Lipase-mediated production of defensive toxins in the marine mollusc Oxynoe olivacea

Metabolites related to caulerpenyne (1), a toxic sesquiterpene featured by two enol-acetate residues, play a major role in the chemical defence of both algae of the genus Caulerpa and a few molluscs of the order Sacoglossa. Here we report the direct evidence that cell-free preparations of Oxynoe olivacea, a Mediterranean sacoglossan, transform efficiently the algal metabolite 1 to oxytoxin-2 (3), the main defensive metabolite of the mollusc. The process implies two distinct hydrolytic activities, here named LIP-1 and LIP-2, able to operate either hydrolysis of the acetyl residue at C-1 or concerted elimination of the acetyl groups at C-4 and C-13. Incubation experiments with tissue homogenates of O. olivacea or with commercially available lipases suggest a two-step mechanism that involves, in vitro, an unstable metabolite characterized as preoxytoxin-2 (4). The course of the entire process can be easily monitored by reverse phase HPLC/ESI-MS, as well as by NMR measurements, which provides direct evidence of the enzymatic mechanism leading to the formation of this last compound (4). In agreement with the literature, both fresh and frozen tissues of Mediterranean Caulerpa prolifera also have the capability to transform 1 into aldehydic derivatives, namely oxytoxin-1 (2) and oxytoxin-2 (3), through hydrolysis of the acetyl groups. However, differently from experiments with mollusc homogenates, the conversion is not complete and caulerpenyne (1) can be detected in the algal suspension for a few hours. HPLC/ESI-MS monitoring of this transformation suggests that the hydrolytic route involves different activities in the mollusc and seaweed.