Role of arginine-304 in the diphosphate-triggered active site closure mechanism of trichodiene synthase

Abstract

The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PP(i)) and aza analogues of the bisabolyl carbocation intermediate are reported. The R304K substitution does not cause large changes in the overall structure in comparison with the wild-type enzyme. The complexes with (R)- and (S)-azabisabolenes and PP(i) bind three Mg2+ ions, and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change is only slightly attenuated compared to that of the wild-type enzyme complexed with Mg2+(3)-PP(i), in which R304 donates hydrogen bonds to PP(i) and D101. In R304K trichodiene synthase, K304 does not engage in any hydrogen bond interactions in the unliganded state and it donates a hydrogen bond to only PP(i) in the complex with (R)-azabisabolene; K304 makes no hydrogen bond contacts in its complex with PP(i) and (S)-azabisabolene. Thus, although the R304-D101 hydrogen bond interaction stabilizes diphosphate-triggered active site closure, it is not required for Mg2+(3)-PP(i) binding. Nevertheless, since R304K trichodiene synthase generates aberrant cyclic terpenoids with a 5000-fold reduction in kcat/KM, it is clear that a properly formed R304-D101 hydrogen bond is required in the enzyme-substrate complex to stabilize the proper active site contour, which in turn facilitates cyclization of farnesyl diphosphate for the exclusive formation of trichodiene. Structural analysis of the R304K mutant and comparison with the monoterpene cyclase (+)-bornyl diphosphate synthase suggest that the significant loss in activity results from compromised activation of the PP(i) leaving group.

Figure 1

(a) Superposition of the active sites of trichodiene synthase, unliganded (green) and complexed with diphosphate (yellow) reveals that D101 and R304 break hydrogen bonds with R62 and T69, respectively, to form a new salt link with each other, thus capping the active site. Diphosphate is shown in red and magenta. Solvent atoms are omitted for clarity and Mg²⁺ ions appear as spheres. (b) Superposition of C_α traces of trichodiene synthase: unliganded (green) and Mg²⁺₃-PP_i complex (yellow). The PP_i anion (red) illustrates the location of the active site; helices and loops that undergo significant diphosphate-induced conformational changes are indicated.

Figure 2

(a) Postulated mechanism for the cyclization of farnesyl diphosphate (FPP) to trichodiene by trichodiene synthase; OPP = diphosphate, NPP = nerolidyl diphosphate (18). (b) R-azabisabolene, a cationic analogue of the bisabolyl cation in the trichodiene synthase mechanism, is a strong competitive inhibitor in the presence of inorganic diphosphate (PP_i) with K_i = 0.51 μM (22); similarly, the enantiomer S-azabisabolene binds in the presence of PP_i with K_i = 0.47 μM (22), suggesting that the stereochemical discrimination is weak at the corresponding step in catalysis.

Figure 3

Active site of unliganded R304K trichodiene synthase. (a) Simulated annealing omit maps of K304 and Mg²⁺ ion are shown in cyan (6.0 σ) and maroon (8.0 σ), respectively. The mutated side chain is well-defined by clear electron density. Metal coordination interactions are represented by black broken lines. (b) Superposition with the active site of unliganded wild-type trichodiene synthase (purple).

Figure 4

Active site of R304K trichodiene synthase complexed with Mg²⁺₃-PP_i and R-azabisabolene (yellow carbon skeleton). (a) Simulated annealing omit maps of K304, PP_i, metal ions, and R-azabisabolene are shown in cyan (4.6 σ), green (5.4 σ), maroon (5.2 σ), and blue (4.0 σ), respectively. Metal coordination interactions are shown as black dotted lines. (b) Superposition with the activesite of wild-type trichodiene synthase complexed with Mg²⁺₃-PP_i (purple). Hydrogen bonds and metal coordination interactions in the mutant are shown asgray and black dotted lines, respectively.

Figure 5

Active site of R304K trichodiene synthase complexed with Mg²⁺₃-PPi and S-azabisabolene. (a) Simulated annealing omit maps of PP_i, metal ions and S-azabisabolene are shown in green (7.0 σ), maroon (4.6 σ), and blue (4.5 σ), respectively. Note that the electron density is sufficiently ambiguous that S-azabisabolene cannot be modelled into the density. Metal coordination interactions are shown as black dotted lines. (b) Superposition with the active site of wild-type trichodiene synthase complexed with Mg²⁺₃-PP_i (purple). Hydrogen bonds and metal coordination interactions in the mutant are shown as gray and black dotted lines, respectively.

Figure 6

(a) Active site of (+)-bornyl diphosphate synthase from Salvia officinalis complexed with Mg²⁺₃ and product bornyl diphosphate (yellow hydrocarbon moiety). Metal ions are represented by gray spheres. Hydrogen bonds and metal coordination interactions are shown as gray and black dotted lines, respectively. (b) Active site of wild-type trichodiene synthase complexed with Mg²⁺₃ and co-product PP_i. Metal ions are represented by gray spheres. Hydrogen bonds and metal coordination interactions are shown as gray and black dotted lines, respectively. The view corresponds to that in (a), from within the active site.