Identification and characterization of esterases involved in the conversion of oleosides in Olea europaea

Since the epidemiological long-term survey of the ‘Seven Countries Study’, olive oil as a health-promoting food has gained increasing attention in pharmacological research. In this context, there was a particular interest in the so-called olive polyphenols, including the secoiridoid derivatives oleocanthal and oleacein, the regular intake of which has been associated with a protective effect against cancer as well as cardiovascular and neurodegenerative diseases. Both substances can be detected in relatively high concentrations in virgin olive oil, whereas they are usually not detectable in intact fruits or leaves. Only upon cell disruption, they are formed from the precursor molecules ligstroside and oleuropein by endogenous β-glucosidases and esterases. This mechanism is part of the plant defense against herbivores and pathogens by generating highly reactive intermediates and reaction products of dialdehydic structure that feature protein-crosslinking and denaturing activities. Despite an increasing interest in the research of these supposedly pharmaceutically active secondary metabolites, little is known about the endogenous enzymes involved in their production. In addition to the already described oleuropein specific β-glucosidase, OeGLU, three esterases, OeEst030, OeEst228 and OeEst679, from Olea europaea acting on deglycosylated oleosides have been identified and characterized. Accordingly, for the first time the enzymatic biotransformation of ligstroside and oleuropein to oleocanthal and oleacein, respectively, was proven using an enzyme mixture. Moreover, two further esterases from O. europeae, OeEst391 and OeEst905, were identified which showed no activity against oleosides and their aglycones, but accepted methylated phytohormones as substrates. For all tested olive enzymes esterase activity against the plant hormones methyl jasmonate (MeJA) and methyl indoleacetic acid (MeIAA) was shown. Those results indicate a possible regulatory role of the enzymes in plant hormone signaling and their descent from respective esterases. Moreover, all tested olive esterases were also capable of catalyzing transesterification in the presence of ethanol yielding ethyl jasmonate (EtJA) and ethyl indole-3-acetate (EtIAA), a feature previously not known for plant esterases. The physiological relevance of this activity, however, has not been clarified. Further investigations focused on the protein structures of OeEst030 and OeEst228. Thus, homology modeling studies suggested that the three-dimensional structures of both proteins show a high similarity to the crystal structure of PNAE and, therefore, supported an assignment to the α/β hydrolase superfamily. Accordingly, it can be assumed that in addition to a core domain consisting of five α helices and six β sheets, there is also a so-called cap domain which regulates the access of substrate to the active site. Furthermore, two of the three supposedly catalytic amino acids, namely serine and histidine, could be localized. The position of the acidic member of the catalytic triad, in contrast, could not be determined. Initially assumed amino acids could be excluded by appropriate mutagenesis studies. In summary, three novel esterases could be identified and characterized, expanding our knowledge of oleoside turnover in damaged tissue of O. europaea