Structure of epi-isozizaene synthase from Streptomyces coelicolor A3(2), a platform for new terpenoid cyclization templates

Abstract

The X-ray crystal structure of recombinant epi-isozizaene synthase (EIZS), a sesquiterpene cyclase from Streptomyces coelicolor A3(2), has been determined at 1.60 A resolution. Specifically, the structure of wild-type EIZS is that of its closed conformation in complex with three Mg(2+) ions, inorganic pyrophosphate (PP(i)), and the benzyltriethylammonium cation (BTAC). Additionally, the structure of D99N EIZS has been determined in an open, ligand-free conformation at 1.90 A 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 of 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 A resolution crystal structure of F198A EIZS in a complex with three Mg(2+) ions, PP(i), and BTAC reveals an 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 Hg(2+) ions has been determined at 1.90 A resolution, showing that metal binding triggers a significant conformational change of helix G to cap the active site.

Figure 1

(a) Ribbon plot of the EIZS-Mg²⁺₃-PP_i-BTAC complex showing the aspartate-rich motif (red) and the NSE motif (orange) flanking the mouth of the active site. The Mg²⁺ ions are shown as magenta spheres, PP_i and BTAC molecules are color coded by atom [carbon (yellow), nitrogen (blue), oxygen (red), and phosphate (orange)]. Helices are labeled according to the convention first established for FPP synthase (12). (b) The quaternary ammonium group of the benzyltriethylammonium cation (BTAC) serves as a mimic of a carbocation intermediate in catalysis.

Figure 2

(a) Simulated annealing omits maps (black) of the PP_i anion, Mg²⁺ ions, and BTAC, contoured at 5σ. Note the cation-π interactions between the positively charged quaternary ammonium group of BTAC and the aromatic rings of F95, F96 and F198 (red dashed lines). (b) Metal coordination interactions (black dashed lines) and hydrogen bond interactions (red dashed lines) in the EIZS-Mg²⁺₃-PP_i complex. (c) Simulated annealing omit maps (black) of the PP_i anion, Mg²⁺ ions, and BTAC in the active site of F198A EIZS, contoured at 5σ. Note the alternative position of BTAC resulting from the F198A mutation.

Figure 3

The Mg²⁺₃-PP_i binding motif is conserved among the bacterial and fungal sesquiterpene cyclases otherwise related by <20% amino acid sequence identity: (a) EIZS from S. coelicolor A3(2) (PDB 3KB9), (b) aristolochene synthase from A. terreus (PDB 2OAB, monomer D), and (c) trichodiene synthase from F. sporotrichioides (PDB 1JFG, monomer B).

Figure 4

(a) Superposition of ribbon plots of EIZS-Mg²⁺₃-PP_i-BTAC (green) and EIZS-Hg²⁺₄ (cyan; Hg²⁺ ions appear as red spheres). Helices D, G, H and J, which undergo the largest changes, are labeled. (b) Salt bridges (black dashes) between helix G and helices D and F stabilize the closed ligand-free structure. (c) Superposition of the EIZS-Mg²⁺₃-PP_i-BTAC complex (green) and D99N EIZS (purple), illustrating the structural changes in helix H and the H-α-1 and J–K loops that accompany active site closure.

Figure 5

(a) A stereoview of the active site surface contour encapsulated by the closed conformation of EIZS is shown as magenta meshwork. The aspartate-rich motif (red) and the NSE motif (orange) are oriented as in Figure 1. (b) The cyclization product, epi-isozizaene, is modeled into the enclosed active site contour of EIZS (magenta meshwork), and the location of the Mg²⁺₃-PP_i cluster is shown as a visual reference. (c) The enclosed active site contour of F198A EIZS (light brown meshwork) into which the new cyclization product β-acoradiene is modeled. The remolded active site contour in this mutant prevents epi-isozizaene formation but permits the formation of new or alternative sesquiterpene products predominantly derived from the bisabolyl carbocation intermediate.

Figure 6

The biosynthetic versatility of EIZS can be manipulated by site-directed mutagenesis, as illustrated for sesquiterpene products identified for wild-type (WT) and mutant cyclases. In general, more diverse sesquiterpene product arrays result from the substitution of aromatic residues defining the active site contour (red labels) than from substitution of residues that coordinate the Mg²⁺ ions required for catalysis (blue labels (14)). For example, F198A EIZS does not generate epi-isozizaene at all, but instead generates a mixture of sesquisabinene-A, Z-α-and Z-γ-bisabolenes, sesquiphellandrene, and β-acoradiene as its major cyclization products. Remolding the active site contour permits the generation of alternative products as long as they can be accommodated within the remolded template, as illustrated for β-acoradiene in Figure 5. It is interesting to note that many of these reactions have been examined in theoretical and computational studies (33).