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

• Published in: Biochemistry (Easton) 2009-06, Vol.48 (26), p.6175-6183
• Main Authors: Gennadios, Heather A, Gonzalez, Veronica, Di Costanzo, Luigi, Li, Amang, Yu, Fanglei, Miller, David J, Allemann, Rudolf K, Christianson, David W
• Format: Article
• Language: English
• Subjects: Gossypium arboreum
• ISSN: 0006-2960
• DOI: 10.1021/bi900483b

(+)-*d-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 A 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 A 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 D307DTYD311 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, D451DVAE455, 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 D307DTYD311 and D451DVAE455 segments reveal the contributions of these segments to catalysis.