Purification and properties of kaurene synthetase from Fusarium moniliforme.

Abstract Kaurene synthetase has been purified approximately 170-fold from cell-free extracts of the fungus Fusarium moniliforme Sheld. The two catalytic activities associated with kaurene synthetase, namely the cyclization of trans-geranylgeranyl-PP to copalyl-PP (activitiy A) and the cyclization of copalyl-PP to (-)-kaurene (activity B), copurify and have not been resolved by any procedures tested. Both activities are associated with a major band of protein after the purified enzyme is subjected to polyacrylamide gel electrophoresis in a pH 8 system. Gel electrophoresis at pH 10.2 of the purified enzyme or in the presence of sodium dodecyl sulfate results in the appearance of many protein bands, none of which retains either catalytic activity. The molecular weight of both the crude and purified enzyme was estimated to be 4.3 to 4.9 x 105 from gel filtration chromatography and sucrose density gradient centrifugation. Prenyl transferase, squalene synthetase, squalene 2,3-oxide cyclase, and kaurene oxidase activities were absent from the purified enzyme. These general properties suggest that kaurene synthetase may be associated with a complex of proteins, although the nature of other polypeptides in the complex is unknown. Activities A and B showed pH optima of 7.5 and 6.9, respectively. Both activities required divalent metal ions with Mg2+ the most active. Ni2+ at 0.1 mm to 1.0 mm supported activity A but not activity B; advantage was taken of this in the biosynthesis of substrate quantities of copalol in crude extracts of the fungus. In the presence of EDTA, dithiothreitol stimulates activity B but not activity A. Several reagents which react with sulfhydryl groups were effective in blocking both activities, but to different extents; in general, activity B is more sensitive to these agents than activity A. The Km for geranylgeranyl-PP in activity A was determined to be approximately 0.7 µm. The Km for copalyl-PP in activity B was approximately 1.0 µm, although this determination was complicated by substrate inhibition at higher substrate concentrations. Several potential inhibitors of both activities were tested. 2'-Isopropyl-4'-(trimethylammonium chloride)-5'-methylphenyl piperidine-1-carboxylate (Amo 1618) and tributyl-2,4-dichlorobenzylphosphonium chloride (Phosfon D) are representative of plant growth retardants which strongly inhibit activity A (Ki for Amo 1618 = 2.3 x 10-7 m), but not activity B. These substances are noncompetitive inhibitors with respect to geranylgeranyl-PP. 2-(N,N-Dimethyl-N-octylammonium bromide)-p-menthan-1-ol (Q-64) and N,N-dimethylaminoethyl 2,2-diphenylpentyl ether (SKF 3301A) are representative of substances which are reasonably effective inhibitors of both activities. Deoxycholate was an inhibitor of activity B, whereas PPi and gibberellic acid were not appreciably active as inhibitors of either activity. Kinetic studies in the presence of both substrates by dual isotope techniques have indicated a preferential utilization in the conversion to kaurene of the copalyl moiety generated from geranylgeranyl-PP in comparison with the pool of added copalyl-PP. These studies also demonstrated an inhibition of activity A by copalyl-PP which is competitive with respect to geranylgeranyl-PP.