Mechanistic insights from the binding of substrate and carbocation intermediate analogues to aristolochene synthase

Abstract

Aristolochene synthase, a metal-dependent sesquiterpene cyclase from Aspergillus terreus, catalyzes the ionization-dependent cyclization of farnesyl diphosphate (FPP) to form the bicyclic eremophilane (+)-aristolochene with perfect structural and stereochemical precision. Here, we report the X-ray crystal structure of aristolochene synthase complexed with three Mg(2+) ions and the unreactive substrate analogue farnesyl-S-thiolodiphosphate (FSPP), showing that the substrate diphosphate group is anchored by metal coordination and hydrogen bond interactions identical to those previously observed in the complex with three Mg(2+) ions and inorganic pyrophosphate (PPi). Moreover, the binding conformation of FSPP directly mimics that expected for productively bound FPP, with the exception of the precise alignment of the C-S bond with regard to the C10-C11 π system that would be required for C1-C10 bond formation in the first step of catalysis. We also report crystal structures of aristolochene synthase complexed with Mg(2+)3-PPi and ammonium or iminium analogues of bicyclic carbocation intermediates proposed for the natural cyclization cascade. Various binding orientations are observed for these bicyclic analogues, and these orientations appear to be driven by favorable electrostatic interactions between the positively charged ammonium group of the analogue and the negatively charged PPi anion. Surprisingly, the active site is sufficiently flexible to accommodate analogues with partially or completely incorrect stereochemistry. Although this permissiveness in binding is unanticipated, based on the stereochemical precision of catalysis that leads exclusively to the (+)-aristolochene stereoisomer, it suggests the ability of the active site to enable controlled reorientation of intermediates during the cyclization cascade. Taken together, these structures illuminate important aspects of the catalytic mechanism.

Figure 1

Proposed mechanism of (+)-aristolochene generation as catalyzed by A. terreus aristolochene synthase (PPO = diphosphate; PPO^– = inorganic pyrophosphate; PPOH = protonated inorganic pyrophosphate). Note that the germacrene A intermediate must reorient itself relative to protonated inorganic pyrophosphate (which is rigidly positioned by metal coordination and hydrogen bond interactions) to enable subsequent protonation and deprotonation steps in catalysis. Certain sequences might proceed in concerted rather than stepwise fashion, in which case carbon atoms bearing full positive charges would only develop partial positive charges. Carbon atoms are numbered in all structures based on the numbering scheme shown for FPP to better follow relevant carbon atoms during the course of the reaction. Analogues of substrate and possible carbocation intermediates in the cyclization cascade are shown in green.

Figure 2

Structural comparison of unliganded ATAS (left) with the ATAS-FSPP complex (right) illustrating conformational changes triggered by the binding of FSPP and 3 Mg²⁺ ions in the active site. The aspartate-rich DDXXD metal-binding segment on helix D is red and the “NSE” metal-binding segment on helix H is orange. In the ATAS-FSPP complex, Mg²⁺ ions are magenta and FSPP is shown as a stick-figure. Selected secondary structure elements are labeled as discussed in the text.

Figure 3

(a) Simulated annealing omit map of FSPP (contoured at 4.0σ) bound to monomer A in the ATAS-Mg²⁺₃-FSPP complex. Atoms are color-coded as follows: C = yellow (protein) or gray (FSPP), O = red, N = blue, P = orange, S = yellow, Mg²⁺ ions = silver spheres, solvent molecules = red spheres. Metal coordination interactions are shown as red dotted lines; hydrogen bond interactions are shown as black dotted lines. Water molecule “w” is trapped in the active site along with FSPP. (b) Superposition of the ATAS-Mg²⁺₃-FSPP complex (color-coded as in (a)) and the ATAS-Mg²⁺₃-PP_i complex (all atoms pale cyan).

Figure 4

(a) Simulated annealing omit map of iminium cation 1 (contoured at 4.0σ) bound to monomer A in the ATAS-1 complex. Atoms are color-coded as follows: C = yellow (protein) or gray (1), O = red, N = blue, P = orange, S = yellow, Mg²⁺ ions = silver spheres, solvent molecules = red spheres. Metal coordination and hydrogen bond interactions are shown as red and black dotted lines, respectively. Water molecule “w” is trapped in the active site along with 1. (b) Superposition of the ATAS-1 complex (color-coded as in (a)) and the ATAS-FSPP complex (all atoms pale cyan).

Figure 5

(a) Simulated annealing omit map of tertiary ammonium cation 2 (contoured at 2.6σ) bound to monomer A in the ATAS-2 complex. Atoms are color coded as follows: C = yellow (protein) or gray (2), O = red, N = blue, P = orange, S = yellow, Mg²⁺ ions = silver spheres, solvent molecules = red spheres. Metal coordination and hydrogen bond interactions are shown as red and black dotted lines, respectively. Water molecule “w” is trapped in the active site along with 3. (b) Superposition of the ATAS-2 complex (color coded as in (a)) with the ATAS-1 complex (all atoms pale cyan).

Figure 6

(a) Simulated annealing omit map of tertiary ammonium cation 3 (contoured at 3.5σ) bound to monomer B in the ATAS-3 complex. Atoms are color coded as follows: C = yellow (protein) or gray (3), O = red, N = blue, P = orange, S = yellow, Mg²⁺ ions = silver spheres, solvent molecules = red spheres. Metal coordination, hydrogen bond, and cation-π interactions are shown as red, black, and green dotted lines, respectively. Water molecules “w” and “ww” are trapped in the active site along with 3. (b) Superposition of the ATAS-3 complex (color coded as in (a)) with the ATAS-FSPP complex (all atoms pale cyan).

Figure 7

(a) Simulated annealing omit map of tertiary ammonium cation 4 (contoured at 3.0σ) bound to monomer A in the ATAS-4 complex. Atoms are color-coded as follows: C = yellow (protein) or gray (4), O = red, N = blue, P = orange, S = yellow, Mg²⁺ ions = silver spheres, solvent molecules = red spheres. Metal coordination, hydrogen bond, and cation-π interactions are shown as red, black, and green dotted lines, respectively. Water molecule “w” is trapped in the active site along with 4. (b) Superposition of the ATAS-4 complex (color coded as in (a)) with the ATAS-FSPP complex (all atoms pale cyan).

Figure 8

(a) Simulated annealing omit map of tertiary ammonium cation 5 (contoured at 3.1σ) bound to monomer A in the ATAS-5 complex. Atoms are color coded as follows: C = yellow (protein) or gray (5), O = red, N = blue, P = orange, S = yellow, Mg²⁺ ions = silver spheres, solvent molecules = red spheres. Metal coordination, hydrogen bond, and cation-π interactions are shown as red, black, and green dotted lines, respectively. Water molecules “w” and “ww” are trapped in the active site along with 5. (b) Superposition of the ATAS-5 complex (color coded as in (a)) with the ATAS-4 complex (all atoms pale cyan).