc 77900 Czech Republic

Czech Agrifood Research Center Olomouc 77900 Czech Republic

Department of Analytical Chemistry Faculty of Sciences Palacký University Olomouc 77900 Czech Republic

Department of Biochemistry Faculty of Sciences Palacký University Olomouc Olomouc 77900 Czech Republic

Department of Botany Faculty of Sciences Palacký University Olomouc Olomouc 77900 Czech Republic

Fakultät für Chemie Institut für Biophysikalische Chemie Universität Wien Wien 1090 Austria

Laboratory of Growth Regulators Faculty of Sciences Palacký University Olomouc Olomouc 77900 Czech Republic

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