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

Cyclic terpenoids, natural products derived from acyclic isoprenoid precursors such as geranyl diphosphate (C10 monoterpenes), farnesyl diphosphate (C15 sesquiterpenes), and geranylgeranyl diphosphate (C20 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 the trichothecane class of antibiotics (6). In comparison, plants produce C10, C15, and C20 cyclic terpenoids for defense against fungi and insects. For example, Abies grandis (Grand fir) secretes multiple cyclic terpenoids in response to stem wounds and insect attack (7). Interestingly, cyclic terpenoids contribute to the desirable taste and aroma of plants such as Salvia officinalis (sage) (8), leading to their use as culinary flavorings. A more significant health-related benefit of plant terpenoids is in their potential pharmaceutical utility, e.g., the cancer chemotherapy drug paclitaxel (Taxol)™ (9), antimalarials like artemisinin from Artemisia annua (10), anti-microbials like linalool and citranellal from Melissa officinalis (balm) (11), and antifungal agents such as α- and β-pinenes from tea tree oil (12). In bacteria, plants, and fungi, cyclic sesquiterpenes (C15) derive from the acyclic, linear precursor farnesyl diphosphate (FPP) through multi-step reactions catalyzed by sesquiterpene synthases (also known as sesquiterpene cyclases) (2, 13, 14). The highly reactive carbocation intermediates of terpenoid cyclization reactions are protected from bulk water during catalysis by a diphosphate-triggered conformational change that caps the cyclase active site. The molecular recognition of the substrate diphosphate group is achieved by metal coordination interactions with divalent metal ions (Mg2+, or sometimes Mn2+) (15, 16) and by multiple hydrogen bonds with enzyme residues. For example, in Fusarium sporotrichiodes trichodiene synthase complexed with inorganic pyrophosphate (PPi), the PPi anion coordinates to 3 Mg2+ ions (liganded by D100, N225, S229, E233, and PPi) and also accepts hydrogen bonds from R182, K232, R304, and Y305 (17). Of these residues, R304 appears to play a key role in the diphosphate-triggered active site closure mechanism: upon PPi binding, R304 breaks a hydrogen bond with T69 in order to form hydrogen bonds with PPi and D101 (which breaks a hydrogen bond interaction with R62) (Figure 1a) (17). These conformational changes, plus those involving the N-terminus (M1-P5), helices 1, D, H, K, and L, and loops 1-A, D-D1, H-α-1, J-K, and K-L (Figure 1b) are likely triggered by the diphosphate group of the FPP substrate as well. The trichodiene synthase mechanism is summarized in Figure 2a (18). 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 ... 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 ... Mutagenesis studies indicate that conservative mutations of R304 (loop J-K), D101 (helix D), or D100 (helix D) compromise activity (19, 20). For example, consider D100, which coordinates to Mg2+A and Mg2+C (17). The D100E mutation results in a 22-fold decrease in kcat/KM and the formation of five aberrant products in addition to trichodiene (20). The crystal structure of D100E trichodiene synthase shows that the diphosphate-triggered conformational change is highly attenuated, partly due to compromised metal binding (21). D101E trichodiene synthase shows a 5-fold decrease in kcat/KM and the generation of five aberrant products in addition to trichodiene (20). In comparison, the R304K mutation results in a 5,000-fold decrease in kcat/KM as well as the generation of at least two aberrant products in addition to trichodiene (19). To probe the importance of R304 as a hydrogen bond donor to diphosphate and its structural role in the diphosphate-triggered conformational change that caps the active site cavity, we now report the X-ray crystal structures of R304K trichodiene synthase and its complexes with both (4R)-7-azabisabolene (designated “R-azabisabolene”), a cationic analogue of the natural bisabolyl carbocation intermediate, and its enantiomer (4S)-7-azabisabolene (designated “S-azabisabolene”) (Figure 2b) (22).