Substrate Folding Modes in Trichodiene Synthase: A Determinant of

Our results advocate a fatalism that the destiny of substrate is determined once it is folded by the active pocket, and each folding mode corresponds ...
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Substrate Folding Modes in Trichodiene Synthase: A Determinant of Chemo- and Stereoselectivity Yong-Heng Wang,† Hujun Xie,‡ Jingwei Zhou,† Fan Zhang,† and Ruibo Wu*,† †

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, P. R. China Department of Applied Chemistry, Zhejiang Gongshang University, Hangzhou 310035, P. R. China

‡

S Supporting Information *

ABSTRACT: The folding mode of substrate FPP in sesquiterpene cyclases/synthases is key to the chemo- and stereoselectivity of the ultimate sesquiterpene products. However, the precise substrate folding modes in most sesquiterpene cyclases are still elusive, and it is challenging for theoretical simulations due to the high flexibility of FPP. Herein, by DFT/ MM MD simulations, we obtain the optimal folding mode of FPP in the 1,6-closure trichodiene synthase and illuminate the whole catalytic mechanism for the biosynthesis of trichodiene. Furthermore, a simple and practical rule is proposed to decipher the relationship between the diverse FPP folding modes and chemical selectivity toward 1,6and 1,10-ring closure, which are common pathways in all sesquiterpene cyclases.

KEYWORDS: folding modes, sesquiterpene cyclases/synthase, QM/MM, chemical control, retro-biosynthesis analysis arnesyl diphosphate (FPP, Figure 1a), a flexible 15-carbon isoprenoid substrate, can be cyclized into more than 300 known monocylic, bicyclic, and tricyclic sesquiterpene natural products with a wide variety of structures and stereochemistry, providing abundant sources for new drug discovery, catalyzed by sesquiterpene cyclases (also called as sesquiterpene synthase) mainly via an 1,6- or 1,10-ring closure pathway.1 In general, a given sesquiterpene cyclase catalyzes either of the two pathways solely, although the promiscuous one that mainly catalyzes 1,10-closure with 1,6-closure as a minor pathway has been reported.2 However, little is known regarding how the enzymes control the two distinct pathways with high fidelity and in what situation the promiscuity emerges. It is believed that the precise folding of substrate highly correlates with the structure and stereochemistry of the ultimate sesquiterpene products.3 Unfortunately, the exact FPP folding modes in most sesquiterpene cyclases are not clear, and especially for 1,6-cyclases, no catalytically productive substrate folding mode has been reported.4 Meanwhile, multiple folding modes, instead of a uniform one, have been detected experimentally in the sesquiterpene 1,10-cyclases.5 In addition, it is challenging for building computational models due to the high flexibility of the substrate.6 These severely hamper the understanding of the correspondences between the substrate folding modes and the main biocyclization pathways in sesquiterpene cyclases (1,6 vs 1,10). Fusarium sporotrichioides trichodiene synthase (FSTS), which catalyzes the conversion of FPP into the sesquiterpene product trichodiene (TD, Figure 1a), the parent compound of the trichothecane family of antibiotics and mycotoxins, is the

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© 2017 American Chemical Society

widely studied 1,6-cyclase in terms of mechanistic enzymology and structural biology.1d,7 The potential conformation of farnesyl chain at the active site of FSTS was inferred by Cane based on his initial proposed mechanism.7a,8 However, this mechanism involved an unsatisfactory anti-Markovnikov secondary carbocation, which could be avoided through a proton transfer pathway as clarified by Tantillo et al. using quantum chemical methods. 9 Based on the Tantillo’s mechanism, we deduced two possible conformations of farnesyl chain with different orientations of C10−C11 double bond (Figure 1a), namely, C11-up and C11-down (the conformation that Cane employed), via retro-biosynthesis conformational analysis from the TD’s absolute configuration validated by experiments.10 However, it is not enough to determine the exact folding mode without considering the relative orientation of diphosphate group (PPi or OPP) against the farnesyl chain. According to the available crystal structures of 1,10-closure sesquiterpene cyclases complexed with substrate analogues,2,5,11 in which the PPi group is anchored by Mg2+-coordination and hydrogen-bond interactions at the hydrophilic region of the active site while the farnesyl hydrocarbon chain is cradled in the hydrophobic region of the active pocket, we summarized 4 kinds of relative orientations between PPi group and farnesyl chain as shown in Figure 1b (also see Figure S1). The farnesyl chain is forced into “U-shape” by the contour of hydrophobic pocket, like a bent palm and represented by an arc arrow; Received: May 4, 2017 Revised: July 27, 2017 Published: August 3, 2017 5841

DOI: 10.1021/acscatal.7b01462 ACS Catal. 2017, 7, 5841−5846

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ACS Catalysis

Figure 1. (a) Retro-biosynthesis conformational analysis of trichodiene based on Tantillo’s mechanism. From intermediate E down, each intermediate has two possible conformations. If not considering the interchange between the two conformations, up or endo conformation will directly lead to the formation of trichodiene; however, down or exo conformation will lead to 7-epi-trichodiene. (b) Four possible orientations of FPP. Straight lines and arc arrows represent OPP group and farnesyl chain, respectively, whereas R, L, C, and O mean right, left, closed and opened, respectively. (c) Four stable folding modes of FPP in FSTS after classical MD simulations, folding modes with O orientation could not be accommodated due to the space limitation in the active pocket of FSTS, the C orientations were not stable and would turn into R or L orientation spontaneously during the classical MD simulations.

however, the PPi group is like the thumb and noted by a straight line. The PPi group may be either perpendicular to or coplanar with the U-shaped farnesyl chain, approximately. Accordingly, the former (vertical) has R and L orientations, which abides by the right-hand and left-hand rule, respectively, and the latter (coplanar) has C (closed) and O (opened) orientations. The combination of 2 conformations of farnesyl chain and 4 relative orientations between PPi group and farnesyl chain

makes 8 possible folding modes for FPP. Since the active pocket of FSTS is not spaciously enough to accommodate the folding mode with O orientation, we used the rest of 6 folding modes to carry out classical molecular dynamics (MD) simulations and obtained 4 stable folding modes as shown in Figure 1c (also see Figure S2 and S3). To further determine the catalytically productive folding mode, more than 2.5 ns quantum mechanics/molecular mechanics (QM/MM) MD simulations on the complete FSTS enzyme systems were 5842

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Figure 2. Relative energy profiles of the first step (PPi cleavage: C1−O1 bond breakage) for different models (a) and structures of key intermediates (b, c, and d); the structure of mode Ldown, similar to mode Lup except for the orientation of C10−C11, is not given.

Figure 3. Free energy profiles for the biosynthesis of trichodiene catalyzed by FSTS. (a) 1,3-PPi transfer; (b) from cisoid 3R-NPP to trichodiene.

1,6-ring closure pathway, will be formed spontaneously; however, this distance is increased with L orientation (see details in Figure S4). The structure of cis farnesyl carbocation A2 derived from mode Rup is ready for 1,6-cyclization by attacking C6 at Si face to form 6R-bisabolyl cation B with the absolute configuration demanded for the formation of TD (Figure 2b); while 6S-bisabolyl cation B will be formed by attacking C6 at Re face from mode Rdown (Figure 2c). Regarding the L orientation, it may lead to the 1,10-cyclization instead of the 1,6-cyclization as the steric hindrance between C15 methyl (colored in yellow, Figure 2d), and the PPi group would impede the formation of NPP. In a word, each folding mode of substrate may correspond to one reaction pathway with specific chemoselectivity (such as 1,6- or 1,10-cyclization) and stereoselectivity (such as Re- or Si-cyclization), and thus, a specific enzyme has to choose one unique folding mode to realize the chemical control over various reaction pathways. Recently, Major et al. theoretically investigated the reaction pathway from cisoid 3R-NPP to TD derived from folding mode Rdown,14 and the energetics seemed to be reasonable. However, the modeling was based on the docking pose of intermediate D with exo conformation instead of the endo conformation derived from mode Rup of substrate FPP, and the rate-limiting step (PPi cleavage), an important testimony for the rationality of modeling, was not considered. Our simulations indicated

performed at the M06-2X(6-31G*)/Amber99sb level, and the relationships between the four stable folding modes and the two reaction pathways (1,6 vs 1,10) were discussed. It is generally accepted that the rate-limiting step for FPP cyclization is the initial cleavage of PPi group (namely, C1−O1 bond breakage) to form farnesyl carbocation,12 which was verified by our previous QM/MM MD simulations.13 In addition, our previous studies also pointed out that the protonation state of PPi has significant effects on this step. In current systems, only one oxygen atom of PPi (O7) can be protonated as the other terminal oxygen atoms coordinated to Mg2+ ions. Herein, we considered 8 models, that is, deprotonated and protonated states of the aforementioned 4 folding modes, and their reaction energy profiles of the PPi cleavage have been determined (see Figure 2a). All the protonated models are energetically more favorable than the deprotonated models. Pronated mode Rup is the most favorable one with a relatively low barrier of about 15 kcal/mol; while the energy paths of other models are always uphill along the reaction coordinate. Hence, mode Rup is the most likely productive folding mode. Additionally, along the reaction coordinate involving the elongation of C1−O1 bond, the distance between C3 and O1 atoms is decreased with R orientation, indicating the nerolidyl diphosphate (NPP, see Figure 1a), a necessary intermediate for 5843

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Figure 4. Conformational analysis of FPP. R and L represent relative orientations between PPi group and farnesyl chain, Re and Si means the PPi group is above the Re and Si face of C3, respectively.

difference (21.1−18.8 = 2.3 kcal/mol) between the initial PPi cleavage process and the following serials of intramolecular reaction is also in a good agreement with the experimentally validated data (17−15 = 2 kcal/mol), and thus, we believe that our predicted free energy landscape of the whole reaction process is very credible. In addition, we also found the sulfur−carbocation interaction between Met73 and C7 of bisabolyl cation intermediate B as previously reported by Major et al.,14 and this interaction stabilizes intermediate B by 1.7 kcal/mol (Figure S6). From the intermediate B, a concerted but asynchronous process of 1,5proton transfer and 6,11-cyclization leads to the generation of spirocycle intermediate G, followed by 1,2-alkyl transfer to form intermediate D (see Figure S7 for details), which is in contrast to the previous mechanisms without the intermediacy of G.9,14 Since the carbocation in intermediate G is far away from the dropped PPi group and no basic residue is near to it in the active pocket, intermediate G will not be deprotonated to form α- or β-chamigrene.9b,16 In addition, the metastable nature of intermediate C effectively avoids the formation of iso-γbisabolene by deprotonation of C12 methyl group.9a It is noted that, from cisoid 3R-NPP to product, the C13 methyl is gradually close to the PPi group along the reaction pathway and finally deprotonated by donating the proton to the O1 of the PPi group (Figure S7), and no obvious conformational adjustment was observed; that is, the conformation of each intermediate is preorganized for the next step (actually, the preorganization starts from the substrate state, see Figure S8). Hence, once the cisoid NPP was formed, it converted into the final product TD immediately, well explaining why the NPP intermediate state was not detected experimentally.12 From the above discussions, we concluded that each folding mode of substrate corresponds to one specific reaction pathway. For current system, R orientation prefers 1,6-ring closure, while L orientation prefers 1,10-ring closure. Is this true when applied for other sesquiterpene cyclases? Our

that the barrier of PPi cleavage from mode Rup is lower than that from mode Rdown, and more importantly, mode Rdown would directly lead to the formation of bisabolyl cation B with S configuration conflicted with the configuration demanded for the product9 if not considering the conformational change of the initial FPP. This scenario reminds us that the aza analogues of bisabolyl cation B adopted a thermodynamically stable instead of productive conformation in the crystal structures of FSTS complexes reported by Christianson et al.7 Similarly, theoretical modeling starting from reaction intermediates instead of the native substrate may also obtain a thermodynamically stable conformation, not corresponding to the correct conformation (folding mode) of substrate. Thus, theoretical modeling starting from the native FPP substrate is more warrantable than that from a hypothetical intermediate. To further confirm the rationality of the folding mode Rup, free energy profiles for the formation of transoid 3R-NPP and the pathway from cisoid 3R-NPP to product TD were evaluated. Free energy barrier of PPi cleavage is 21.1 kcal/ mol (Figure 3a and also see Figure S5 for details), comparable to the experimental result of about 17 kcal/mol.12 The formed intermediate A1, trans farnesyl cation (namely, nerolidyl cation), is unstable and ready to form transoid 3R-NPP nearly barrierless. Hence, the cleavage of PPi and the formation of 3RNPP can be viewed as a concerted but highly asynchronous step, and this effectively avoids side reaction pathways, such as 1,10-ring closure or the formation of farnesene by deprotonation.7f Subsequently, by rotation of C2−C3 single bond, transformation from transoid 3R-NPP to cisoid 3R-NPP is very facile.15 From cisoid 3R-NPP to the final product TD, the free energy barrier of each single step is no more than 13.1 kcal/mol and the overall free energy barrier is 18.8 kcal/mol, comparable to the experimental result of about 15 kcal/mol,12 and the overall process is exothermic by 19.8 kcal/mol to yield a two-ring product finally (Figure 3b). Meanwhile, the predicted barrier 5844

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nonproductive ones, corresponding to side pathways, are filtered by the high energy barrier of PPi cleavage, ensuring the high fidelity of enzymatic catalysis. In other words, it is the selective rate enhancement that picks out the productive folding mode of substrate from nonproductive ones. Therefore, we have to point out that the folding-mode-selective rate enhancement of the first ionization reaction is likely to be an alternative sophisticated strategy exerted by terpene synthases to achieve chemical control over various reaction pathways, not just the posterior carbocation rearrangement reactions. In summary, the unique substrate folding mode in the trichodiene synthase is determined for the first time by elaborative DFT/MM MD simulations. Our results advocate a fatalism that the destiny of substrate is determined once it is folded by the active pocket, and each folding mode corresponds to a reaction pathway with specific chemo- and stereoselectivity. Meanwhile, the terpene synthase will selectively activate one of the folding modes to realize chemical control over a variety of reaction pathways. This new insight will shed light on protein engineering by utilizing the fidelity/promiscuity features of sesquiterpene cyclases. Additionally, we found a general rule (Figure 4) that summarizes the relationships between substrate folding modes and the two distinct reaction pathways (1,6 vs 1,10-ring closure) catalyzed by sesquiterpene cyclases with the common substrate FPP. This practical rule could guide us to infer the unclear folding modes of FPP in other sesquiterpene cyclases. Research on other sesquiterpene cyclases are now ongoing in our laboratory.

previous QM/MM MD simulations on two similar 1,10-closure sesquiterpene cyclases indicated that the productive folding mode of substrate is L orientation in ATAS (Aspergillus terreus aristolochene synthase) while it is R orientation in TEAS (Nicotiana tabacum 5-epiaristolochene synthase).13a These seemingly ruleless phenomenon inspire us if supplementary stereochemistry factors should be also considered to elaborate the relationships between the folding modes and the two distinct reaction pathways (1,6 vs 1,10-ring closure). Accordingly, another factor, Re or Si, was introduced to define the conformation of FPP, as shown in Figure 4. Re means that the PPi group is above the Re face of C3, and this conformation will lead to the formation of 3R-NPP through Re face 1,3-PPi transfer; while Si means that PPi is above the Si face and form 3S-NPP through Si face 1,3-PPi transfer. The 1,3PPi transfer is forbidden due to the steric hindrance between the PPi group and the C15 methyl in Si-R or Re-L modes. Hence, more exactly, Re-R (such as FSTS) and Si-L folding modes prefer 1,6-ring closure, whereas Si-R (such as TEAS) and Re-L (such as ATAS) prefer 1,10-ring closure, and this rule should be appropriate for all sesquiterpene cyclases. Noticeably, this rule can work only in the system that the rotation of C1−O1 bond is limited; otherwise, there is always a proper orientation leading to 1,3-PPi transfer. The fidelity of chemoselectivity depends on the strict control over the conformation of substrate. In other words, if an enzyme controls the FPP to form the NPP with a strict “staggered” conformation, it mostly catalyzes 1,6-ring closure pathway with high fidelity; however, if NPP has a strict “eclipsed” conformation, 1,10-closure is expected (Figure 4). On the contrary, if the conformation control is not so strict, the promiscuity of enzyme will emerge, such as TEAS that mainly catalyzes the 1,10-pathway but also has a side branch to 1,6pathway (see more discussions in Figure S9). Two intriguing features of enzymatic reaction are rate enhancement and chemical control.14,17 In most terpene synthases, the rate enhancement function of protein environment seems to only play a critical role in the first ionization step (herein PPi cleavage) as the subsequent intramolecular carbocation rearrangement reactions are usually very fast even without the help of enzyme environment.9,16 Furthermore, the chemical control, which determines the chemo- and stereoselectivity of product, is thought to be highly dependent on interactions between key residues in the active pocket and the later formed carbocation, such as cation-π interaction, C−H···X and C+···X interaction (X is a heteroatom), and steric interaction, among others.7b,17 In the current system, we did find a C+···S interaction (between Met73 and C7 of bisabolyl cation intermediate B) that could stabilize the key intermediate B, but the attendant chemoselectivity is directly related to the conformational preorganization instead of this interaction (see Figures S7 and S8 for detail). In other words, the chemical control is predefined by the rate-determining ionization step, which was ignored in the previous studies of terpene synthases.14,17 Actually, due to the inherent carbocation reactivity, not all steps need the intervention of enzyme,16b and the enzyme-aided rate enhancement of the ionization step is selective toward folding mode, corresponding to a reaction pathway with specific chemo- and stereoselectivity. As illuminated in this work, the active pocket of FSTS can accommodate 4 possible folding modes of substrate (maybe more),18 but only the productive one has a feasible energy barrier of PPi cleavage (the rate-limiting step) and other

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscatal.7b01462. Computational details, Scheme S1, Table S1, Figures S1−S9 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Hujun Xie: 0000-0002-0035-2634 Ruibo Wu: 0000-0002-1984-046X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Key R&D Program of China (2017YFB0202600), Guangdong Natural Science Founds for Distinguished Young Scholars (2016A030306038), and the Pearl River S&T Nova Program of Guangzhou (2014J2200062). We thank the Guangzhou Supercomputer Center for the Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund.

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REFERENCES

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