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 wildtype enzyme. The complexes with R-and S-azabisabolenes and PP i bind 3 Mg 2+ 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 Mg 2+ 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 only to 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 Mg 2+ 3 -PP i binding. Nevertheless, since R304K trichodiene synthase generates aberrant cyclic terpenoids with a 5000-fold reduction in k cat /K M , 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 suggests that the significant loss in activity results from compromised activation of the PP i leaving group. AbbreviationsFPP, farnesyl diphosphate; GPP, geranyl diphosphate; PP i , inorganic pyrophosphate Cyclic terpenoids, natural products derived from acyclic isoprenoid precursors such as geranyl diphosphate (C 10 monoterpenes), farnesyl diphosphate (C 15 sesquiterpenes), and geranylgeranyl diphosphate (C 20 diterpenes), are found in myriad life forms where they serve a wide variety of physiological and ecological functions (1,2,3). Bacteria and fungi produce numerous cyclic sesquiterpene antimicrobial and antifungal agents that target competing organisms and thereby confer a selective advantage to the host organism. For example, various Streptomyces species secrete terpene antibiotics (4,5) and certain species of Fusarium secrete † This work was supported by National Institutes of Health Grants GM56838 (D.W.C.) and GM30301 (D.E.C.). ‡ Atomic coordinates of R304K trichodiene synthase and its complexes with R-azabisabolene-Mg 2+ 3 -PP i and S-azabisaboleneMg 2+ 3 -PP i have been deposited in the Protein Data Bank with accession codes 2AEK, 2AEL, and 2AET, respectively. * To whom correspondence should be addressed at the Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, 231 South 34 th St., Philadelphia, PA 19104-6323 [215-898-5714 Mutagenesis studies indicate that conservative mutations of R304 (loop J-K), D...

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].…”

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

“…diterpene synthases demonstrated a common H-bond network (SI Appendix, Fig. S1 D and E) (27,29,(33)(34)(35)(36)(37)(38)(39)(40)(41). This suggests that a conserved basic amino acid-rich triad sets and holds PP i in place during the complete catalytic cascade.…”