Reply to Comment on “Substrate Folding Modes in Trichodiene

Jan 22, 2018 - Major et al. claimed that the patterns of proton transfer (Re- vs Si-face) and methyl transfer (C12 or C13) in our recent work [Wang et...
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Correspondence/Rebuttal Cite This: ACS Catal. 2018, 8, 1363−1370

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Reply to Comment on “Substrate Folding Modes in Trichodiene Synthase: A Determinant of Chemo- and Stereoselectivity” Yong-Heng Wang,† Fan Zhang,† Jingwei Zhou,† Hujun Xie,‡ 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

‡

ACS Catal. 2017, 7 (9), 5841−5846. DOI: 10.1021/acscatal.7b01462 ACS Catal. 2017, 7. DOI: 10.1021/acscatal.7b02823 ABSTRACT: Major et al. claimed that the patterns of proton transfer (Re- vs Si-face) and methyl transfer (C12 or C13) in our recent work [Wang et al. ACS Catal. 2017, 7, 5841−5846] on biocatalysis in trichodiene synthase (FSTS) contradict the socalled “well-established” spectrometric data and make “incorrect statements” on their previous work [Major et al. ACS Catal. 2017, 7, 812−818]. This response aims to clarify that the proton/methyl transfer styles are not yet validated experimentally, at least from their citations in the rebuttal paper [Major et al. DOI: 10.1021/acscatal.7b02823] and other studies we have found. Furthermore, the main conclusion (we proposed a general rule) in our previous work indeed is not dependent on those two reaction steps. In addition, we also compare the different views on reaction energetics, chemo-selectivity, and theoretical modeling for FSTS between Major’s and our work, which can serve as a framework for citation by other research groups and academic discussion in the future.

1. PROLOGUE Fusarium sporctrichioides trichodiene synthase (FSTS) catalyzes the conversion of farnesyl diphosphate (FPP) into the sesquiterpene product trichodiene (TD, Scheme 1). Major and our group both had interests in this enzyme system and published works in this journal this year. Then Major wrote a rebuttal comment1 and pointed out that “the findings and key reaction steps” in our work2 contradict some “well-established” NMR and mass spectrometry data. However, the so-called “well-established” experimental proofs are insufficient to support their arguments, at least from the citations in ref 1 and other literatures we have learned. Besides, we received the authors criticism for our statements (in ref 2) on the stereochemistry of the key intermediate validated in their previous work.3 In fact, we did not make “incorrect statements”, and their misunderstanding is likely to be caused by ignoring a conditional adverbial clause, “...if not considering the conformational change of the initial FPP”, which is unambiguously defined in ref 2 and is the prerequisite to the inference of stereochemistry of the subsequent cyclization steps. In ref 2, via retro-biosynthesis conformational analysis, we inferred two possible conformations of hydrocarbon chain of FPP, namely, C11-up and C11-down, which differ in the orientation of C10−C11 (Scheme 1). Although both conformations give the same absolute configuration of product if not considering enzyme environment, the patterns of proton transfer and methyl transfer are different as highlighted in blue in Scheme 1. Then we consider the enzyme environment and perform quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) simulations to investigate the folding modes of substrate FPP in the active site of FSFS and illuminate the catalytic mechanisms; in addition, a practical rule is summarized to predict the substrate folding modes for 1,6- and 1,10-cyclases, which catalyze the 1,6- and 1,10© XXXX American Chemical Society

cyclization of FPP, respectively. When building theoretical models from the substrate FPP, expect for the two conformations of hydrocarbon chain we also consider relative orientations between the hydrocarbon chain and the diphosphate (OPP) group, which makes several combinations corresponding to different substrate folding modes as defined in ref 2. Our simulations determine the optimal substrate folding mode Rup with a C11-up configuration of the hydrocarbon chain, which leads to a Re-face proton transfer and a C13 methyl transfer (there is a typo that inverts the numbering of C12/C13 atom in ref 2, and we appreciated that Major pointed out this typo in ref 1). This contradicts the Major’s result reported in ref 3, in which a Si-face proton transfer and a C12 methyl transfer are adopted. Before point-by-point response, one more thing we have to point out is that the main contributions of our work (ref 2) is summarizing the regulatory laws of chemo- and stereoselectivity in the cyclization reactions catalyzed by sesquiterpene cyclases, and most of the content of ref 2 is devoted to this issue, but not merely determining which conformation of substrate is correct as stated in the Comment (ref 1). Moreover, the different Re-/ Si-face proton transfer and C12/C13 methyl migration process would lead to different conformations of FPP, but it would not defeat the main conclusions, namely the practical rule proposed in ref 2, because our proposed general rule is not sensitive to the tail conformation difference from C12/C13 but mostly based on the head folding mode (conformation) of substrate FPP, that is, the steric hindrance between diphosphate group and the head C15 methyl group. (vide infra) Received: November 15, 2017

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Scheme 1. Two Possible Conformations of FPP Can Both Give the Correct Configuration of Product with Different Patterns of Proton Transfer and Methyl Transfer If Not Considering Enzyme Environment

2. RESPONSE 2.1. The So-Called “Well-Established” Experimental Data Are Insufficient. In Major’s rebuttal, they claim that the patterns of hydrogen transfer (Re- or Si-face transfer) and methyl transfer (C12 or C13 methyl transfer) were “wellestablished” by experimental NMR and mass spectrometry data, and they cite a lot of references4−12 (refs 4−12 herein, correspond to the refs 6, 8, 18−24 of their Comment) as the grounds of arguments, while by our careful checking, the cited nine references were not directly related to this problem, except for refs 6 and 7. In both refs 6 and 7, only isotope radioactive assays were performed to determine the position of isotopelabeled atoms instead of the stereochemistry. The directly related proof may only come from the “Scheme 2” in ref 7 (corresponding to ref 19 in Major’s Comment. To be clear and convenient for readers, we represent it as Figure 1a herein, because it is an old literature with low resolution for this figure). Regarding Proton Transfer. Scheme 2 in ref 7 did schematically show the 3H-labeled hydrogen atom on C6 was transferred to the Si-face of C10, and the formed 3H-substitued C10 was chiral with S configuration (see Figure 1a), contradicting the configuration used in our work (ref 2). However, the original sentence in ref 7 is that “The almost complete loss of tritium radioactivity associated with the conversion of 13 into 15 serves to locate the tritium label specifically at C(2) or, less likely at C(3), in verrucarol (3)...” (The C2 in verrucarol corresponds to the C10 in trichodiene). That is, it is experimentally validated that the hydrogen atom was most likely to be transferred to the C10 site. Furthermore, we did not find any words about the absolute configuration (S or R) of the C10 (namely, C2 in verrucarol) in ref 7; that is, the pattern of hydride transfer (Re- or Si-face transfer) was

uncertain. Accordingly, the mechanism schematically shown in the Scheme 2 in ref 7 is just a proposed mechanism with one possibility but not confirmed stereochemistry style of the hydride transfer process (Figure 1a), not as claimed by Major in the Comment (Figure 1b, namely, Scheme 2 in ref 1): “...wellestablished stereochemistry from NMR and mass-spectrometry isotope labeling experiments carried out by several groups: it is the Si-face, not Re, of C10 to which the proton on C6 migrates.” It is worthy to note the differences between the primary mechanism7 shown in Figure 1a and modified mechanism summarized in Scheme 1, which is adopted by Major,3 Tantillo,13 and us1 with minor differences. In the former mechanism (Figure 1a), the closure of the 5-membered ring precedes the hydrogen transfer, and the transferred hydrogen atom is a hydride (so noted as hydride transfer in Figure 1a). In this mechanism, the hydride can only transfer to the Si-face of the C10 in the 5-membered ring (Re-face transfer is impossible due to the relative orientation of the 5-membered ring and the 6-membered ring), which is by chance consistent with Major’s work in ref 3. Therefore, the exclusive Si-face hydride transfer for this step is owing to the proposed reaction mechanism, but not established by experiments in ref 7. On the contrary, in the newest mechanism (Scheme 1), the hydrogen transfer precedes the closure of 5-memebered ring and the transferred hydrogen atom is a proton instead of hydride; moreover, the proton can transfer either to the Re-face or to the Si-face of the C10 site at the double bond, depending on the conformation of the hydrocarbon chain. Regarding Methyl Transfer. First of all, in both refs 6 and 7, 14C label was used, instead of 13C label as stated in Major’s Comment. Moreover, no literary description definitely pointed out the transferred methyl was C12 or C13 by our careful double-checking from refs 6 and 7, merely the Scheme 2 in ref 1364

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Scheme 2. Both C12 (trans) and C13 (cis) Methyl of FPP Are Possible To Be Labeled from Mevalonate

determine the patterns should be experimental data such as crystal structure and ECD spectrum that could determine the absolute configuration (S or R) of the 3H-substitued C10 in trichodiene and which methyl is labeled by 14C (C12 or C13 methyl). Unfortunately, we cannot find these persuasive grounds of arguments from the citations in ref 1. 2.2. Misunderstanding of Our Statement on the Configuration of Key Intermediate. In ref 2, we mentioned that “mode Rdown would directly lead to the formation of bisabolyl cation B with S configuration conflicted with the configuration demanded for the product if not considering the conformational change of the initial FPP”. However, it does not mean the cyclization step reported in ref 3 would lead to the formation of bisabolyl cation intermediate with incorrect S configuration. In ref 3, it was explicitly wrote that R is the correct configuration of “(6R)-bisabolyl cation”, and we would not intentionally distort their descriptions. Actually, the folding mode Rdown of substrate could lead to the correct R configuration of bisabolyl cation via a conformational change; conversely, the incorrect S configuration would be obtained without the conformational change (Figure 2). However, Major ignored this prerequisite which was unambiguously expressed

Figure 1. (a) Possible mechanism for biosynthesis of verrucarol schematically shown in Scheme 2 of ref 7 (corresponding to ref 19 in Major’s Comment). Note that it is just a schematic diagram (mechanism model), while the absolute configuration (S or R, corresponding to Si- or Re-face hydride transfer) of C10 and which methyl (C12 or C13) is transferred are not determined in ref 7; (b) patterns of methyl transfer and proton transfer shown in Scheme 2 of the Comment, and Major et al. proclaimed that the patterns and the “stereochemistry” are “well established” by experimental data.

7 (namely Figure 1a herein) schematically shows the labeled carbon atom is the trans methyl (C12 methyl), indicating the transferred methyl is C12,6 which appears to agree with Major’s work (Figure 1b) while contradicting the pattern of methyl transfer in our work (ref 2). However, we also find another labeling pattern (cis methyl label) schematically shown in the related experimental literatures14,15 which were not mentioned in Major’s Comment. To be clear and convenient for readers, we reproduce it and highlight the key atoms from the original literature (ref 6) to be Scheme 2a herein; it indicates that the transferred methyl is C13,6 which is consistent with our work (ref 2). Furthermore, the possible mechanism for the formation of FPP from mevalonate is shown in Scheme 2b.16 From theoretical analysis, both C4,C8,C13-labeled and C4,C8,C12labeled FPP are possible from C2-labeled mevalonate, indicating both C12 and C13 methyl transfer are possible. In a word, both the patterns of hydrogen transfer and methyl transfer were not definitely determined experimentally, at least from the citations in Major’s Comment and the studies we have learned. As far as we know, the most convinced evidence to

Figure 2. Possible connection between the substrate folding mode Rdown in our models starting from substrate state (a) and Major’s model starting from intermediate state (b). 1365

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Figure 3. Correspondences between folding modes and main reaction pathways (1,6- and 1,10-ring closure) in sesquiterpene synthases, summarized in ref 2 (original Figure 4). FSTS: Fusarium sporctrichioides trichodiene synthase; TEAS: Nicotiana tabacum 5-epiaristolochene synthase; ATAS: Aspergillus terreus aristolochene synthase.

in the conditional adverbial clause “...if not considering the conformational change of the initial FPP” in ref 2, and subjectively assumed that we were pointing out their cyclization step led to the incorrect configuration: “These authors created and illustrated their Rdown conformation that is duplicated in Figure 1c, based on which they proclaimed that the cyclization step in our study ‘would directly lead to the formation of bisabolyl cation B with S configuration conflicted with the configuration demanded for the product” (see ref 1). In our work (ref 2), the substrate FPP was used as a starting point to build computational models, in contrast to Major’s work that the intermediate D (cation 6 in ref 3) was employed. After classical molecular dynamics (MD) simulations, we obtained four stable folding modes of the substrate, namely, mode Rup, Rdown, Lup, and Ldown. To further determine which one is the catalytically productive folding mode, we used the energy barrier of chemical rate-limiting step, namely, the C−O bond dissociation, as a testimony, which was frequently used in our previous work to determine the protonation state of diphosphate group.17,18 By elaborative QM/MM MD simulation, we determined the optimal substrate folding mode Rup as it gives the most reasonable energy profile for the ratelimiting step and the whole reaction energy profiles are in agreement with the presteady-state kinetic experiment (see below “Discussion” part).19 By careful structural analysis we found that, if not considering conformational change of the initial substrate, the folding mode Rdown would directly lead to the incorrect S configuration of bisabolyl cation (note that the conformation of hydrocarbon chain in mode Rdown is slightly different from the C11-down conformation schematically shown in Scheme 1a, and after MD simulations the orientation of C6−C7 double bond is changed, see Figure S3 in ref 2 for details). As shown in Figure 2, obviously, starting from the substrate of folding mode Rdown, there are two choices either with no conformational change to form the incorrect S configuration of bisabolyl cation or with conformational change

to form the correct R configuration. Since the conformational change usually requires the consumption of energy, starting from mode Rdown will most likely lead to the incorrect S configuration. Moreover, the energy barrier of the rate-limiting step for mode Rdown was higher than mode Rup. Thus, we thought the folding mode Rdown of substrate FPP was irrational (ref 2). Differently, Major et al. employed the intermediate structure as a starting point for modeling and did not consider the prior steps including the first rate-limiting step. Obviously, the computational models employed in Major’s and our work are different, it is meaningless to state that “the methods and level of theory used in that work are nearly identical to our own recent work”, without considering the model differences. Accordingly, it is impossible for them to investigate the various substrate folding modes and observe the possible conformational change from substrate FPP to the key intermediate bisabolyl cation B. Thus, we speculate that Major’s misunderstanding (ref 1) lies in quoting our words out of context in our work (ref 2) and the different starting points (substrate vs intermediate) to study the enzymatic reaction.

3. DISCUSSION 3.1. Main Contributions of Our Work (Ref 2). In ref 1, Major stated that the “main result” of our work is “based on energetic considerations” to reach a conclusion that mode Rup is the most likely productive substrate folding mode, and prejudge our conclusion contradicts the so-called “wellestablished” experimental data. Actually, the conclusion is based on multiple considerations, including the relative orientations between the diphosphate group and hydrocarbon chain, the conformations of hydrocarbon chain inferred by “retro-biosynthesis conformational analysis”, the energy barrier of the rate-limiting step and then the energy profiles of the whole reaction pathway. Moreover, this is just a small part of the “main result” of our work in ref 2, and the core content and 1366

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Figure 4. Comparison of free-energy profiles for FSTS biocatalysis from ref 2 (a and b, original Figure 3) and ref 3 (c, original Figure 2), intermediates 1, 2, 3, 4, 6, 7, 8, and 9 in ref 3 correspond to cisoid 3R-NPP, A2, B, C, D, E, F1, and trichodiene in ref 2, respectively.

the highlight of our work are to summarize a general law of chemo- and stereoselectivity regulations in the reactions catalyzed by sesquiterpene cyclases. Much text of ref 2 is devoted to discussing the types of substrate folding (R, L, C, and O, see Figure 1b in ref 2), and the relationships between the substrate folding modes and the two main reaction pathways catalyzed by sesquiterpene cyclases (Figure 3, original Figure 4 in ref 2), namely 1,6- and 1,10-ring closure; these might be, however, the universal and predictive rule, and thus this is the most important “main result” in our previous work (ref 2) but not the results for identifying the optimal substrate folding mode and illuminating its possible catalytic mechanism in FSTS. Additionally, we found that the fidelity and promiscuity of enzymatic reactions is likely to be highly dependent on whether the control exerted by the enzyme over the substrate folding is strict or not, in sesquiterpene cyclases. Notably, the only difference between the optimal mode Rup in ref 2 and the model employed by Major et al. in ref 1, which can be derived from mode Rdown in ref 2 via a conformation change, is the orientation of terminal double bond C10−C11. The above-mentioned experimentally uncertain stereochemistry is on the C10 and C12/C13 migration. While our rule is based on the steric hindrance between diphosphate group and the head C15 methyl group (see details in ref 2), but not sensitive to the conformation of the tail C10/C12/C13 group. Therefore, whichever theoretical model (Major’s or ours) is correct, and even if the so-called experimental proof is established, does not af fect the main conclusions and the practical rule (Figure 3). 3.2. Different Views on Reaction Energetics. In ref 1, Major made a comment on our free-energy profile in ref 2 (Figure 4b). They stated that “the conversion of bisabolyl cation (3) to the subsequent intermediates has a significant

barrier according to Wu and co-workers. This is problematic because this bottleneck appears after the key intermediate (3), which branches into pathways leading to many byproducts” (the “significant barrier” is 11.8 kcal/mol in ref 2, and the intermediate 3 in ref 3 corresponds to B in ref 2). Indeed, from the bisabolyl cation, an intermediate may branch into many pathways, but these pathways correspond to different conformations of this key intermediate. Tantillo et al. had classified the conformations of bisabolyl cation and summarized the correspondences between the conformations of bisabolyl cation and different products.20 From this point of view, the chemoselectivity of the enzymatic reaction depends greatly on the conformational preorganization of the key intermediate. In ref 2, we analyzed the conformation of bisabolyl cation and found that the conformation is preorganized/ready for the next 1,5-proton transfer step (Figure S7 in ref 2). Because the carbocation is located at C7 site, far away from the dropped diphosphate group (the generally accepted base for deprotonation),21 and no other alkaline residues are near to it in the active pocket, the bisabolyl cation is less likely to be deprotonated to form bsiabolene.22 The more likely side reaction is via 1,2-hydride transfer to form homobisabolyl cation.20 The potential energy scan along the reaction coordinate of 1,2-hydride transfer indicated that a conformational change is required, and before this conformational change, the barrier is as high as 15 kcal/mol (unpublished works), while the barrier for 1,5-proton transfer, the main reaction pathway, is only 11.8 kcal/mol, so the possibility of 1,2-hydride transfer is ruled out in the view of energetics. It should be noted that to evaluate the chemoselectivity toward dif ferent reaction pathways, the absolute value of a single reaction 1367

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Patterns of Cyclization Are Different Following the 1,5Proton Transfer. In our simulations (ref 2), following the 1,5proton transfer, the intermediate C is a metastable state and it will spontaneously form intermediate G through 6,11cyclization nearly with no free-energy barrier (eq 1, Figure 4b

barrier is incapable, instead the relative value of the barrier dif ference between dif ferent pathways should be used. In the following, we will carefully compare the related computational results obtained by the two works in refs 2 and 3. Predicted Absolute/Relative Barriers in Ref 2 Are More Reasonable Compared with the Experiments. Presteady-state kinetic studies19 shown that the ionization step of the substrate FPP in the active site is the slowest chemical step (rete-limiting step) with a first-order rate constant of 4 s−1, converted to a reaction barrier of about 17 kcal/mol; while the cyclization of NPP to the product trichodiene has a net rate constant of 200 s−1, converted to a reaction barrier of about 15 kcal/mol. The rate-limiting step in our calculations is the same as experimental results, and the calculated energy barriers for these two processes are 21.1 and 18.8 kcal/mol, respectively (Figure 4a and 4b), which differ from the experimental values by 3−4 kcal/mol. Considering the errors of fitting experimental data and the errors caused by theoretical formula and computational method, the differences between experiment and computation are acceptable. When we consider the relative barrier between these two processes, the computational value is very close to the experimental value (21.1−18.8 = 2.3 kcal/mol vs 17−15 = 2 kcal/mol). Because the relative energy barrier will offset the systematic error (both computational and experimental data), so is more creditable. In ref 3 (Figure 4c), Major did not consider the FPP-to-NPP process (including the initial ratelimiting step), and the overall energy barrier of the cyclization of NPP to product is only 9.8 kcal/mol, which is 5 kcal/mol lower than the experimental value of 15 kcal/mol. Since the conformation of NPP in ref 3 is derived reversely from intermediate D (namely 6 in Figure 4c) instead of forwardly from the substrate FPP, it may be trapped in a higher-energy local minimum near the carbocation intermediate A2 (2 in Figure 4c), so the reaction barrier will be lower if using this local minimum as a starting point of energy. As mentioned above (section 2.2), a conformational change is required to connect the key intermediate in ref 3 with the mode Rdown in ref 2 (see Figure 2), so if this conformational change takes place at the NPP state, the energy for the conformational change of NPP intermediate (1 in Figure 3c) should be considered to reestimate the reaction barrier reported in ref 3. The 1,6-Cyclization Step Is Exothermic in Ref 2 and Endothermic in Ref 3. It is well-known that the cyclization reactions are usually exothermic.20,23−28 However, in ref 3, the 1,6-cyclization of cis farnesyl cation A2 (2 in Figure 4c) to form bisabolyl catin B (3 in Figure 3c) is endothermic by 3−4 kcal/ mol with a barrier of 4.9 kcal/mol, indicating the intermediate A2 (2 in Figure 4c) is a relatively stable state. This is problematic because the allylic cation (the charge is delocalized at C1, C2, and C3 site) in intermediate A2 is close to the diphosphate group dropped from the C3 site in the last step, and the dissociative diphosphate group, a generally accepted base for deprotonation in class I terpene cyclases,21 is very likely to deprotonate the C4 or C15 (next to C3) to form farnesene, which actually is a side product in the mutants of FSTS.22 On the contrary (Figure 4b), the 1,6-cyclization in ref 2 is exothermic by 3.6 kcal/mol with a little barrier of 1.4 kcal/ mol, indicating the intermediate A2 is a metastable state and the NPP-to-B process can be viewed as a concerted but highly asynchronous step, which effectively avoids the formation of side product farnesene by deprotonation.22

and see Figure S7 in ref 2 for details), instead of intermediate D directly through 7,11-cyclization. This is reasonable, because the C11 atom is closer to C6 atom than to C7 atom, after the 1,5-proton transfer from C6 site to C10 site. That is, the conformation is preorganized/read for this cyclization. The metastable nature of intermediate C effectively avoids the deprotonation of C12 or C13 methyl group by the dissociative OPP group (near the carbocation at C11 site) to form iso-γbisabolene.22 Since the carbocation in intermediate G is far away from the dissociative 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;23,29,30 instead the conformation is preorganized/read for the next 1,2-alkyl transfer (the C6−C11 single bond is perpendicular to the plane of C7 carbocation, see Figure S7 in ref 2 for details) and hence it is readily to form intermediate D. It is inspiring to note that the intermediates with carbocation near the dropped OPP group (the general base), including A1, A2 and C, are all metastable states, while the following intermediates from them, including 3R-NPP, B and G, are all stable states with no cation in hydrocarbon chain or the carbocation far away from the dropped OPP group. Obviously, this may be a sophisticated strategy exerted by terpene synthases to avoid the various deprotonated side products. In ref 3 (Figure 4c), the intermediate C (4 in ref 3) will directly lead to the formation of D (6 in ref 3) through 7,11cyclization. Note that the conformations of intermediate C in ref 2 and ref 3 are different (C11-up and C11-down, respectively, see Scheme 1), and this may be the main reason for the different patterns of cyclization (6,11- vs 7,11cyclization). Though adopting similar conformation of intermediate C with the gas-phase calculations by Hong and Tantillo,13 Major et al. obtain different results. In the gas-phase calculations, the intermediate C undergoes several conformational changes to reach a suitable conformation for the 7,11cyclization, while the 7,11-cyclization from C (4 in ref 3) to D (6 in ref 3) is barrierless in ref 3 (Figure 4c). Because the authors in ref 3 did not provide sufficient structural information on key intermediates at active site of FSTS and the potential of mean force (PMF) profile for this step, we have no more comments. 3.3. Different Views on Building Computational Model. In ref 1, Major et al. pointed out that theoretical modeling starting from substrate is “far from trivial” and “it is not clear which one may be considered as the “native FPP substrate” for a given enzyme configuration”, while starting from the rigid intermediate structure is “simpler”. Indeed, modeling from the substrate FPP is much more difficult due to its high flexibility. To solve this problem, we used several limiting conditions, including the retro-biosynthesis conformational analysis and the relative orientations between the hydrocarbon chain and the diphosphate group, to reduce the 1368

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easily obtained from an intermediate structure. Many crystal structures are devoted to decipher the binding of protein with substrate analogues. Unfortunately, the substrate analogues can only give approximate binding states, and in many cases multiple binding modes are detected,33−35 which cannot reflect the real reactive state of substrate; while the native substrate is so reactive that the modern crystallography are still impossible to capture the reactive state of enzyme−substrate complex. We knew that theoretical modeling starting from substrate state is also challenging to detect all possible reactive conformations of substrate. But it does not mean we should not have an adventurous attempt as done in ref 2. Fortunately, our results are consistent with experimental data including kinetic measurements19 and isotope radioactive assays6,7 we have learned, and the practical rule summarized in ref 2 will continue to guide us to infer the unclear substrate folding in other sesquiterpene synthases. The related work is actually ongoing in our laboratory.

number of the possible substrate folding modes. By doing this, we obtain only 8 possible folding modes and after classical MD simulations 4 ones are left for the next QM/MM MD simulations. Actually, this is based on the rational analysis instead of the docking methods as stated in Major’s Comments. In the QM/MM MD simulations, we consider an important result, the reaction barrier of the rate-limiting step, to determine the optimal folding mode of substrate. It is hard to believe a folding mode with an unfeasible reaction barrier of the rate-limiting step will give a correct reaction mechanism. Though “far from trivial” it is, we would like to take the substrate state as a starting point for modeling. Every enzymatic reaction starts from substrate state and ends with product state. The substrate and product state are usually connected by several intermediate states. For biochemical reactions catalyzed by terpene synthases, the intermediate states (i.e., carbocations) are so reactive that conformational relaxation becomes little possible, and thus the conformation of intermediate state is usually under kinetic rather than thermodynamic control.21,31,32 As shown in Figure 5, for example, though conformer 2 is

4. EPILOGUE Finally, we appreciate any public and private academic discussion from Prof. Major and others in future, while we have to emphasize again that the so-called “well-established” experiment is not validated from the literatures cited in ref 1. It is our responsibility to discuss more details on the complicated enzymatic reaction catalyzed by FSTS, because the inappropriate comments proclaimed in ref 1 would absolutely misguide readers’ prejudgments on our previous work (ref 2) if we do not give a response in public. Additionally, our aim is not to prove which modeling (Major’s or ours) is “right”. In our opinion, it is not yet possible to make definite answers for so many unsolved problems in FSTS catalysis. The combination of theoretical modeling and experimental investigation, as well as the development of new experimental and computational strategies might be more feasible and promising. Thus, we will make an end of the pointless quarrel which does not change anything. In other words, it is unnecessary to continue on debating which theoretical modeling is better, and the more significant point is whether we solved the scientific problems that currently exist in the field, as well as the citations from both experimental and computational scientists in the future. This is the most persuasive criterion by which to judge the contribution of Major’s and our work (ref 3 and ref 2).

Figure 5. Example for kinetic control of intermediate (I) conformation. Though conformer 2 is thermodynamically more stable than conformer 1, the barrier to reach conformer 2 is higher than conformer 1 and thus substrate (S) and product (P) are connected by conformer 1.

thermodynamically more stable than conformer 1, the barrier to reach conformer 2 is higher than conformer 1, so substrate (S) and product (P) are connected by the kinetically favorable conformer 1 instead of the thermodynamically stable conformer 2. Therefore, using a thermodynamically stable intermediate structure as a starting point for modeling cannot always guarantee its reaction pathway is genuine. Moreover, the biggest drawback for using the intermediate structures as a starting point is that the rate-determined step would not be investigated, because the initial cleavage of OPP group is thought to be the rate-determined step in many sesquiterpene synthases, such as this FSTS enzyme. The terpene enzyme catalysis is so complicated that no one can guarantee his/her theoretical model is definitely correct to explain all experimental phenomena. It is hard to say who is more sensible to choose starting point for theoretical modeling. Obviously, theoretical modeling starting from a rigid intermediate structure is much simpler, but our intention is to investigate the correspondence between substrate folding and various reaction pathways catalyzed by terpene synthases, which attracted many concerns. So we need to investigate various substrate folding modes, not just the correct one (actually no one knows the correct one), but these cannot be

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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. 1369

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(35) 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 (PDB codes: 4OKZ).

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DOI: 10.1021/acscatal.7b03906 ACS Catal. 2018, 8, 1363−1370