Catalytic Control in the Facile Proton Transfer in ... - ACS Publications

Sep 25, 2017 - and Dan Thomas Major*,†. † ... verticillen-8-yl cation (cation D, Scheme 2), remains a .... of Taxadiene by TXS Proceeding via Dire...
0 downloads 0 Views 2MB Size
Article

Subscriber access provided by RYERSON UNIVERSITY

Catalytic Control in the Facile Proton Transfer in Taxadiene Synthase Yehoshua Freud, Tamar Ansbacher, and Dan T. Major ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b02824 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 25, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Catalysis is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

Catalytic Control in the Facile Proton Transfer in Taxadiene Synthase Yehoshua Freud,1 Tamar Ansbacher1,2 and Dan Thomas Major*1 1

Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel

2

Hadassah Academic College, 7 Hanevi'im St. Jerusalem, 9101001, Israel

ABSTRACT: Enzymes are highly efficient and usually very specific biocatalysts. However, some enzyme families, such as terpene synthases, are inherently promiscuous due to the extremely challenging chemistry they have evolved to tackle. Here we focus on one such enzyme, taxadiene synthase (TXS), which produces taxa-4(5),11(12)-diene, a key precursor to the chemotherapy agent taxol. A central chemical step in the biosynthesis of taxa-4(5),11(12)-diene by TXS is an intramolecular proton transfer. The inherent out-of-enzyme energetics for this facile proton transfer dictates a two-step proton transfer as the most favorable pathway, raising the question why an enzyme would prefer an indirect pathway that leaves it prone to side-product formation. In the current work, we employ hybrid quantum and molecular mechanical classical and path-integral simulations to address the nature of the intramolecular proton transfer in TXS, and we find that in the enzyme the direct proton transfer is slightly preferred over the indirect two-step pathway. This suggests that the enzyme might have evolved to favor a simpler, direct mechanistic pathway, thereby asserting chemical control by reducing its promiscuity. Understanding the underpinnings of such chemical control is likely to be important when attempting to design natural products in non-enzymatic environments. KEYWORDS: Proton transfer, Enzyme catalysis, Lyase, Chemical control, QM/MM simulations

INTRODUCTION Enzymes catalyze chemical reactions in organisms with exquisite rate enhancements1 and remarkable specificity. However, some enzymes produce non-negligible quantities of unwanted sideproducts, despite being subjected to evolutionary pressure.2 Examples of such promiscuous enzymes include ribulose-1,5biphosphate carboxylase,3 triose phosphate isomerase,4 enoylthioester reductases,5 and terpene synthases.6 This latter class of enzymes is responsible for the synthesis of over 60% of all natural products. Terpene synthases generate complex hydrocarbon scaffolds from highly reactive carbocation intermediates, employing a rich chemical toolbox including ring-formations, rearrangements, methyl migrations, and proton and hydride transfers.7-9 The extreme reactivity of carbocations presents an unusual challenge for these enzymes: how to control chemistry, rather than how to accelerate chemistry. Hence, a fundamental question is how these enzymes guide the reaction flux towards the desired products, away from competing pathways leading to sideproducts.

TXS O OO O P O P O GGPP O

H

H Taxadiene

Scheme 1. Biosynthesis of taxadiene from GGPP by TXS.

A terpene synthase of particular interest is taxadiene synthase (TXS), which catalyzes the formation of the diterpene taxa-4(5),11(12)-diene (henceforth, taxadiene), from an acyclic C20 precursor geranylgeranyl diphosphate (GGPP) (Scheme 1). taxadiene may subsequently serve as a substrate for the formation of taxol,10-12 which is an important natural anti-cancer agent.13-14 Therefore, understanding the mechanism for the formation of taxadiene in TXS may have substantial pharmaceutical implications. The detailed mechanism for the taxadiene formation, which contains a sequence of cyclizations and proton transfer steps, has been studied extensively, using experimental15-17 and computational18-19 tools. The product distribution profile for this reaction has showed that the main product, taxa-4,11-diene, is formed with 93.2% yield, while 4.7% of the isomer taxa-4(20),11diene is also formed. Additionally, the side-product verticillene is formed in 2.1% yield.16, 20 Detailed mechanistic aspects, such as the presumed low-barrier proton transfer step moving from verticillen12-yl cation (cation C, Scheme 2) to verticillen-8-yl cation (cation D, Scheme 2), remains a mechanistic conundrum, although the transfer is known to be intramolecular.17 The proton may be transferred directly from C10 to C6 in cation C, forming cation D (Scheme 2). Alternatively, as suggested by Tantillo and coworkers,18 this transfer may occur via a two-step mechanism, where the proton is transferred from C10 to C2 forming the verticillen-4yl cation (cation F in Scheme 2), followed by a proton transfer from C2 to C6, forming cation D. The latter indirect mechanism was shown to be energetically favored in the gas phase,18-19, 21 and has been adopted as the most likely pathway.20

ACS Paragon Plus Environment

ACS Catalysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

7 6

10 H 11

11

10

7

C

H 2

3

F

3

2

6

11

7

10

6 H 2 D

Page 2 of 6

The latter of the two Arg residues closes the active site. Hence, in our model, R578 interacts directly with the PP moiety as in BPPS, whereas R580 closes the active site via interactions with neighboring loops. The role of the Arg residues in the RXR motif in our model is also in excellent agreement with experimental data on the diterpene cyclase, abietadiene synthase.33 Following model construction, cation C was docked into the active TXS site, prior to commencing mechanistic simulations (Figure 1, see SI for Docking details). Two configurational possibilities were considered.13 Below we only present the lowest free energy pathway; the general conclusions are the same for the alternative conformation. In both configurations, the cation at position C11 possibly forms a p-cation interaction with Trp753 (Table S1).

3

Scheme 2. Proton transfer in the biosynthesis of taxadiene by TXS might proceed via direct (C→D) or indirect (C→F→D) pathways.

Although gas-phase calculations shed light on the inherent reactivity of the substrate,18-19, 22-23 the enzymatic TXS reaction mechanism cannot be understood without taking into consideration the protein-solvent environment.6, 24-27 In the current work, we address the crucial proton transfer step in TXS, and using multiscale simulation methods we show that the enzyme might have evolved to prefer a simpler, direct mechanistic pathway, thereby reducing its promiscuity.

COMPUTATIONAL DETAILS Despite the importance of TXS, its three-dimensional active structure is currently not known, as the reported X-ray crystal structure is in a catalytically inactive form due to lack of Nterminal residues. These residues should both cap the active site and hold a nearby loop (J-K loop) in a position that blocks off the mouth of the active site.11, 13, 28 Consequently, the first step en route to understanding the TXS mechanism, is to build a catalytically active model of TXS, employing theoretical tools, such as homology modeling.29-30 Modeling the missing N-terminal residues, the J-K loop (Lys836-Asp850), and an additional loop near the Nterminus (Ser569-Val581), is crucial in order to cap the active site of TXS. Sequence alignment followed by structure superposition, homology and loop modeling were employed (see SI for detailed procedure), leading to an active model based on bornyl diphosphate synthase (BPPS) from salvia officinalis (PDB code 1N23, 2.40 Å resolution).31 In this BPPS based model,32 the first Nterminal residue in TXS is Arg84, while the catalytically active version contains five additional residues. Therefore, the sequence MDDIP was added to the N-terminus in an extended conformation and allowed to relax during molecular dynamics (MD) simulations. Our model is somewhat different to another previously suggested TXS active conformation,20 mainly in the positioning and role of R578 and R580, which are conserved among class I terpene synthases. These residues are part of a RXR motif, where X is often a polar or charged residue. In three available crystal structures of related mono- and sesquiterpene cyclases in their active form (5epi-aristolochene synthase from nicotiana tabacum (PDB code 3M02), limonene synthase from mentha spicata (PDB code 2ONG), and the aforementioned BPPS, the former of the two Arg residues interacts directly with the PP moiety. This interaction is likely crucial to activate the initial C-O heterolytic bond cleavage.

(a)

PP

(b)

Arg578

C

Figure 1. (a) Model of the active form of TXS (grey) superimposed on the original crystal structure (PDB ID: 3P5R; blue) as well as the BPPS crystal structure (PDB ID: 1N23; pink). (b) Zoom-in on the active site. The figure highlights the pyrophosphate, carbocation C, and Arg578.

We adopt a computational protocol like that employed in our multiscale modeling study of monoterpene and sesquiterpene systems.6, 24-27 Specifically, we employ a combined quantum mechanics-molecular mechanics (QM/MM) potential to model the proton transfer steps of the multistep cascade to form taxadiene (Scheme 2).34-35 Similarly to our previous studies, the substrate hydrocarbon framework, as well as the metal-pyrophosphate cluster PP-(Mg2+)3, are treated quantum mechanically, while the remaining enzyme-solvent system is represented by the CHARMM22/27 MM force field.36 The QM region is treated by density functional theory (DFT), using the M06-2X functional.6, 2427, 37 The three-point charge TIP3P model is used for water.38 Free energy MD simulations were performed as previously described, to obtain the potential of mean force for the chemical step of interest.6 Statistical errors were obtained with the bootstrapping approach.39 We note that the current error bars are somewhat lower than what is often observed (±1 kcal/mol). This is possibly due to the very rigid active site framework, which is a result of the tight binding of the pyrophosphate moiety. Path-integral simulations were performed to quantize the transferring protons, as well as the carbon donor-acceptor pair for each reaction studied.40-42 In this description, each classical particle is replaced by a set of quasiparticles (i.e. beads), allowing a delocalized quantum behavior.43-45 We employed a higher-order factorization scheme to reduce the number of required beads to six per atom,46 coupled with enhanced sampling algorithms. 50 classical configurations were employed in conjunction with 20 Monte Carlo sampling steps for reactant and transition states for each of the proton transfer pathways. All simulations used the CHARMM simulation platform combined with the Q-Chem quantum chemistry package.47-49

ACS Paragon Plus Environment

Page 3 of 6

RESULTS We studied the two proton transfer pathways (C→D and C→F→D) for the TXS catalyzed reaction, using hybrid QM/MM classical free energy simulations, in conjunction with Feynman path-integral (PI) quantum simulations to account for zero-point energy and tunnelling effects on the proton transfer. The free energy profiles are presented in Table 1, Figure 2 and Figures S1S3). The reaction free energy for the C→D formation is -15.8 kcal/mol, which is significantly lower than the value of 1.8 kcal/mol obtained in the gas-phase. The barrier for the direct C→D proton transfer is 4.0 kcal/mol, which is also lower than the value of 11.2 kcal/mol in the absence of the enzyme. Hence, the enzyme significantly changes the free energy profile, and importantly reduces the free energy barrier. The indirect proton transfer, C→F→D, is associated with a sequential downhill process. The first barrier (C→F) is 4.9 kcal/mol, and the second barrier (F→D) is 6.6 kcal/mol. These values are somewhat similar to the gas-phase values of 7.9 kcal/mol and 8.0 kcal/mol. The effect of nuclear quantum effects is to reduce the barrier height in the enzyme by ca. 2 kcal/mol for all three steps studied, which suggests that zeropoint effects dominate, while tunneling is limited.40 The relative stability of the intermediate, F, is -9.9 kcal/mol, while in the gasphase the comparable value is 0.1 kcal/mol, underscoring the significant effect of the enzyme environment . In conclusion, there is a slight preference for the direct proton transfer in TXS, contrary to what we32 and others18-19 obtain for the reaction in absence of the enzyme. However, considering the small difference, both pathways might be possible.

10 TSC→F

5

0

DG (kcal/mol)

C6, involves cation migration from C11 to C7, i.e. towards the pyrophosphate (Fig. 3). In contrast, in the first step of the indirect pathway, the proton is transferred from C10 to C2, with a slightly reduced concurrent cation charge migration towards the PP moiety than in the direct mechanism. Table 1. Activation and reaction free-energies (kcal/mol) for the taxadiene forming proton transfer in the gas-phase and in TXS. & Δ"#→%

Gasphasea Enzymeb

‡ Δ"#→%

1.8

& Δ"#→%

11.2

‡ Δ"#→%

0.1

‡ Δ"#→%

7.9

8.0

-15.8 4.0 -9.9 4.9 6.6 ± 0.6 ± 0.6 ± 0.5 ± 0.5 ± 0.6 a M06-2X/6-31+G(d,p). Nuclear quantum effects were included using harmonic normal mode analysis. b QM(M06-2X)/MM free energy simulations. Nuclear quantum effects were included using Feynman path integrals. Statistical uncertainty was obtained employing the bootstrapping method.39

(a)

(b)

(c)

(d)

(e)

(f)

Figure 3. Snapshots of intermediate cations (a) C, (b) F, (c) D and transition states (d) C→D, (e) C→F, (f) F→D generated during QM(M06-2X)/MM MD simulations. Color coding: red, oxygen; orange, phosphorus; magenta, Mg2+ ions; green, carbocation carbon; gray, protein carbon; blue, nitrogen; white, hydrogen. In cation C, the hydrogen is located on C10. In cation F, the hydrogen is located on C2. In cation D, the hydrogen is located on C6.

TSC→D

C TSF→D

-5

10

F

Positive charges

-10

-15

D

-20

Figure 2. Free energy profiles for the generation of cation D directly from C (black curve) or via cation F (grey curve). Profiles were generated from free energy simulations using QM(M06-2X)/MM and Feynman path integrals.

Inspecting the relative distances between the carbocation locations and the diphosphate provides insight into how TXS manages to change the relative mechanistic preference from an indirect to a direct proton transfer process. The ensemble averaged distances between the PP moiety (O3/O7 atoms) and the C11 (cation C), C7 (cation D), and C3 (cation F) positions are 9.5/7.6, 6.2/4.5, and 6.1/5.5 Å, respectively (Table S1, Fig. 3, 4). The direct proton transfer pathway, which entails proton transfer from C10 to

Radial Distribution Function

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

Negative charges

8

Asp613 Mg2+,Arg578,Arg754

PP 6

Asp614,Asp617, Glu691,Glu765

4

2

0 0

ACS Paragon Plus Environment

5

10 Distance (Å)

15

ACS Catalysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Radial Distribution Function for the C7 cation (defined as the origin) of D in Taxadiene Synthase. All marked residues are located in the highly charged active site region region.

This electrostatic steering is facilitated by the unique active site architecture of terpene synthases, wherein a highly polar region is flanked by a hydrophobic pocket, and such a binary active site cavity facilitates charge migration control.6, 24-27, 32, 50-51 The polar region in TXS is arranged in an approximately layered manner relative to the cation binding pocket (Fig. 4), similar to that observed for BPPS and trichodiene synthase.27 Since the pyrophosphate group composes the inner layer, the charge migration control is largely dictated by this moiety. On the other hand, p-cation interactions with Trp753 are seemingly weakened during formation of either cation D or F (Table S1), and such interactions are unlikely to control the proton transfer step. We note that such electrostatic steering has been suggested by Peters and co-workers, and is likely a general feature in terpene synthases.50-51 The current view of the chemistry in TXS, where the enzyme plays an active role in controlling chemistry, contrasts that of earlier studies were the enzyme was assigned a more passive role.18-20 Additionally, premature quenching of either of the C6 protons or C7 exocyclic protons is avoided, as neither are in proton abstraction distance from the pyrophosphate, active site water molecule, or other alternative bases. On the other hand, the indirect pathway, which entails an intermediate with a carbocation at the C3 position, may be deprotonated at the C3 exocylic position, by the PP moiety, or at the C4 position by an active site water molecule. This may explain the observed verticillene sideproduct in TXS.16, 20 Indeed, the only slight preference for the direct C→D pathway suggests that the indirect pathway may compete with the preferred pathway.

Page 4 of 6

pyrophosphate cofactor and amino acid residues. Such a realization is likely to be important when attempting to design terpenes in non-enzymatic environments, as in the absence of enzymatic control elements, product specificity will be challenging.54-55

CONCLUSIONS In some enzyme families, the main catalytic challenge is how to control chemistry, rather than how to accelerate chemistry. The inherent out-of-enzyme energetics for the presumed lowbarrier proton transfer in the terpene synthase taxadiene synthase dictates a two-step proton transfer as the most favorable pathway, raising the question why an enzyme would prefer an indirect pathway that leaves it prone to side-product formation. In the current work, we employ hybrid quantum and molecular mechanical classical and path-integral simulations to address the nature of the facile intramolecular proton transfer in TXS, and we find that in the enzyme the direct proton transfer is slightly preferred over the indirect two-step pathway. This suggests that the enzyme might have evolved to favor a simpler, direct mechanistic pathway, reducing its promiscuity. Understanding the underpinnings of the chemical control in enzymes is crucial when attempting to design natural products in non-enzymatic environments.

ASSOCIATED CONTENT Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. (1) Free energy profiles for proton transfer steps. (2) Details of taxadiene modeling (PDF).

DISCUSSION

AUTHOR INFORMATION

The current TXS simulation studies indicate that the direct proton transfer mechanism is slightly preferred in the enzyme over a previously proposed indirect mechanism.18-19 Hence, evolution might have selected the direct proton transfer pathway over an indirect one, to reduce the likelihood of side-product formation, although this evolutionary pressure is seemingly only moderate, as in the plant world some promiscuity is tolerated. We note that the free energy barrier reductions reported here due to the enzyme environment have no implication for the overall rate of the catalytic cascade, as the rate limiting step in class I terpene synthases is typically the initial C-O bond cleavage or product release.26 Therefore, the effect of the enzyme on the reaction cascade that is responsible for some of the most intricate molecular architectures known, is to provide a correct initial substrate fold and to control the subsequent chemistry, not to accelerate it. A crucial aspect in our understanding of enzyme’s function is how chemical control is obtained. In terpene synthases, it has been assumed that following initial substrate binding and concomitant correct folding in the active site, the product formation is essentially governed by the inherent reactivity of carbocations.52-53 We argue based on a growing body of experimental50-51 and theoretical data,6, 24-27 that terpene synthases actively control chemistry via traditional catalytic elements. A crucial such element is electrostatic interactions of intermediate carbocations with the

Corresponding Author *[email protected]

Author Contributions D.T.M designed the research, Y.F., T.A., and D.T.M. performed the studies and wrote the paper.

ACKNOWLEDGMENT This work has been supported by the Israel Science Foundation (Grant # 2146/15).

REFERENCES 1. Warshel, A.; Sharma, P. K.; Kato, M.; Xiang, Y.; Liu, H.; Olsson, M. H. M. Chem. Rev. 2006, 106, 3210-3235. 2. Khersonsky, O.; Tawfik, D. S. Annu. Rev. Biochem. 2010, 79, 471-505. 3. Zhu, X. G.; Long, S. P.; Ort, D. R. Curr. Opin. Biotechnol. 2008, 19, 153–159. 4. Richard, J. P. Biochemistry 1991, 30, 4581–4585. 5. Rosenthal, R. G.; Vögeli, B.; Wagner, T.; Shima, S.; Erb, T. J. Nat. Chem. Biol. 2017, 13, 745-749. 6. Major, D. T.; Freud, Y.; Weitman, M. Curr. Opin. in Struct. Biol. 2014, 21, 25-33. 7. Croteau, R. Chem. Rev. 1987, 87, 929-954.

ACS Paragon Plus Environment

Page 5 of 6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

8. Cane, D. E. Chem. Rev. 1990, 90, 1089-1103. 9. Christianson, D. W. Chem. Rev. 2006, 106, 3412-3442. 10. Hefner, J.; Rubenstein, S. M.; Ketchum, R. E. B.; Gibson, D. M.; Williams, R. M.; Croteau, R. Chem. Biol. 1996, 3, 479-489. 11. Croteau, R.; Hezari, M.; Hefner, J.; Koepp, A.; Lewis, N. G., Paclitaxel biosynthesis - the early steps. In Taxane Anticancer Agents: Basic Science and Current Status, Georg, G. I.; Chem, T. T.; Ojima, I.; Vyas, D. M., Eds. 1995; Vol. 583, pp 72-80. 12. Koepp, A. E.; Hezari, M.; Zajicek, J.; Vogel, B. S.; Lafever, R. E.; Lewis, N. G.; Croteau, R. J. Biol. Chem. 1995, 270, 8686-8690. 13. Köksal, M.; Jin, Y.; Coates, R. M.; Croteau, R.; Christianson, D. W. Nature 2010, 469, 116-120. 14. Ganguly, A.; Yang, H.; Cabral, F. Mol. Cancer Ther. 2010, 9, 2914-2923. 15. Jin, Q. W.; Williams, D. C.; Hezari, M.; Croteau, R.; Coates, R. M. J. Org. Chem. 2005, 70, 4667-4675. 16. Jin, Y.; Williams, D. C.; Croteau, R.; Coates, R. M. J. Am. Chem. Soc. 2005, 127, 7834-7842. 17. Williams, D. C.; Carroll, B. J.; Jin, Q.; Rithner, C. D.; Lenger, S. R.; Floss, H. G.; Coates, R. M.; Williams, R. M.; Croteau, R. Chem. Biol. 2000, 7, 969-977. 18. Gutta, P.; Tantillo, D. J. Org. Lett. 2007, 9, 1069-1071. 19. Hong, Y. J.; Tantillo, D. J. J. Am. Chem. Soc. 2011, 133, 18249-18256. 20. Schrepfer, P.; Buettner, A.; Goerner, C.; Hertel, M.; van Rijn, J.; Wallrapp, F.; Eisenreich, W.; Sieber, V.; Kourist, R.; Brück, T. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E958-E967. 21. Tokiwano, T.; Endo, T.; Tsukagoshi, T.; Goto, H.; Fukushi, E.; Oikawa, H. Org. Biomol. Chem. 2005, 3, 2713-2722. 22. Tantillo, D. J. Nat. Prod. Rep. 2011, 28, 1035-1053. 23. Tantillo, D. J. Angew. Chem. Int. Ed. 2017, 56, 1004010045. 24. Weitman, M.; Major, D. T. J. Am. Chem. Soc. 2010, 132, 6349-6360. 25. Major, D. T.; Weitman, M. J. Am. Chem. Soc. 2012, 134, 19454-19462. 26. Dixit, M.; Weitman, M.; Gao, J.; Major, D. T. ACS Catal. 2017, 7, 812-818. 27. Major, D. T. ACS Catal. 2017, 7, 5461-5465. 28. Williams, D. C.; Wildung, M. R.; Jin, A. Q. W.; Dalal, D.; Oliver, J. S.; Coates, R. M.; Croteau, R. Arch. Biochem. Biophys. 2000, 379, 137-146. 29. Webb, B.; Sali, A. Meth. Mol. Biol. 2014, 1137, 1-15. 30. Marti-Renom, M. A.; Stuart, A. C.; Fiser, A.; Sanchez, R.; Melo, F.; Sali, A. Annu. Rev. Biophys. Biomol. Struct. 2000, 29, 291– 325. 31. Whittington, D. A.; Wise, M. L.; Urbansky, M.; Coates, R. M.; Croteau, R. B.; Christianson, D. W. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 15375-15380. 32. Freud, Y. Understanding the Biosynthetic Mechanism in Taxadiene Synthase using Computational Methods. Bar-Ilan University, Israel, 2014. 33. Peters, R. J.; Croteau, R. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 580-584. 34. Warshel, A.; Levitt, M. J. Mol. Biol. 1976, 103, 227-249. 35. Gao, J., Methods and Applications of Combined Quantum Mechanical and Molecular Mechanical Potentials. VCH: New York, 1995; Vol. 7.

36. MacKerell, A. D., Jr.; Bashford, D.; Bellott, R. L.; Dunbrack, R. L., Jr.; Evanseck, J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.; Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F. T. K.; Mattos, C.; Michnick, S.; Ngo, T.; Nguyen, D. T.; Prodhom, B.; Reiher, W. E., III; Roux, B.; Schlenkrich, M.; Smith, J. C.; Stote, R.; Straub, J.; Watanabe, M.; Wiorkiewicz-Kuczera, J.; Yin, D.; Karplus, M. J. Phys. Chem. B 1998, 102, 3586-3616. 37. Zhao, Y.; Truhlar, D. G. Theor. Chem. Acc. 2007, 120, 215241. 38. Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. J. Chem. Phys. 1983, 79, 926-935. 39. Hub, J. S.; de Groot, B. L.; van der Spoel, D. J. Chem. Theory Comput. 2010, 6, 3713-3720. 40. Major, D. T.; Eitan, R.; Das, S.; Mhashal, A.; Singh, V., Nuclear Quantum Effects in Enzymatic Reactions. In Simulating Enzyme Reactivity, Tunon, I.; Moliner, V., Eds. RCS Publishing: Cambridge, UK, 2017; pp 340-374. 41. Hwang, J. K.; Chu, Z. T.; Yadav, A.; Warshel, A. J. Phys. Chem. 1991, 95, 8445-8448. 42. Hwang, J. K.; Warshel, A. J. Phys. Chem. 1993, 97, 1005310058. 43. Major, D. T.; Gao, J. L. J. Mol. Graph. Model. 2005, 24, 121127. 44. Major, D. T.; Garcia-Viloca, M.; Gao, J. L. J. Chem. Theory Comput. 2006, 2, 236-245. 45. Major, D. T.; Gao, J. L. J. Chem. Theory Comput. 2007, 3, 949-960. 46. Azuri, A.; Engel, H.; Doron, D.; Major, D. T. J. Chem. Theory Comput. 2011, 7, 1273-1286. 47. Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S.; Karplus, M. J. Comput. Chem. 1983, 4, 187-217. 48. Brooks, B. R.; Brooks, C. L., III; MacKerell, A. D., Jr.; Nilsson, L.; Petrella, R. J.; Roux, B.; Won, Y.; Archontis, G.; Bartels, C.; Boresch, S.; Caflisch, A.; Caves, L.; Cui, Q.; Dinner, A. R.; Feig, M.; Fischer, S.; Gao, J.; Hodoscek, M.; Im, W.; Kuczera, K.; Lazaridis, T.; Ma, J.; Ovchinnikov, V.; Paci, E.; Pastor, R. W.; Post, C. B.; Pu, J. Z.; Schaefer, M.; Tidor, B.; Venable, R. M.; Woodcock, H. L.; Wu, X.; Yang, W.; York, D. M.; Karplus, M. J. Comput. Chem. 2009, 30, 15451614. 49. Woodcock, H. L., III; Hodoscek, M.; Gilbert, A. T. B.; Gill, P. M. W.; Schaefer, H. F., III; Brooks, B. R. J. Comput. Chem. 2007, 28, 1485-1502. 50. Xu, M.; Wilderman, P. R.; Peters, R. J. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 7397-7401. 51. Zhou, K.; Peters, R. J. Chem. Commun. 2011, 47, 40744080. 52. Hong, Y. J.; Tantillo, D. J. J. Am. Chem. Soc. 2009, 131, 7999-8015. 53. Hong, Y. J.; Tantillo, D. J. Nature Chem. 2014, 6, 104-111. 54. Zhang, Q.; Tiefenbacher, K. Nature Chem. 2015, 7, 197202. 55. Zhang, Q.; Catti, L.; Pleiss, J. r.; Tiefenbacher, K. J. Am. Chem. Soc. 2017, 139, 11482-11492.

ACS Paragon Plus Environment

ACS Catalysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 6

TOC Graphic

11

10

7

F

3

3

2

6

C

H 2

7 6

10 H 11

Out of Enzyme Pathway

11

In Enzyme Pathway

7

10

6 H 2 D

3

6

ACS Paragon Plus Environment