Molecular Recognition of the Substrate Diphosphate Group Governs Product Diversity in Trichodiene Synthase Mutants^,

Abstract

The X-ray crystal structures of Y305F trichodiene synthase and its complex with coproduct inorganic pyrophosphate (PP(i)) and of Y305F and D100E trichodiene synthases in ternary complexes with PP(i) and aza analogues of the bisabolyl carbocation intermediate are reported. The Y305F substitution in the basic D(302)RRYR motif does not cause large changes in the overall structure in comparison with the wild-type enzyme in either the uncomplexed enzyme or its complex with PP(i). However, the loss of the Y305F-PP(i) hydrogen bond appears to be compensated by a very slight shift in the position of the side chain of R304. The putative bisabolyl carbocation mimic, R-azabisabolene, binds in a conformation and orientation that does not appear to mimic that of the actual carbocation intermediate, suggesting that the avid inhibition by R- and S-azabisabolenes arises more from favorable electrostatic interactions with PP(i) rather than any special resemblance to a reaction intermediate. Greater enclosed active-site volumes result from the Y305F and D100E mutations that appear to confer greater variability in ligand-binding conformations and orientations, which results in the formation of aberrant cyclization products. Because the binding conformations and orientations of R-azabisabolene to Y305F and D100E trichodiene synthases do not correspond to binding conformations required for product formation and because the binding conformations and orientations of diverse substrate and carbocation analogues to other cyclases such as 5-epi-aristolochene synthase and bornyl diphosphate synthase generally do not correspond to catalytically productive complexes, we conclude that the formation of transient carbocation intermediates in terpene cyclization reactions is generally under kinetic rather than thermodynamic control.

Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases

“…(vi) This steering and cation-attracting role of R580-PP i leads to direct premature deprotonation in cations C-F. (vii) It further leads to direct or indirect premature deprotonation when the finetuned and electrostatically balanced timeline of cyclization and hydride shifts is perturbed. (viii) Instead, in the absence of counteracting amino acid-assisted electrostatic effects, steric restriction imposed by the active site and steering of R580-PP i provide for cyclization from cation A→E, which corroborates the template model and dominating kinetic instead of thermodynamic control (24,54). (ix) Due to proximity of the positively charged carbocation to R580-PP i and in agreement with docking cluster analyses, cations F and E comprise lower energy levels in TXS compared with cation C in relation to cation A.…”

Exploring biosynthetic diversity with trichodiene synthase

“…Although the structures of trichodiene synthase mutants complexed with the presumably positively charged ligands R-azabisabolene or S-azabisabolene plus Mg 2+ 3 -PP i have previously been reported [11,12], the protonation states of their tertiary amino nitrogen atoms cannot be determined in the 2.5-2.95 Ǻ resolution electron density maps of proteins. In contrast, the four hydrocarbon substituents of the quaternary ammonium group of BTAC are readily visible in the electron density map, thereby conclusively confirming that the ligand bound in the active site of trichodiene synthase is positively charged.…”

“…These aza compounds exhibit a 100-fold range of binding affinities in the presence of PP i (Table 2). Structural studies show that both the R-and the S-azabisabolene analogues are bound in a variety of orientations in the active sites of Y305F and R304K trichodiene synthases [11,12]. Interestingly, while BTAC binds ∼15-fold less tightly than either R-or S-azabisabolene (Table 2, Fig.…”

“…Thus, it appears that electrostatic interactions in the Mg 2+ 3 -PP i cluster are weaker when a positive charge is bound or generated in catalysis. The weakening of electrostatic interactions in the Mg 2+ 3 -PP i cluster, which "locks" the active site template in the closed conformation, could contribute to the anomalous binding modes observed for R-and S-azabisabolenes in complexes with Y305F and R304K trichodiene synthases [11,12]. Moreover, it is possible that metal coordination interactions are weakened upon initial carbocation formation in the cyclization cascade.…”

“…2b) require PP i for measurable inhibition with K I = 0.51 μM and an induced inhibition constant of 2.6 μM, with the magnitude of inhibition proportional to the PP i concentration [10]. X-ray crystallographic analyses of azabisabolene complexes with trichodiene synthase mutants show that the binding of these positively charged inhibitors in multiple conformations is stabilized by Mg 2+ 3 -PP i complexation [11,12]. A similar Mg 2+ 3-PP i cluster also stabilizes the binding of certain aza analogues to the monoterpene synthase, bornyl diphosphate synthase [13].…”