Purification and kinetic properties of elisabethatriene synthase from the coral Pseudopterogorgia elisabethae

The Bahamian octocoral Pseudopterogorgia elisabethae is the source of pseudopterosins, diterpene glycosides with potent anti-inflammatory activity. The first committed step in pseudopterosin biosynthesis comprises the cyclisation of the universal diterpene precursor geranylgeranyl diphosphate to elisabethatriene. This reaction is catalysed by elisabethatriene synthase, which was purified to homogeneity from a crude coral extract. This represents the first purification to apparent homogeneity of a terpene cyclase from any marine source. The reaction kinetics of elisabethatriene synthase was examined using a steady state approach with 3 H-labelled isoprenyldiphosphates varying in carbon chain length (C 10 , C 15 , C 20 ). For the reaction of elisabethatriene synthase with its natural substrate geranylgeranyl diphosphate, values of K m (2.3 × 10 − 6 M), V max (3.4 × 10 4 nM elisabethatriene * s − 1 ) and the specificity constant ( k cat / K m = 1.8 × 10 − 10 M − 1 * s − 1 ) were comparable with diterpene cyclases from terrestrial plants. Elisabethatriene synthase also catalysed the conversion of C 15 and C 10 isoprenyldiphosphate analogues to monoterpene and sesquiterpene olefins, respectively. Kinetic parameters indicated that substrate specificity and K m of elisabethatriene synthase decreased with decreasing isoprenoid carbon chain length. Furthermore, GC–MS analysis showed increased product diversity with decreasing isoprenoid carbon chain length.