Crystal structure of F95Q epi-isozizaene synthase, an engineered sesquiterpene cyclase that generates biofuel precursors β- and γ-curcumene

Abstract

The saturated hydrocarbon bisabolane is a diesel fuel substitute that can be derived from sesquiterpene precursors bisabolene or curcumene. These sesquiterpenes are generated from farnesyl diphosphate in reactions catalyzed by eponymous terpenoid cyclases, but they can also be generated by engineered terpenoid cyclases in which cyclization cascades have been reprogrammed by mutagenesis. Here, we describe the X-ray crystal structure determination of F95Q epi-isozizaene synthase (EIZS), in which the new activity of curcumene biosynthesis has been introduced and the native activity of epi-isozizaene biosynthesis has been suppressed. F95Q EIZS generates β- and γ-curcumene regioisomers with greater than 50% yield. Structural analysis of the closed active site conformation, stabilized by the binding of 3 Mg²⁺ ions, inorganic pyrophosphate, and the benzyltriethylammonium cation, reveals a product-like active site contour that serves as the cyclization template. Remolding the active site contour to resemble curcumene instead of epi-isozizaene is the principal determinant of the reprogrammed cyclization cascade. Intriguingly, an ordered water molecule comprises part of the active site contour. This water molecule may also serve as a final proton acceptor, along with inorganic pyrophosphate, in the generation of curcumene regioisomers; it may also contribute to the formation of sesquiterpene alcohols identified as minor side products. Thus, the substitution of polar side chains for nonpolar side chains in terpenoid cyclase active sites can result in the stabilization of bound water molecules that, in turn, can serve template functions in isoprenoid cyclization reactions.

Keywords: Diesel fuel; Energy biotechnology; Enzyme; Protein engineering; X-ray crystallography.

Figure 1

Proposed mechanism for F95Q EIZS-catalyzed cyclization of farnesyl diphosphate to form β- and γ-curcumene regioisomers. C3-C2 bond rotation indicated by magenta arrow, 1,2-hydride shift indicated by green arrow. Red and blue arrows correspond to proton elimination reactions yielding β- and γ-curcumene, respectively. The deprotonation reaction leading to γ-curcumene could be mediated by water #372 trapped in the active site as indicated, or it could be mediated by metal-bound inorganic pyrophosphate (^–OPP) as discussed in the text. Although the stereochemistry of β- and γ-curcumene has not been established, it is presumed to derive from the 7(S)-homobisabolyl cation definitively established in the biosynthesis of epi-isozizaene. Even if the 7(R)-homobisabolyl cation is formed, either enantiomer of β- and γ-curcumene could be chemically hydrogenated to yield diesel fuel substitute bisabolane.

Figure 2

(Left) Ribbonplot of F95Q EIZS color-coded as follows: backbone atoms, blue; DDXXD metal-binding motif, red; NSE metal-binding motif, orange; BTAC, stick figure with pink C atoms and blue N atom; magnesium ions, green spheres; PP_i, stick figure with orange P atoms and red O atoms. (Right) Superposition of the F95Q EIZS-Mg²⁺₃-PP_i-BTAC complex with the wild-type EIZS-Mg²⁺₃-PP_i-BTAC complex (all atoms yellow; PDB 3KB9).

Figure 3

(a) Stereoview of F95Q EIZS-Mg²⁺₃-PP_i-BTAC complex, showing Polder omit maps for BTAC and Q95 (contoured at 6σ and 4σ, respectively). Color codes are as follows: C (protein) = blue, C (BTAC) = cyan, N = dark blue, O = red, P = orange, Mg²⁺ ions = green spheres. Water #372 is shown as a small red sphere. (b) Complex in (a) superimposed on the wild-type EIZS-Mg²⁺₃-PP_i-BTAC complex (gold; Mg²⁺ ions = magenta spheres). Hydrogen bond interactions are indicated by red dashed lines. Other interactions are shown as black dashed lines.

Figure 4

(a) Stereoview of the EIZS-Mg²⁺₃-PP_i-BTAC complex, less the BTAC molecule. Color coding is as follows: Mg²⁺ ions = large magenta spheres; F95 and PP_i are shown with C = aqua, O = red, P = orange. The active site contour is represented by firebrick red meshwork and is fit by a model of epi-isozizaene (gold). (b) Stereoview of the F95Q EIZS-Mg²⁺₃-PP_i-BTAC complex, less the BTAC molecule. Color coding is as follows: Mg²⁺ ions = large green spheres, water #372 = small red sphere; Q95 and PP_i are shown with C = green, N = dark blue, O = red, P = orange. The Q95---water #372 hydrogen bond is represented by a blue dashed line. The active site contour is represented by purple meshwork and is fit by models of β- and γ-curcumene and the 7(S)-homobisabolyl cation (red, blue, and gold, respectively).