Molecular and Biochemical Characterization of Olive 4-Hydroxyphenyl Pyruvate Dioxygenase Involved in the Biosynthesis of Tocopherols Present in Virgin Olive Oil

Olive (Olea europaea) fruit contains high amounts of tocopherols that are responsible, along with secoiridoid phenolic compounds, for most of the antioxidant and anti-inflammatory properties of virgin olive oil. This study focuses on the molecular and biochemical characterization of olive 4-hydroxyphenyl pyruvate dioxygenase (OeHPPD) catalyzing the biosynthesis of homogentisic acid, which constitutes the phenolic residue in the tocopherol molecule. OeHPPD is a cytoplasmic enzyme with a molecular weight of 49.8 kDa and a predicted tertiary structure very similar to the Arabidopsis enzyme that suggests similar catalytic mechanisms. OeHPPD has an estimated K_cat of 75.26 s^–1 and catalytic efficiency (K_m/K_cat) of 0.145 μM^–1 s^–1 with 4-hydroxyphenyl pyruvate as the substrate. The expression analysis in fruits from selected olive cultivars harvested at different ripening stages indicates that the OeHPPD gene is temporally regulated and cultivar-dependent. Moreover, the analysis of OeHPPD expression in fruits affected by drought stress suggests that HPPD is involved in olive environmental adaptation.