Structure of Epi-Isozizaene Synthase from Streptomyces coelicolor A3(2), a Platform for New Terpenoid Cyclization Templates

The X-ray crystal structure of recombinant epi-isozizaene synthase (EIZS), it sesquiterpene cyclase from Streptomyces coelicolor A3(2), has been determined at 1.60 angstrom resolution. Specifically, the structure of wild-type EIZS is that of its closed conformation in complex with three Mg2+ ions, inorganic pyrophosphate (PPi), and the benzyltriethylammonium cation (BTAC). Additionally, the structure of D99N EIZS has been determined in an open, ligand-free conformation at 1.90 angstrom resolution. Comparison of these two structures provides the first view of conformational changes required for substrate binding and catalysis in a bacterial terpenoid cyclase. Moreover, the binding interactions of BTAC may mimic those of a carbocation intermediate in catalysis. Accordingly, the aromatic rings of F95, F96, and F198 appear to be well-oriented to stabilize carbocation intermediates in the cyclization cascade through cation-pi interactions. Mutagenesis or aromatic residues in the enzyme active site results in the production of alternative sesquiterpene product arrays due to altered modes of stabilization of carbocation intermediates as well as altered templates for the cyclization of farnesyl diphosphate. Accordingly, the 1.64 angstrom resolution crystal structure of F198A EIZS in a complex with three Mg2+ tons, PPi, and BTAC reveals in alternative binding orientation of BTAC; alternative binding orientations of a carbocation intermediate could lead to the formation of alternative products. Finally, the crystal structure of wild-type EIZS in a complex with four Hg2+ ions has been determined at 1.90 angstrom resolution, showing that metal binding triggers a significant conformational change of helix G to cap the active site.