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

Olive (<i>Olea europaea</i>) 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 <i>Arabidopsis</i> enzyme that suggests similar catalytic mechanisms. OeHPPD has an estimated <i>K</i>_cat of 75.26 s^-1 and catalytic efficiency (<i>K</i>_m/<i>K</i>_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 <i>Oe</i>HPPD gene is temporally regulated and cultivar-dependent. Moreover, the analysis of <i>OeHPPD</i> expression in fruits affected by drought stress suggests that HPPD is involved in olive environmental adaptation.