Protonation-Dependent Diphosphate Cleavage in FPP Cyclases and

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Protonation Dependent Diphosphate Cleavage in FPP Cyclases and Synthases Fan Zhang, Nanhao Chen, Jingwei Zhou, and Ruibo Wu ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b02096 • Publication Date (Web): 07 Sep 2016 Downloaded from http://pubs.acs.org on September 7, 2016

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Protonation Dependent Diphosphate Cleavage in FPP Cyclases and Synthases Fan Zhang†1, Nanhao Chen†‡1, Jingwei Zhou†, Ruibo Wu†*

† School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, P.R. China

‡ Department of Chemistry, University of California, Davis, California 95616, USA 1

F. Zhang and N. Chen contributed equally to this work

*E-mail: [email protected]

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Abstract The cleavage of the magnesium-assisted diphosphate group (the PPi group) is one significant and prevalent rate-limiting step to trigger the enzyme catalysis synthesis of terpenoids natural products. However, the PPi cleavage procedure has been rarely studied in most theoretical researches of terpenoid biosynthetic mechanism. In this work, QM(DFT)/MM MD simulations were employed to illuminate the detailed PPi cleavage mechanism in the three different enzyme systems (ATAS, TEAS and FPPS). We found that the most rational protonation state of PPi group is highly dependent on the Mg2+ coordination modes and the enzyme classes. The de-protonation PPi is favorable for triggering the catalysis reaction in ATAS while mono-protonation in FPPS and bi-protonation in TEAS are advantageous. As a result, similar PPi cleavage occurs by means of nucleophilic substitution reaction in TEAS/FPPS/ATAS, but presents SN1, SN2 and borderline mechanism, respectively. Finally, the alternative functions of PPi protonation and Mg2+ coordination modes are discussed.

Keywords: farnesyl diphosphate cyclase (FPPC); farnesyl diphosphate synthase (FPPS); diphosphate (PPi) cleavage; protonation state; QM/MM


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Introduction Natural products are the most important sources of new drug discovery.1-5 Terpenoids, the largest class of natural products, include about 75 thousands diverse molecules up to date.6 They are all initiated from the common C5 building unit namely isopentenyl diphosphate (IPP, see Figure 1) and its isomer dimethylallyl diphosphate (DMAPP, see Figure 1). One of the bottlenecks in modern drug discovery is the limitation of chemical diversity for drug screening.7 Sesquiterpenes, originated from three C5 units namely the farnesyl diphosphate (FPP, see Figure 1), have a large amount of scaffold diversity with about 22 thousand members and more than 300 known monocyclic, bicyclic, and tricyclic sesquiterpene natural products with different carbon skeletons.8-10 Additionally, FPP is also the direct precursor of triterpenoids, which are another class of terpenoids with lots of diverse structures. Apparently, FPP is a pivotal intermediate to enhance the diversity of terpenoids.11 Therefore, it is essential to understand the biosynthetic mechanism of FPP and its derivative cyclic products, sesquiterpenes. Lots of biological researches have been done to understand the structures and function of the FPP synthases (FPPS) and cyclases (FPPC, also called as sesquiterpenes synthases).12-17 As shown in Figure 1, the FPPS catalyzes the condensation of the IPP with DMAPP to form C10 isoprenoid geranyl diphosphate (GPP), and then condenses with a second IPP to form the C15 isoprenoid FPP, by “head to tail” style.18 Subsequently, the FPPC can cyclize the FPP to various sesquiterpene products with different cyclic skeletons. Our current research is based on the Escherichia Coli FPPS and two representative kinds of FPPCs, namely Aspergillus Terreus aristolochene synthase (ATAS) and Nicotiana tabacum 5-epi-aristolochene synthase (TEAS), as shown in Figure 2. All of these three enzymes have lots of crystal structures in high resolution.6, 13, 19-26 As observed from Figure 2, they all contain three magnesium ions (Mg2+) in the active pocket. For either the substrates in FPPS (namely DMAPP/GPP/IPP) or the substrate in FPPC (namely FPP), their diphosphate group (PPi, namely OPP group shown in Figure 1) are coordinated with the Mg2+. And all of these substrates are linear amphipathic compounds with a polar diphosphate group and a nonpolar unsaturated hydrocarbon chain. However, different metal coordination shells (different coordination ligands) and substrate conformational states (different molecule shape) among FPPS/ATAS/TEAS are observed (see Figure 2). Interesting, FPPS is a sole functional enzyme and


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ATAS also has a high fidelity with aristolochene as the only product,27 while TEAS has enzymatic promiscuity with many different minor products including 1-10 closure and 1-6 closure pathway products,24 as described in Figure 1. Meanwhile, it is clearly that these two FPPCs adopt almost the same reaction pathway but produce the major products with different chiralities. The diversity and chiral differences of products in ATAS and TEAS are thought to be originated from different substrates reactive conformations adopted in the enzymes that take advantage of the multiple chemical reactivity inherent in FPP.8 As shown in Figure 2, for both ATAS and FPPS, the PPi is bi-dentate coordinated with Mg2+B and Mg2+C while mono-dentate bound with Mg2+A, but it is quite different in TEAS, with Mg2+A&C coordinating to the PPi while Mg2+B not.

Figure 1. The brief biosynthetic pathways of FPPS, ATAS and TEAS. FPPS catalyze carbon-chain elongation to form FPP is rendered in blue and its further cyclization pathways under the catalysis of ATAS/TEAS are rendered in red. The main reaction pathway in TEAS shares the same reaction steps in ATAS, and its 1-6 closure side reaction pathway is shown in grey.


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Regarding to the reaction mechanisms, it is well-known that the initial reaction step is similar and commonly existed in the FPPCs and FPPS in light of the terpenoid biogenesis pathway, that is, the cleavage of PPi (C-O bond breakage) to form carbenium intermediate. Indeed, this kind of PPi cleavage reaction is not only existed in FPPCs and FPPS, but also prevalent in some other terpenoid elongases and cyclases.28-31 And many theoretical studies on the substrate or active pocket model by means of static electronic structure methods have been done to understanding the subsequent reaction steps,32-39 whereas the initial PPi cleavage procedure is neglected in most case. Nevertheless, the PPi cleavage step has been widely regarded as the important rate-limiting step in the whole reaction experimentally and theoretically.10, 40 Despite the PPi cleavage is thought to be significant, the protonation state of the PPi group is rare discussed. As is well known, both deprotonated PPi(PPi3-) and mono-protonated PPi (PPi2-) may exist in solution at physiological pH (~7).41 Moreover, since the local pH value would be quite different in the condense system due to the fluctuation of enzyme environment and polarization of surrounding water molecules, it is necessary to re-estimate which kind of FPP protonation states (FPP3-, FPP2-, FPP-, FPP) is dominant in these enzyme systems, and it is expectable that different protonation states may affect the following PPi cleavage mechanism. For example, the deprotonation state FPP (FPP3-) is thought to be the active form in various FPP cyclases35, 42 and native product state in FPP synthases. In contrast, Merz found that the FPP2(namely mono-protonated PPi) is the most likely productive form in FPP transferase several years ago.43 We discovered that the tri-protonated PPi would trigger the enzymatic PPi cleavage and subsequent C-C bond formation reaction in GPPS very recently.44 Furthermore, as much as we known, it is still very difficult to directly identify the exact protonation state of diphosphate or phosphate by current available computational tools.45-54 Since protonation state of the PPi is a so critical question but rarely discussed in detail in the previous studies of terpenoids biogenesis, the powerful computational tool to studying enzymatic catalytic mechanism, QM(DFT)/MM, has been apply herein to elucidating the cleavage mechanisms of PPi in ATAS, TEAS, and FPPS. Finally, we reveal the critical effects of the PPi protonation states and the Mg2+ coordination interaction on promoting the PPi cleavage.


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Figure 2. The active site comparison of (a) ATAS, (b) TEAS and (c) FPPS. Reaction coordinates (RC) are noted in blue (on the left 3D views). Corresponding 2D views are shown on the right with the QM atoms in black and others in grey. The potential protonation sites are highlighted in blue and red respectively, while C-O bond for cleavage is noted by wavy line. The electron transfer diagram for the initial step and detailed RCs are also labeled on the right 2D views.


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Methods Preparation of the Enzyme-Ligand Complexes The X-ray crystal structures of ATAS were all collected and downloaded from RCSB PDB. It indicates that its crystal structures often contain substrate analogues or diphosphate group only (Table S1).19, 26 Take the resolution and substrate binding modes into consideration, the PDB entry 4KUX with substrate analogue FSPP binding in the active site is selected. The sulfur atom of the FSPP was replaced with oxygen to regenerate the original substrate FPP and the coordination mode between substrate and the Mg2+ ions was maintained. Because the missing residues (residue 1-7 and 312-314) are not near to the active site, the original protein structures without adding these missing residues are employed as our initial protein structures. There is a large contact area between the two homologous chains and their active site pockets were close to each other, thus, the two chains of ATAS in the crystal structure were maintained in the simulations. Based on the crystal structure, all the six marginal oxygen atoms of the PPi group are coordinated with the three magnesium ions (Mg2+A/Mg2+B/Mg2+C) except one “free” oxygen atom (labeled as O7 in Figure 2(a)) that could be protonation or deprotonation. Therefore, two models, deprotonated and single protonated on O7, respectively, were constructed to investigate which protonation state is preferable and reasonable. Although many X-ray crystal structures of TEAS have been reported to date, as summarized in Table S2, only a few solved the J-K loop residues with side chains (PDB entry 5EAT and PDB 4RNQ).23, 25 Some structures contained substrate analogs or protein mutations (PDB entry 3M00, 3M01 and 3M02). Because the 3M02 contained all residues except side chains of J-K loop residues and revealed the binding mode between all the three Mg2+ ions and the substrate analogue, we chose the PDB entry 3M02 and the J-K loop residues 521-534 of the recently released PDB entry 4RNQ to rebuild the protein model of TEAS, which had been employed successfully in our previous study.55 Since the ligand in the 3M02 was a cis-trans substrate analogue, we modify the substrate analogue (2-trans,6-trans)-2-fluorofarnesyl diphosphate of the PDB entry 3M01 to regenerate the original substrate FPP by replacing the fluorine substituent group with hydrogen and superposing it into the rebuild TEAS protein. In TEAS crystal structures, as shown in Figure 2(b), only two magnesium ions (Mg2+A/Mg2+C) directly coordinated with the oxygen atoms of the PPi group but another one (Mg2+B) indirectly connected with the PPi group by coordinating with


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one water molecule. Hence, two “free” oxygen atoms (O3 and O7) of the PPi group could be (de-) protonation and thus four models (both deprotonation, single protonation on O3 or O7 respectively, and both protonation) were constructed. For FPPS, the crystal structure of Ec-FPPS (PDB entry 1RQI),13 a protein complexed with IPP and substrate analogue DMASPP, was selected for rebuilding the FPPS enzyme-substrate complex which could catalyze the assemble of the isoprenoid building blocks DMAPP and IPP to yield the GPP intermediate. The sulphur atom of the allylic substrate (DMASPP) was replaced by oxygen atom to regenerate the original substrate. The His98 was determined as singly protonated at ε position to form hydrogen bond with oxygen of the IPP. Since metal coordination interaction with PPi is similar to ATAS model, two models (deprotonated and single protonated on O7, see Figure 2(c)) were constructed for FPPS modeling. Classical MD Simulations For the above mentioned eight constructed models of the three enzyme-substrate systems, the AMBER99SB force field56 was employed for the protein and the TIP3P model was used for solvent water molecules57. The force field parameters of different ligands (FPP, DMAPP and IPP) were generated from AMBER GAFF force field58. The restrained electrostatic potential (RESP) charge59 was used to calculate the partial atomic charge of substrates from HF/6-31G* calculation with the Gaussian 09 package60. The initial coordinates and topology files were generated by the tleap program in AMBER1261. The MD simulations were carried out using the AMBER12 molecular simulation package, and all models were treated with the same MD protocols via employing the periodic boundary condition with cubic models (ATAS is 113 × 86 × 111 Å, TEAS is 90 × 100 × 78 Å and FPPS is 89 × 86 × 71 Å, respectively.). First, minimizations were carried out to relax the solvent and optimize the system. After the optimization, each system was heated from 0 to 300 K gradually under the NVT ensemble for 100 ps, followed by another 100 ps NPT ensemble MD simulations at 300 K and the target pressure of 1.0 atm. In the NPT ensemble, the Berendsen thermostat method was used to control the system temperature62. Afterward, a 5 ns MD simulation under the NVT ensemble was carried out. The SHAKE algorithm63 was applied to constrain all hydrogen-containing bonds during the MD simulations. A cutoff of 12 Å was set for both van der Waals and electrostatic interactions. All simulations were accomplished with the pmemd program in AMBER12. Finally, snapshots of each system from the stable trajectories were


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used to build the initial structures for the subsequent QM/MM simulations. Setup for QM/MM MD Simulations All QM/MM models were prepared by deleting the solvent molecules beyond 32 Å from the C1 atom in the substrate FPP or DMAPP. The QM/MM regions of all models consist of more than 20000 atoms. As shown in Figure 2, the substrate FPP and the three Mg2+ ions together with some Mg2+ coordination residues were considered as the QM area for ATAS and TEAS, the detailed benchmark tests on TEAS with QM region choice had been performed and the results were discussed in Figure S14. Since the B3LYP method often underestimates the barrier of the cyclization process,64 all of these atoms in the QM subsystem were described with the M06-2X,65, 66

using the 6-31G(d) basis set which is widely used in studying the cyclization reaction,67, 68 and

every model adds up to about 700 basis functions in total. For FPPS, the DMAPP, IPP, Asp105, Asp111, Asp244, three Mg2+ ions and eight water molecules which directly coordinate with the Mg2+ were included in QM subsystem. Since computational accuracy is warranted based on previous benchmark investigation on the similar DMAPP and IPP elongation reaction catalyzed by GPPS,44 and the benchmark tests on FPPS also showed warrantable results as discussed in Figure S15, thus, all of the QM subsystems in FPPS were treated by B3LYP/6-31G* to reduce the computational cost69,

70

,. The QM/MM boundary was treated by the improved pseudo bond

approach.71-73 Same molecular mechanical force field in previous classical MD simulations was used for all the remaining atoms. The spherical boundary condition was employed, and atoms more than 25 Å away from the spherical center were fixed. The 12 and 18 Å cutoffs were employed for van der Waals and electrostatic interactions, respectively. The QM/MM systems were minimized again for several iterations and more than 5 ps QM/MM MD simulations were performed. The resulting conformations were used to map out the minimum energy path with the reaction coordinate driving method.74 After scanning the reaction path of different models, the MM subsystems were further equilibrated with 500 ps classical MD simulations with the frozen QM subsystem. Finally, the resulting snapshots were treated as the starting structures for umbrella sampling at the QM/MM level. Every window was calculated for 20 ps with 1 fs time step. The system temperature was controlled by the Berendsen thermostat method at 300 K, and the Newton equations of motion were integrate by the Beeman algorithm.75 After biased-potential-based QM/MM MD umbrella sampling,76 the WHAM77, 78 program was employed to calculate the free


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energy profile with data collected from each window. All of these QM/MM calculations were performed with the modified QChem79 and Tinker80 programs.

Figure 3. The relative energy profiles of the first step (PPi cleavage namely C1-O7 bond breakage) for different protonation states in the ATAS(a), TEAS(b), and FPPS(c). All these profiles were obtained after minimizing the energy pathway forward and backward several times.

Results The critical protonation effect on PPi cleavage As shown in Figure 2, there are two main kinds of coordination modes in the FPPS and FPPCs. One is adopted by most of FPPCs and FPPS while the other is only employed by TEAS. These different coordination modes imply the possible different protonation states of the PPi group. As discussion in the above Method section, the potent protonation sites of the PPi group are analyzed, and only one oxygen atom (O7) can be protonated in ATAS and FPPS whereas two (O3 and O7) in TEAS. Thus, the eight models including all possible protonation styles are considered and the primary results are depicted in Figure 3. The relative energy profiles of PPi cleavage process (C1-O1 bond breakage) in ATAS, TEAS and FPPS are compared to figure out the most rational protonation state. A lower barrier indicates a higher feasibility of PPi cleavage. Therefore, as shown in Figure 3(a) and 3(c), the deprotonation state (PPi3-) is more reasonable for ATAS whereas the single protonation sate (PPi2-) is more reasonable for FPPS. Otherwise, for ATAS, the barrier becomes very high, and the energy curve is not smooth for the mono-protonated PPi2- state because the protonation of the O7 would destroy the connection between the PPi group and the two positively charged residues (Lys220 and Arg308, as shown in Figure S1). In contrast, both Lys213 and Arg301 form strong hydrogen bonds with the PPi group and can neutralize the negative charge of the PPi group in view of the ATAS crystal structures. Nevertheless, the hydrogen bond interaction


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between Tyr309 and O7 is well maintained in both protonation and deprotonation states, and the binding pose of PPi is also well preserved. Regarding the FPPS, as shown in Figure S2, due to the larger active pocket and much more water molecules surrounding the PPi group, the protonation of O7 would not affect the PPi group and the two conserved positively charged residue (Lys258 and Arg116), and several water molecules would escape from the PPi binding site thus, the binding modes of PPi are well preserved by the metal coordination interaction and the saturated hydrogen bond network. Although similar Mg2+ coordination modes are observed both in ATAS and FPPS, it is found that their most reasonable protonation states are quite different. TEAS, compared to ATAS and FPPS, displays a distinctive metal-PPi binding mode, and four possible protonation states are classified in Figure 2, the corresponding relative energy profiles of the C1-O1 breakage are plotted in Figure 3(b). Apparently, only the protonation on both O3 and O7 could make the C-O cleavage kinetically feasible while other three protonation states were less likely due to the high reaction barrier. Moreover, we also examine the concerted mechanism to promote the PPi cleavage as shown in Figure S3, although unfeasible thermodynamically, the free energy barrier of the PPi cleavage concerted with the 1-10 cyclization would be only 23.4 kcal/mol, thus further proving that bi-protonation of PPi would facilitate the C1-O1 breakage in TEAS. In summary, our primary calculations indicated that the same PPi cleavage reaction is promoted by means of totally different protonation states in ATAS/TEAS/FPPS, whether the Mg2+ coordination interaction with PPi group is similar in ATAS/FPPS or different in TEAS. To confirm that the de-protonation of the PPi group in ATAS, the mono-protonation in FPPS and bi-protonation in TEAS are reasonable, powerful QM(DFT)/MM free energy simulations were performed to illuminate the possible distinguishable enzymatic catalysis mechanisms of the PPi cleavage reaction in ATAS/TEAS/FPPS (see below).


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Figure 4. The free energy profile of the GA cyclization in ATAS. Herein the relative free energies and their standard deviation values of each state is shown in parentheses. The GA cyclization consists two procedures: PPi cleavage concerted with 1-10 cyclization, and the proton transfer. The 10~15ps and 15~20ps snapshots (structures) of the QM/MM trajectories are chosen to evaluate the standard deviations of free energy. Concerted but Asynchronous in ATAS Following the identification of the deprotonation state of PPi (PPi3-), the two reaction coordinates (RC) have been considered. First, the free energy curve along the r1 (as shown in Figure 2) indicates that the product state after this type of PPi cleavage style is not stable, because the curve is rising steadily (see Figure S4), thus, the stepwise mechanism has been ruled out. Then, we have also considered the Npp reaction pathway which is the well-known side reaction in TEAS (see Figure 1) by driving along RC=r1-d(O1-C3). According to the free energy profile presented in Figure S5, it is impossible to go through the 1-3 Npp pathway in TEAS due to the barrier that is as high as 37 kcal/mol and the unstable product state, attributed to the cis-trans isomerization around the C2-C3 delocalized double bond and the steric hindrance effect of the C1-C2 double bond at Npp state (see detailed discussion in Figure S6). Subsequently, the concerted mechanism has been considered instead, with its free energy profile along RC1 shown in Figure 4 here, the PPi cleavage and GA cation cyclization are concerted with a ~23 kcal/mol reaction barrier that is comparable to the experimental results27 (about 21 kcal/mol). This indicates that the cleavage of the PPi group would be the rate-limiting step. Thermodynamically, this is an endothermic reaction that absorbs ~22 kcal/mol. However, the following proton transfer reaction would occur spontaneously and release a considerable amount of energy (discussed later). All of these characteristics make the Germacrene A (GA) cation be a metastable state. According to the key structures in Figure 5 and the C1-O1/C1-C10 distance


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evolutions shown in Figure S7, this concerted reaction is highly asynchronous kinetically, because the PPi cleavage and the GA cation cyclization procedures occur in series. Starting from the substrate, the C1-O1 bond breaks first (the C1-O1 distance increases strikingly from 1.4 Å to 2.7 Å) and then the C1-C10 bond is still unformed (the C1-C10 distance is still approximately 2.7 Å), leading to a highly charge-separate zwitter-ion state (namely Pro-TS1 in Figure 5), with a highly negative charge on the free PPi group and a positive charge on the un-cyclized hydrophobic fragment, as listed in Table S3. After this state, the reaction proceeds though the TS1 and the C1-C10 distance decreases sharply and the 10-member ring yields to the GA cation state. From the zwitter-ion state to the GA cation state, the positive charge would transfer from the delocalized carbocation C1-C2-C3 to the C11 atom intensively (see Table S3). Furthermore, the less positive charge on C3 (0.36) relative to that on C1 (0.56) in the zwitter-ion state ensures that the reaction will go through the 1-10 cyclization instead of through the 1-3 Npp pathway.

Figure 5. The key structures during the PPi cleavage concerted with the 1-10 cyclization in ATAS. The hydrogen bond interaction is shown in black dash line and the key distances are shown in blue dash line. The average values of distances and its standard deviation (in parenthesis) are given.

As shown in Figure 4, with the cyclization of the GA cation, one of the hydrogen atoms on the nonpolar C12 atom will spontaneously transfer to the polar O1 atom of the PPi group,


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triggered by the high negative charge on O1 (see table S3). The proton transfer process is energetically feasible with a very low reaction barrier of 2 kcal/mol and exothermicity of ~13 kcal/mol. The detailed proton transfer steps have been shown in Figure 6, initially, one clearly local conformation regulation of the C11-C12-C13 isopropyl group occurred to the IM1 state. Then, the proton starts to transfer from C12 to O1 to yield the stable neutral intermediate, GA. Although the formation of the GA intermediate is endothermic with an overall heat of approximately 9.3 kcal/mol, many more steps are still necessary to achieve the final product aristolochene, including the second protonation, cyclization, hydride and methyl transfers, and so on (in Figure 1). Based on the calculations of Tantillo, most of these subsequent reaction steps are exothermic.32, 33, 81, 82 Thus, it is predictable that the reaction following the GA formation will release much energy to make the entire reaction pathway thermodynamically feasible. Therefore, taking these results together with the free energy profile, it is concluded that the PPi cleavage is the most important impetus for triggering the entire reaction.

Figure 6. The key structures of the proton transfer in ATAS. The corresponding RC (namely RC2 shown in Figure 2) values of different states have been marked in parentheses. The hydrogen bonds have been colored in black while key distances in blue. The average distance and the SD values have been shown beside the key distances.


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Stepwise in TEAS The most reasonable bi-protonation of PPi (PPi-) has been suggested in TEAS and the concerted mechanism is excluded due to the high barrier (23.4 kcal/mol) and endothermicity (see Figure S3). Accordingly, the stepwise mechanism of the PPi cleavage and GA cation cyclization has been elucidated with one stable intermediate. The computational stepwise rate-determining reaction free energy barrier of PPi cleavage is about 22 kcal/mol (see Figure 7), matching the experimental data (about 19.3 kcal/mol)24 better than the concerted mechanism. Additionally, the zwitter-ion intermediate could be stabilized well by the stepwise mechanism, whereas it is unstable via the concerted mechanism (see details in Figure S3). As summarized in Figure 7, the stepwise reaction starts from the C-O bond weakening (increase of C1-O1 distance), and then IM1 is formed proceeding through TS1 after the complete breaking of the C1-O1 bond, the IM1 is then quite stable with the C1-C10 distance of more than 3.0 Å. Subsequently, the hydrophobic carbenium part goes through a low barrier (about 5 kcal/mol) TS2 to achieve the 1-10 closure cyclization of the GA cation.

Figure 7. The free energy profile and the key structures of the GA cation cyclization in TEAS. Hydrogen bonds have been colored in black and key distances in blue. All the average values and SD values of the key distances have been marked.


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Interestingly, as observed from the data in Table S4, the positive charge of the carbenium is delocalized in the C1-C2-C3 conjugative area at the TS1 state and is then localized on the C3 atom at the IM1 state. As a result, both the C1-C2 and C2-C3 bonds have the property of a double bond from the TS1 to IM1 state, with the C1-C2 and C2-C3 distance fluctuating at the 1.35~1.40 Å and 1.36~1.44 Å ranges, respectively (in Figure S8). Moreover, we propose that this conjugative C1-C2-C3 subgroup in the IM1 state may play a crucial role in diversifying the products by triggering the 1-6 cyclization Npp pathway (See Figure 1) and not only the 1-10 cyclization GA pathway. The relative energy of the pathways is mapped in Figure S9 to demonstrate the possibility of such a 1-6 cyclization side reaction pathway in TEAS. It is found that the formation of Npp is very facile while the following PPi cleavage from C3 atom in the 1-6 cyclization side reaction becomes unfeasible if the following reaction steps are not considered. Accordingly, it is feasible for the cisoid products obtained through the NPP pathway to exist in TEAS, while this side reaction only represents a tiny portion of minor products as experimental observation.24, 83

Figure 8. The free energy profile and the key structures of the GA formation in TEAS.

In addition to the different the cyclization mechanism, the subsequent proton transfer in TEAS differs from that in ATAS. As observed from the GA cation structure shown in Figure 8, the isopropyl group on C11 is far away from the O1 of the PPi group (approximately 6 Å) making the methyl (C12/C13) hydrogen transfer to the O1 atom more difficult so that, our computationally predicted relative reaction energy is higher than 11 kcal/mol. The previous experimental results


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showed that the Tyr520Phe mutation will result in the termination of GA formation84. However, the proton transfer to Tyr520 is excluded by our simulations due to the higher than 17 kcal/mol relative barrier (see Figure S10). Considering that the distance between the carboxyl oxygen atom of Asp444 and the proton is very short (approximately 2Å, see Figure 8) based on the QM/MM MD trajectories of IM2 state, the proton transfer to Asp444 proceeds through the TS3 state and finally yields the GA intermediate reaction pathway proposed herein. The reaction is very facile with a lower free energy barrier of 6.6 kcal/mol and moreover it will release a large amount of energy (in Figure 8) to promote the subsequent series of steps after GA formation. Considering the above mentioned experimental Tyr520Phe mutation results, Tyr520 may act as the proton-shuttle accepting the proton on Asp444 to promote the subsequent reaction steps investigated in further ongoing computational simulations in our group that are beyond the scope of the present study. Both ATAS and TEAS have the same function of producing the major product aristolochene with different chiralities (epimers) from the same FPP substrate, and they adopt almost the similar reaction pathway as shown in Figure 1. However, it is revealed herein that the PPi cleavage occurs by different mechanisms to yield the same intermediate molecule GA. In ATAS, the FPP will adopt the deprotonation state and undergo a concerted reaction to cyclize the GA intermediate. However, double protonation of FPP and a stepwise mechanism is dominant in TEAS. Meanwhile, the destinations of the proton transfer in the two reactions are also distinct from each other. An intramolecular-like proton transfer (to the PPi group) occurs in ATAS whereas an intermolecular reaction (to Asp444) occurs in TEAS. As observed from Figure 9, energetically, while the PPi cleavage barriers are both approximately 22 kcal/mol, the thermodynamic properties of the two processes are quite different. The entire GA formation procedure in ATAS is endothermic while it is exothermic in TEAS. The tiny difference between the barriers in TEAS and ATAS (22.3 and 23.9 kcal/mol, respectively) indicates that the reaction velocity of ATAS would be slightly slower than that of TEAS, in agreement with the experimental data showing the kcat value27 of ATAS isslightly smaller than that of TEAS24 (kcat=0.0173±0.0007s-1 at 30℃ vs kcat=0.0416±0.012s-1 at 25℃).


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Figure 9. The whole free energy profiles of the GA formation in ATAS and TEAS. Herein the structures of ATAS have been rendered in blue and TEAS in black.

Figure 10. The free energy profile comparison between two protonation states of FPPS. Concerted and Synchronous in FPPS As observed in ATAS, FPPS exhibits the similar Mg2+ coordination mode to PPi. However, as discussed above, the most rational protonation states of FPPS and ATAS are different. In order to determine the effects of the different protonation states of the PPi group in FPPS, their corresponding free energy profiles are similarly mapped and are depicted in Figure 10. Apparently, the deprotonation state is less possible because the barrier of 24.9 kcal/mol is much higher than the experimental results (about 18.4 kcal/mol).85 Meanwhile, the entire reaction process is endothermic. Therefore, the deprotonation state of the PPi group (PPi3-) is excluded. Instead, the corresponding free energy profile for the O7 single protonation state is also shown in Figure 10 and displays a much lower barrier (18.5 kcal/mol) that in good agreement with the experimental result (18.4 kcal/mol). Furthermore, the protonation of the PPi group will change the


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thermodynamic properties of the whole reaction, making the reaction exothermic. Based on the free energy curve and the structures presented in Figure 11, the reaction is concerted for the PPi group cleavage and the C-C bond formation. From the substrate to TS1 (Figure 11(a, b)), the C-O bond distance changes from 1.51 Å to 2.29 Å while the C-C bond distance fluctuates from 3.37 Å to 2.54 Å. Together with the curve representing the change of these two distance along the RC (Figure S13), all of this evidence strongly demonstrates the concerted and synchronous property of the C-O bond cleavage and the C-C bond formation. The positive charge transfer could be observed clearly from the charge information in Table S5. The cleavage of the PPi group promotes the charge separation and leads to the concentration of the positive charge on C1 at TS. Then, this positive charge transfers to the C3’ atom of IPP during the C-C bond formation. After the formation of IM1, the proton on C3’ of IPP transfers to the O1 atom of the PPi group to achieve the product state. Thus, it is reasonable to regard it as the Bronsted base proton acceptor. This reaction is very facile with a barrier of approximately 6.4 kcal/mol barrier and would release a large amount of energy for a total energy release of about 10 kcal/mol. While both stepwise mechanisms have been revealed to be independent on the protonation state of the PPi group in FPPS, the key IM1 intermediate is totally different. As shown in Figure 11(c) and (f), due to the protonation of O7, the proton on C2’ is much closer to O1 of the PPi group in DMAPP (2.89 Å for O1 vs 4.33 Å for O7, in Figure 11(c)). Subsequently, the proton transfer from C2 to O1 would release a large amount of energy making the whole reaction exothermic. In contrast, unop deprotonation of the PPi group, in the IM1 state, the proton on C2’ is closer to O7 of the PPi group than to O1 (2.63 Å for O7 vs 4.18 Å for O1, in Figure 11(f)), thus the proton would transfer to O7 but not O1, leading to an unstable IM1 intermediate and a smaller heat release during the proton transfer. Therefore, the protonation of PPi in DMAPP is essential for the promotion of the elongation reaction catalyzed by FPPS.


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Figure 11. The key structures along the reaction in FPPS at under the PPi single protonation state. (a-e) are the structures of substrate, TS1, IM1, TS2 and product states respectively. (f) is the structure under the deprotonation state and selected herein for comparison to (c). The key distances evolution along the PPi cleavage is summarized in Figure S13.

Discussions The PPi cleavage (C-O bond breakage) and subsequent C-C bond formation in ATAS/TEAS/FPPS is a nucleophilic substitution reaction where PPi acts as the leaving group while C10-C11 (or C3’-C4’) double bond acts as the nucleophile. Since the reaction involves a stable and dissociative carbocation intermediate (IM1) in TEAS, and the whole process is followed by the separation of the leaving group, the nucleophilic attack of C10 and deprotonation of isopropyl in the stepwise, the nucleophilic substitution reaction is characteristic of a standard SN1 mechanism. As shown in Table 1, the transition state (TS1) is highly-dissociative compared to the ATAS/FPPS. The resultant carbocation intermediate can be stabilized well by the protein environment of TEAS because the atomic charge on C1 is the lowest among the data for TEAS/ATAS/FPPS (see Table 1). Meanwhile, the potential carbocation rearrangement side reaction at the IM1 state (namely the 1,6-closure cyclization side pathway starting from the IM1 state) is likely to be responsible for the enzymatic promiscuity of TEAS as shown in Figure 1. In contrast, because the C-O bond breakage and C-C bond formation are synchronous in


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FPPS leading to a penta-coordinate transition state with C1 adopting an approximately sp2 hybridization, the reaction follows a standard SN2 mechanism. As shown in Table 1, the nucleophile (C4’) attacks the carbocation C1 at near linear angle (163°) to the O1 atom of the leaving PPi group at the TS state and its structure is much more associative in light of the C1-O1 and C1-C4’ distances. As is well known, a hydrophobic aprotic solvent environment with low dielectric constant and apolarity, is favorable for the SN2 type of nucleophilic substitution reaction. Hence, as discussed in the results, several solvent water molecule are pushed away from the reaction center by the mono-protonation on the PPi group, facilitating the PPi cleavage with the help of hydrophobic aprotic local protein environment.

Table 1. Summary of the key structural and charge evolution differences in TEAS/FPPS/ATAS. Herein, the C1-chg and O1-chg represent the charge information of C1 and O1 atom while d(C-O) and d(C-C) indicate the distances of C1-O1 and C1-C10 (TEAS/ATAS) or C1-C4’ (FPPS) respectively. Θ(O-C-C) means the angle value. C1-chg (e)

O1-chg (e)

Θ(O-C-C) (deg,°)

d(C-O) (Å)

d(C-C) (Å)

TS1

0.20

-0.64

137

2.31

3.83

IM1

-0.32

-0.56

130

3.72

3.34

TS1

0.55

-0.77

163

2.29

2.54

IM1

0.14

-1.03

51

3.14

1.62

Pro-TS1

0.56

-0.89

131

2.71

2.73

TS1

0.23

-0.90

156

3.00

2.06

MS

0.01

-0.94

168

3.13

1.72

TEAS

SN 1

FPPS

ATAS

Style

SN 2

mix

Based on the key structural data and charge information as shown in Table 1, for ATAS, it is likely to occur by a so-called borderline mechanism (namely the mix of SN1 and SN2 reaction) because the formation and breakage of the bonds around the pro-TSs state is close to an SN2 style while its TS is close to an SN1 type structure. Nevertheless, in light of the bigger charge difference on negative O1 and positive C1 that is similar to the FPPS but distinct form TEAS (see Table 1), and the more linear Near-attack-conformation at the TS1 state, the SN2 mechanism is likely to be more dominant, thus the reaction is concerted but asynchronous in ATAS as discussed above.


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It is intriguing that the PPi group could adopt various types of protonation states (de-/bi-/mono-protonation) to facilitate the terpene cyclization (ATAS/TEAS) or elongation (FPPS) reactions by means of the different PPi cleavage mechanism. In ATAS, the deprotonation state is dominant while the PPi cleavage would be not feasible if not for the double protonation of the FPP in TEAS. As we had noted, the major difference in FPP binding poses between ATAS and TEAS is the metal coordination interaction. The coordination number between Mg2+ and FPP ligand is 5 and 3 in ATAS and TEAS, respectively (see Table 2), as one Mg2+ is unanchored to the FPP in TEAS. Similarly, in our previous research on another elongase, GPPS, only two Mg2+ existed in the active site and tri-protonation on PPi group is required to trigger the C-O bond cleavage (namely PPi cleavage).44 However, the system containing three Mg2+, FPPS, could initiate the reaction by mono-protonation on the PPi group. Therefore, the protonation requirement is highly dependent on the Mg-PPi coordination modes, and we proposed that bi-protonation on the PPi group would be functionally equivalent to two Mg-O coordination bonds. Meanwhile, it indicates that an additional proton on the PPi group is necessary for elongase compared with cyclases. This results maybe due to the existence of two PPi groups (one IPP and one DMAPP or GPP) in the catalytic site of FPPS. In contrast, only one PPi group (only FPP) exists in the catalytic site of FPPCs. Two highly negatively charged PPi groups would reduce the total positive charge of Mg ions less than 4 units (see Table 2) in FPPS, while it is close to 6 units in ATAS/TEAS regardless of the protonation. The high positive charge on magnesium ions is favorable for the stabilization of the transition state and intermediate states because the anchored PPi is more negative in these two states than in the substrate state. Thus, despite sharing the similar three coordinated magnesium ions with 5 coordination bonds to the PPi group in FPPS and ATAS, further mono-protonation is required in FPPS whereas de-protonation is sufficient in ATAS. Similarly, although for both TEAS and GPPS, only two coordinated magnesium ions with 3 coordination bonds with the PPi group are present, tri-protonation is necessary for GPPS while bi-protonation is enough for TEAS. Thus, we propose that the protonation requirement also depends on the enzyme class (elongases or cyclases).


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Table 2. Summary of the key differences for each protonation states in ATAS, TEAS and FPPS. The C1-chg and O1-chg represent the charge information of these two atom while the δ(C1-O1) means the difference between them. The Mg-chg summarized the total charge of the three Mg2+ ions. The d(C1-O1) indicates the C1-O1 bond distance, Mg-Coor means the coordination number between magnesium ions and the PPi group, and Proton No. indicates the proton number on the PPi group. The reasonable protonation states and their key differences have been highlighted by bold and blue. C1-chg (e)

O1-chg (e)

Mg-chg (e)

δ(C1-O1) (e)

d(C1-O1) (Å)

Mg-Coor

Proton No.

De-pro

0.63

-0.64

5.60

1.27

1.48

5

0

mono-Pro

0.23

-0.41

5.55

0.65

1.47

5

1

De-pro

-0.02

-0.57

5.76

0.55

1.45

3

0

bi-Pro

-0.08

-0.24

5.79

0.16

1.47

3

2

De-pro

0.23

-0.67

3.40

0.9

1.48

5

0

mono-Pro

0.46

-0.74

3.79

1.20

1.51

5

1

ATAS

TEAS

FPPS

Furthermore, the C1-O1 bond should be considered a polar covalent bond or even an ionic-like covalent bond in ATAS and FPPS, especially considering that the charge difference between C1 and O1 is more than 1.2 units in the most reasonable protonation state as listed in Table 1. Accordingly, for ATAS at the PPi deprotonation state and FPPS at the PPi mono-protonation state, the positive charge of C1 and negative charge of O1 are highly separated at the substrate and the transition state as discussed above, implying that the polarization effect of C1-O1 is higher and the resultant bond distance increase is tiny (see Table 2), thus, the C-O bond is weaker than the general C-O polar covalent bond, which is advantageous for the C-O polar-covalent bond cleavage. In contrast, since the charges of C1 and O1 are both negative in the PPi deprotonation state, the C1-O1 bond in TEAS could be considered a regular covalent bond. However, by double protonation on the PPi in TEAS, it would more or less increase the distance of the C1-O1 bond. Strikingly, this would neutralize the charge of C1 and O1 atoms and leads to a non-polar-like covalent bond which is also not as stable as the normal C-O polar covalent bond, thus facilitating the PPi cleavage.


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In sum, we strongly suggest that the identification of the exact PPi protonation state in FPPS and FPPCs is critical because it influences the nature of the C-O covalent bond based on the above discussion. The mechanism of PPi cleavage, a universal and prevalent rate-determining reaction step in various terpene elongase and cyclase, is highly dependent on the protonation state of PPi. And the rational protonation state is determined by the magnesium ions coordination modes and the enzyme class (elongase or cyclase). That is, (1) bi-protonation on PPi group has equivalent effect as PPi bi-dentate with magnesium ion; (2) additional protonation on PPi group is necessary in elongase compared with cyclases, which is attributed to the one more positive charged PPi group in elongase in most case.

Conclusion As one common and important nucleophilic substitution reaction step in the terpenoid biosynthesis, the metal-assisted diphosphate (PPi) cleavage has been comparative studied among ATAS/TEAS/FPPS for the first time. And the different reaction mechanisms have been revealed: SN1 in TEAS, SN2 in FPPS, and borderline mechanism in ATAS. Moreover, we found that the magnesium coordination modes and the native active site environment will greatly influence the protonation state of the PPi group, which is determinative for the features of the C-O bond breakage and C-C bond formation. Although extensive studies on many other similar systems are necessary, to some extent, two new hints could be concluded herein to identify the rational protonation state of PPi in FPP-relative terpenoid cyclase and elongase: (a) function of PPi bi-dentate with magnesium ion could be equivalently replaced by bi-protonation on PPi group; (b) protonation on PPi group is likely to be dominant in terpene elongase while it is necessary to re-estimate protonation state of PPi group base on the magnesium ion coordination modes in FPP cyclase.

AUTHOR INFORMATION Corresponding Authors: *E-mail: [email protected]


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Notes The authors declare no competing financial interest.

Acknowledgements This work was supported by the National Natural Science Foundation of China (21272289), Guangdong Natural Science Founds for Distinguished Young Scholars (2016A030306038) and the Pearl River S&T Nova Program of Guangzhou (2014J2200062). We thank the Guangzhou and Shenzhen Supercomputer Center, and Guangdong Province Key Laboratory of Computational Science and Guangdong Province Computational Science Innovative Research Team for providing the computational resources. We also thank Dr. Shenglong Wang at NYU-ITS for his kind help.

ASSOCIATED CONTENT Supporting Information Table S1-S5 and Figure S1-S15. Summary of available crystal structures for ATAS and TEAS; charge transfer in ATAS, TEAS and FPPS; key structures of the deprotonation state and single protonation state in ATAS and FPPS; free energy profile of concerted mechanism in TEAS; free energy profile of the PPi cleavage along the C-O bond in ATAS; free energy profile of the Npp formation; cis-trans isomerization during the 1-3 Npp formation pathway; change of the key distances during the PPi cleavage and the 1-10 cyclization in ATAS and TEAS; relative energy profiles of the Npp formation from IM1 and the following PPi cleavage in TEAS; relative energy profiles of other proton transfer pathway in TEAS; free energy profile and the key structures of the deprotonation state of FPPS; change of two key distances during the PPi cleavage (C-O cleavage) concerted with the C-C formation in deprotonation state and single protonation state of FPPS; QM/MM benchmark tests of TEAS PPi cleavage with different QM regions; benchmark tests on QM methods choice in FPPS. References (1) Over, B.; Wetzel, S.; Grütter, C.; Nakai, Y.; Renner, S.; Rauh, D.; Waldmann, H. Nat. Chem. 2012, 5, 21-28. (2) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2012, 75, 311-335. (3) Parenty, A.; Moreau, X.; Campagne, J. M. Chem. Rev. 2006, 106, 911-939. (4) Driggers, E. M.; Hale, S. P.; Lee, J.; Terrett, N. K. Nature Reviews Drug Discovery 2008, 7, 608-624. (5) Mallinson, J.; Collins, I. Future Med. Chem. 2012, 4, 1409-1438.


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(6) Chen, M.; Chou, W. K.; Al-Lami, N.; Faraldos, J. A.; Allemann, R. K.; Cane, D. E.; Christianson, D. W. Biochemistry 2016, 55, 2864-2874. (7) Asai, T.; Tsukada, K.; Ise, S.; Shirata, N.; Hashimoto, M.; Fujii, I.; Gomi, K.; Nakagawara, K.; Kodama, E. N.; Oshima, Y. Nat. Chem. 2015, 7, 737-743. (8) Miller, D. J.; Allemann, R. K. Nat. Prod. Rep. 2012, 29, 60-71. (9) Davis, E. M., and Croteau, R. Top. Curr. Chem. 2000, 209, 53−95. (10) Christianson, D. W. Chem. Rev. 2006, 106, 3412-3442. (11) Dhar, M. K.; Koul, A.; Kaul, S. New Biotechnol. 2013, 30, 114-123. (12) Lesburg, C. A. Science 1997, 277, 1820-1824. (13) Hosfield, D. J.; Zhang, Y.; Dougan, D. R.; Broun, A.; Tari, L. W.; Swanson, R. V.; Finn, J. J. Biol. Chem. 2004, 279, 8526-8529. (14) Aaron, J. A.; Lin, X.; Cane, D. E.; Christianson, D. W. Biochemistry 2010, 49, 1787-1797. (15) Vedula, L. S.; Jiang, J.; Zakharian, T.; Cane, D. E.; Christianson, D. W. Arch. Biochem. Biophys. 2008, 469, 184-194. (16) Gennadios, H. A.; Gonzalez, V.; Di Costanzo, L.; Li, A.; Yu, F.; Miller, D. J.; Allemann, R. K.; Christianson, D. W. Biochemistry 2009, 48, 6175-6183. (17) Baer, P.; Rabe, P.; Fischer, K.; Citron, C. A.; Klapschinski, T. A.; Groll, M.; Dickschat, J. S. Angew. Chem. Int. Ed. 2014, 53, 7652-7656. (18) Liu, Y. L.; Lindert, S.; Zhu, W.; Wang, K.; McCammon, J. A.; Oldfield, E. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 2530-2539. (19) Chen, M.; Al-lami, N.; Janvier, M.; D'Antonio, E. L.; Faraldos, J. A.; Cane, D. E.; Allemann, R. K.; Christianson, D. W. Biochemistry 2013, 52, 5441-5453. (20) Shishova, E. Y.; Yu, F.; Miller, D. J.; Faraldos, J. A.; Zhao, Y.; Coates, R. M.; Allemann, R. K.; Cane, D. E.; Christianson, D. W. J. Biol. Chem. 2008, 283, 15431-15439. (21) Shishova, E. Y.; Di Costanzo, L.; Cane, D. E.; Christianson, D. W. Biochemistry 2007, 46, 1941-1951. (22) Kersten, R. D.; Diedrich, J. K.; Yates, J. R., 3rd; Noel, J. P. ACS Chem. Biol. 2015, 10, 2501-2511. (23) Rising, K. A.; Crenshaw, C. M.; Koo, H. J.; Subramanian, T.; Chehade, K. A.; Starks, C.; Allen, K. D.; Andres, D. A.; Spielmann, H. P.; Noel, J. P.; Chappell, J. ACS Chem. Biol. 2015, 10, 1729-1736. (24) Noel, J. P.; Dellas, N.; Faraldos, J. A.; Zhao, M.; Hess, B. A., Jr.; Smentek, L.; Coates, R. M.; O'Maille, P. E. ACS Chem. Biol. 2010, 5, 377-392. (25) Starks, C. M. Science 1997, 277, 1815-1820. (26) Shishova, E. Y.; Yu, F.; Miller, D. J.; Faraldos, J. A.; Zhao, Y.; Coates, R. M.; Allemann, R. K.; Cane, D. E.; Christianson, D. W. J. Biol. Chem. 2008, 283, 15431-15439. (27) Felicetti, B.; Cane, D. E. J. Am. Chem. Soc. 2004, 126, 7212-7221. (28) Croteau, R. B.; Satterwhite, D. M.; Wheeler, C. J.; Felton, N. M. J. Biol. Chem. 1989, 264, 2075-2080. (29) Wise, M. L.; Pyun, H.-J.; Helms, G.; Assink, B.; Coates, R. M.; Croteau, R. B. Tetrahedron 2001, 57, 5327-5334. (30) Peters, R. J.; Ravn, M. M.; Coates, R. M.; Croteau, R. B. J. Am. Chem. Soc. 2001, 123, 8974-8978. (31) Liu, Z.; Zhou, J.; Wu, R.; Xu, J. J. Chem. Theory Comput. 2014, 10, 5057-5067. (32) Hong, Y. J.; Tantillo, D. J. Org. Lett. 2006, 8, 4601-4604. (33) Hong, Y. J.; Tantillo, D. J. J. Am. Chem. Soc. 2009, 131, 7999-8015. (34) Hamlin, T. A.; Hamann, C. S.; Tantillo, D. J. J. Org. Chem. 2015, 80, 4046-4053. (35) O'Brien, T. E.; Bertolani, S. J.; Tantillo, D. J.; Siegel, J. B. Chem. Sci. 2016, 7, 4009-4015.


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Protonation-Dependent Diphosphate Cleavage in FPP Cyclases and

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