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Abstract: Putative precursors in pseudopterosin biosynthesis, the hydrocarbons isoelisabethatriene (10) and erogorgiaene (11), have been identified from an extract of Pseudopterogorgia elisabethae collected in the Florida Keys. Biosynthetic experiments designed to test the utilization of these compounds in pseudopterosin production revealed that erogorgiaene is transformed to pseudopterosins A-D. Together with our previous data, it is now apparent that early steps in pseudopterosin biosynthesis involve the cyclization of geranylgeranyl diphosphate to elisabethatriene followed by the dehydrogenation and aromatization to erogorgiaene.

The pseudopterosins are diterpene glycosides isolated from the marine gorgonian, Pseudopterogorgia elisabethae, which exhibit anti-inflammatory and analgesic activity greater than the industry standard, indomethacin. Previously, we isolated the pseudopterosin diterpene cyclase product, elisabethatriene, using a radioactivity-guided isolation. Identification of this metabolite, and the conversion of labeled geranylgeranyl diphosphate to elisabethatriene, provided us with an assay to guide the isolation of the enzyme responsible for this cyclization. The soluble protein preparation from P. elisabethae has been partially purified (∼15,000-fold) using a combination of low-resolution anion-exchange, low-resolution hydrophobic interaction, high-resolution hydroxyapatite, and high-resolution anion-exchange chromatography. The diterpene cyclase was identified by comparing the molecular weight from gel permeation chromatography (∼47,000 Da) with those of protein bands from purified fractions using SDS–PAGE gel electrophoresis. Kinetic analysis and evaluation of amino acid inhibition studies indicated that the enzyme displays similar characteristics to other terpenoid cyclases isolated from terrestrial sources. This report represents the first purification and characterization of a terpene biosynthetic enzyme from a marine invertebrate. [Copyright &y& Elsevier]