The cyclization of farnesyl diphosphate and nerolidyl diphosphate by a purified recombinant delta-cadinene synthase

Abstract

The first step in the conversion of the isoprenoid intermediate, farnesyl diphosphate (FDP), to sesquiterpene phytoalexins in cotton (Gossypium barbadense) plants is catalyzed by delta-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]-alpha-bisabolol, 8.1% [6,6,15,15,15-2H5]-(beta)-bisabolene, 9.8% [4,4,13,13-2H4]-(E)-beta-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 kcat/Km 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)-beta-farnesene, and beta-bisabolene are enzyme dependent, but the formation of alpha-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)-beta-farnesene, and beta-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 alpha-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.

Figure 1

GC-MS analysis of the sesquiterpene products formed from the cyclization of (3RS)-[4,4,13,13,13-²H₅]-NDP by recombinant CDN1-C1.

Figure 2

Mass spectra of the sesquiterpenes formed from the cyclization of (3RS)-[4,4,13,13,13-²H₅]-NDP by CDN1-C1. A, [4,4,13,13-²H₄]-(E)-β-farnesene. B, [6,6,15,15,15-²H₅]-β-bisabolene. C, [8,8,15,15,15-²H₅]-CDN. D, [6,6,15,15,15-²H₅]-α-bisabolol.

Figure 3

Proposed reaction mechanism for the cyclization of (1RS)-[1-²H](E,E)-FDP by CDN1-C1.

Figure 4

Proposed reaction mechanism for the cyclization of (3RS)-[4,4,13,13,13-²H₅]-NDP.

Figure 5

Active site region of CDN1-C1 modeled on the TEAS structure. Amino acids from the crystal structure of TEAS within 0.4 nm of FHP and the MG (II)_n ions are shown in red and those from the modeled CDN1-C1 are shown in CPK coloring. MG (II) ions are shown as blue spheres and FHP is shown in gold. Amino acid numbers are shown for TEAS. A, Identical active site residues for TEAS and CDN1 synthase. B, Active site residues for TEAS and CDN1-C1 that differ in identity or position. The arrow labeled “loop” points to the mobile loop from TEAS residues 519 through 525. The arrow labeled “S-S” points to the disulfide bond between C402 and C440 in the CDN1-C1 model. The figure was constructed with the Swiss Pdb Viewer (Swiss Institute of Bioinformatics, Geneva).

Figure 6

SDS-PAGE of purified recombinant CDN1-C1. A, Protein markers. B, CDN1-C1 synthase after the removal of the GST protein from the GST-CDN1-C1 fusion protein with thrombin and the separation of the CDN1-C1 free of the GST protein by chromatography on a fast-flow Q-Sepharose anion-exchange column.