Crystal Structure of (+)-δ-Cadinene Synthase from Gossypium arboreum and Evolutionary Divergence of Metal Binding Motifs for Catalysis

(+)-delta-Cadinene synthase (DCS) from Gossypium arboreum (tree cotton) is a sesquiterpene cyclase that catalyzes the cyclization of farnesyl diphosphate in the first committed step of the biosynthesis of gossypol, a phytoalexin that defends the plant from bacterial and fungal pathogens. Here, we report the X-ray crystal structure of unliganded DCS at 2.4 angstrom resolution and the structure of its complex with three putative Mg2+ ions and the substrate analogue inhibitor 2-fluorofarnesyl diphosphate (2F-FPP) at 2.75 angstrom resolution. These structures illuminate unusual features that accommodate the trinuclear metal cluster required for substrate binding and catalysis. Like other terpenoid cyclases, DCS contains a characteristic aspartate-rich (DDTYD311)-D-307 motif on helix D that interacts with Mg2+ (A) and Mg2+ (C). However, DCS appears to be unique among terpenoid cyclases in that it does not contain the "NSE/DTE" motif on helix H that specifically chelates Mg2+ (B), which is usually found as the signature sequence (N,D)D(L,I,V)X(S,T)XXXE (boldface indicates Mg2+ (B) ligands). Instead, DCS contains a second aspartate-rich motif, D(451)DVAE(455), that interacts with Mg2+ (B). In this regard, DCS is more similar to the isoprenoid chain elongation enzyme farnesyl diphosphate synthase, which also contains two aspartate-rich motifs, rather than the greater family of terpenoid cyclases. Nevertheless, the structure of the DCS-2F-FPP complex shows that the structure of the trinuclear magnesium cluster is generally similar to that of other terpenoid cyclases despite the alternative Mg2+ (B) binding motif. Analyses of DCS mutants with alanine substitutions in the D-307 DTYD311 and D(451)DVAE(455) segments reveal the contributions of these segments to catalysis.