Plant Physiol, April 2001, Vol. 125, pp. 1754-1765

The Cyclization of Farnesyl Diphosphate and Nerolidyl Diphosphate by a Purified Recombinant -Cadinene Synthase¹

Departments of Biochemistry and Biophysics (C.R.B., J.-L.L., D.W.P.) and Chemistry (H.J.W.), Texas A&M University, College Station, Texas 77843-2128; and United States Department of Agriculture, Agricultural Research Service, Southern Plains Agricultural Research Center, College Station, Texas 77845 (J.L., R.D.S.)

The first step in the conversion of the isoprenoid intermediate, farnesyl diphosphate (FDP), to sesquiterpene phytoalexins in cotton (Gossypium barbadense) plants is catalyzed by -cadinene (CDN) synthase. CDN is the precursor of desoxyhemigossypol and hemigossypol defense sesquiterpenes. In this paper we have studied the mechanism for the cyclization of FDP and the putative intermediate, nerolidyl diphosphate, to CDN. A purified recombinant CDN synthase (CDN1-C1) expressed in Escherichia coli from CDN1-C1 cDNA isolated from Gossypium arboreum cyclizes (1RS)-[1-²H](E, E)-FDP to >98% [5-²H]and [11-²H]CDN. Enzyme reaction mixtures cyclize (3RS)-[4,4,13,13,13-²H₅]-nerolidyl diphosphate to 62.1% [8,8,15,15,15-²H₅]-CDN, 15.8% [6,6,15,15,15-²H₅]--bisabolol, 8.1% [6,6,15,15,15-²H₅]-()-bisabolene, 9.8% [4,4,13,13-²H₄]-(E)--farnesene, and 4.2% unknowns. Competitive studies show that (3R)-nerolidyl diphosphate is the active enantiomer of (3RS)-nerolidyl diphosphate that cyclized to CDN. The k_cat/K_m values demonstrate that the synthase uses (E,E)-FDP as effectively as (3R)-nerolidyl diphosphate in the formation of CDN. Cyclization studies with (3R)-nerolidyl diphosphate show that the formation of CDN, (E)--farnesene, and -bisabolene are enzyme dependent, but the formation of -bisabolol in the reaction mixtures was a Mg²⁺-dependent solvolysis of nerolidyl diphosphate. Enzyme mechanisms are proposed for the formation of CDN from (E,E)-FDP and for the formation of CDN, (E)--farnesene, and -bisabolene from (3RS)-nerolidyl diphosphate. The primary structures of cotton CDN synthase and tobacco epi-aristolochene synthase show 48% identity, suggesting similar three-dimensional structures. We used the SWISS-MODEL to test this. The two enzymes have the same overall structure consisting of two -helical domains and epi-aristolochene synthase is a good model for the structure of CDN synthase. Several amino acids in the primary structures of both synthases superimpose. The amino acids having catalytic roles in epi-aristochene synthase are substituted in the CDN synthase and may be related to differences in catalytic properties.