Catalytic Control in the Facile Proton Transfer in Taxadiene Synthase Yehoshua Freud,† Tamar Ansbacher,†,‡ and Dan Thomas Major*,†

Research Article

pubs.acs.org/acscatalysis

† Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel ‡ Hadassah Academic College, 7 Hanevi’im Street, Jerusalem 9101001, Israel

*S Supporting Information

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 as to 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 nonenzymatic environments. KEYWORDS: proton transfer, enzyme catalysis, lyase, chemical control, QM/MM simulations

■ INTRODUCTION Scheme 1. Biosynthesis of Taxadiene from GGPP by TXS Enzymes catalyze chemical reactions in organisms with exquisite rate enhancements1 and remarkable specificity. However, some enzymes produce non-negligible quantities of unwanted side products, despite being subjected to evolutionary pressure.2 Examples of such promiscuous enzymes include ribulose-1,5-biphosphate carboxylase,3 triose phosphate isomerase,4 enoyl-thioester reductases,5 and terpene synthases.6 This last 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 The detailed mechanism for taxadiene formation, which formations, rearrangements, methyl migrations, and proton and contains a sequence of cyclizations and proton transfer steps, − − hydride transfers.7 9 The extreme reactivity of carbocations has been studied extensively, using experimental15 17 and presents an unusual challenge for these enzymes: how to control computational18,19 tools. The product distribution profile for chemistry rather than how to accelerate chemistry. Hence, a this reaction has showed that the main product, taxa-4,11-diene, fundamental question is how these enzymes guide the reaction is formed with 93.2% yield, while 4.7% of the isomer taxa- flux toward the desired products, away from competing 4(20),11-diene is also formed. Additionally, the side product pathways leading to side products. verticillene is formed in 2.1% yield.16,20 Detailed mechanistic A terpene synthase of particular interest is taxadiene synthase aspects, such as the presumed low-barrier proton transfer step (TXS), which catalyzes the formation of the diterpene taxa- moving from verticillen-12-yl cation (cation C, Scheme 2)to 4(5),11(12)-diene (henceforth, taxadiene) from the acyclic C20 verticillen-8-yl cation (cation D, Scheme 2), remains a precursor geranylgeranyl diphosphate (GGPP) (Scheme 1). mechanistic conundrum, although the transfer is known to be 17 Taxadiene may subsequently serve as a substrate for the intramolecular. The proton may be transferred directly from − formation of taxol,10 12 which is an important natural anticancer agent.13,14 Therefore, understanding the mechanism Received: August 20, 2017 for the formation of taxadiene in TXS may have substantial Revised: September 13, 2017 pharmaceutical implications. Published: September 25, 2017

Scheme 2. Possibility of Proton Transfer in the Biosynthesis synthases. These residues are part of a RXR motif, where X of Taxadiene by TXS Proceeding via Direct (C → D) or is often a polar or charged residue. In three available crystal Indirect (C → F → D) Pathways structures of related mono- and sesquiterpene cyclases in their active form, 5-epi-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. 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

C10 to C6 in cation C, forming cation D (Scheme 2). Alternatively, as suggested by Tantillo and co-workers,18 this transfer may occur via a two-step mechanism, where the proton is transferred from C10 to C2, forming the verticillen-4-yl 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 18,19,21 20 phase and has been adopted as the most likely pathway. Figure 1. (a) Model of the active form of TXS (gray) superimposed Although gas-phase calculations shed light on the inherent 18,19,22,23 on the original crystal structure (PDB ID 3P5R; blue) as well as the reactivity of the substrate, the enzymatic TXS reaction BPPS crystal structure (PDB ID 1N23; pink). (b) Enlargement of the mechanism cannot be understood without taking into fi − active site. The gure highlights the pyrophosphate, carbocation C, consideration the protein−solvent environment.6,24 27 In the and Arg578. current work, we address the crucial proton transfer step in TXS, and using multiscale simulation methods we show that the Supporting Information for docking details). Two the enzyme might have evolved to prefer a simpler, direct configurational possibilities were considered.13 Below we only mechanistic pathway, thereby reducing its promiscuity. present the lowest free energy pathway; the general conclusions are the same for the alternative conformation. In both ■ COMPUTATIONAL DETAILS configurations, the cation at position C11 possibly forms a Despite the importance of TXS, its three-dimensional active π−cation interaction with Trp753 (Table S1 in the Supporting structure is currently not known, as the reported X-ray crystal Information). structure is in a catalytically inactive form due to the lack of N- We adopt a computational protocol like that employed in our terminal residues. These residues should both cap the active site multiscale modeling study of monoterpene and sesquiterpene − and hold a nearby loop (J-K loop) in a position that blocks off systems.6,24 27 Specifically, we employ a combined quantum the mouth of the active site.11,13,28 Consequently, the first step mechanics−molecular mechanics (QM/MM) potential to en route to understanding the TXS mechanism is to build a model the proton transfer steps of the multistep cascade to catalytically active model of TXS, employing theoretical tools, form taxadiene (Scheme 2).34,35 Similarly to our previous such as homology modeling.29,30 Modeling the missing N- studies, the substrate hydrocarbon framework, as well as the 2+ terminal residues, the J-K loop (Lys836-Asp850), and an metal pyrophosphate cluster PP-(Mg )3, are treated quantum additional loop near the N-terminus (Ser569-Val581) is crucial mechanically, while the remaining enzyme−solvent system is in order to cap the active site of TXS. Sequence alignment represented by the CHARMM22/27 MM force field.36 The followed by structure superposition, homology, and loop QM region is treated by density functional theory (DFT), using − modeling were employed (see the Supporting Information for the M06-2X functional.6,24 27,37 The three-point charge TIP3P a detailed procedure), leading to an active model based on model is used for water.38 Free energy MD simulations were bornyl diphosphate synthase (BPPS) from Salvia officinalis performed as previously described, to obtain the potential of (PDB code 1N23, 2.40 Å resolution).31 In this BPPS-based mean force for the chemical step of interest.6 Statistical errors model,32 the first N-terminal residue in TXS is Arg84, while the were obtained with the bootstrapping approach.39 We note that catalytically active version contains five additional residues. the current error bars are somewhat lower than what is often Therefore, the sequence MDDIP was added to the N-terminus observed (±1 kcal/mol). This is possibly due to the very rigid in an extended conformation and allowed to relax during active site framework, which is a result of the tight binding of molecular dynamics (MD) simulations. Our model is somewhat the pyrophosphate moiety in terpene synthases. Path-integral different from another previously suggested TXS active simulations were performed to quantize the transferring conformation,20 mainly in the positioning and role of R578 protons, as well as the carbon donor−acceptor pair for each − and R580, which are conserved among class I terpene reaction step studied.40 42 In this description, each classical

7654 DOI: 10.1021/acscatal.7b02824 ACS Catal. 2017, 7, 7653−7657 ACS Catalysis Research Article particle is replaced by a set of quasi-particles (i.e., beads), process. The first barrier (C → F) is 4.9 kcal/mol, and the − allowing a delocalized quantum behavior.43 45 We employed a second barrier (F → D) is 6.6 kcal/mol. These values are higher-order factorization scheme to reduce the number of somewhat similar to the gas-phase values of 7.9 and 8.0 kcal/ required beads to six per atom,46 coupled with enhanced mol. The effect of nuclear quantum effects is to reduce the sampling algorithms. Fifty classical configurations were barrier height in the enzyme by ca. 2 kcal/mol for all three steps employed in conjunction with 20 Monte Carlo sampling studied, which suggests that zero-point effects dominate, while steps for reactant and transition states for each of the proton tunneling is limited.40 The relative stability of the intermediate, transfer pathways. All simulations used the CHARMM F,is−9.9 kcal/mol, while in the gas phase the comparable simulation platform combined with the Q-Chem quantum value is 0.1 kcal/mol, underscoring the significant effect of the − chemistry package.47 49 enzyme environment. In conclusion, there is a slight preference for the direct proton transfer in TXS, in contrast to what we32 ■ RESULTS and others18,19 obtain for the reaction in absence of the ff We studied the two proton transfer pathways (C → D and C enzyme. However, considering the small di erence, both → F → D) for the TXS-catalyzed reaction, using hybrid QM/ pathways might be possible. MM classical free energy simulations, in conjunction with Inspecting the relative distances between the carbocation Feynman path-integral (PI) quantum simulations to account locations and the diphosphate provides insight into how TXS for zero-point energy and tunnelling effects on the proton manages to change the relative mechanistic preference from an transfer. The free energy profiles are presented in Table 1, indirect to a direct proton transfer process. The ensemble Figure 2, and Figures S1−S3 in the Supporting Information. averaged distances between the PP moiety (O3/O7 atoms) and the C11 (cation C), C7 (cation D), and C3 (cation F) Table 1. Activation and Reaction Free Energies (kcal/mol) positions are 9.5/7.6, 6.2/4.5, and 6.1/5.5 Å, respectively for the Taxadiene-Forming Proton Transfer in the Gas (Table S1 in the Supporting Information and Figures 3 and 4). Phase and in TXS

Δ r Δ ⧧ Δ r Δ ⧧ Δ ⧧ GC→D GC→D GC→F GC→F GF→D gas phasea 1.8 11.2 0.1 7.9 8.0 enzymeb −15.8 ± 0.6 4.0 ± 0.6 −9.9 ± 0.5 4.9 ± 0.5 6.6 ± 0.6 aM06-2X/6-31+G(d,p). Nuclear quantum eﬀects were included using harmonic normal-mode analysis. bQM(M06-2X)/MM free energy simulations. Nuclear quantum eﬀects were included using Feynman path integrals. Statistical uncertainty was obtained by employing the bootstrapping method.39

Figure 3. Snapshots of intermediate cations (a) C, (b) F, and (c) D and transition states (d) C → D, (e) C → F, and (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.

The direct proton transfer pathway, which entails proton transfer from C10 to C6, involves cation migration from C11 to C7: i.e., toward the pyrophosphate (Figure 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 toward the PP moiety than in the direct mechanism. This electrostatic steering is facilitated by the unique active Figure 2. Free energy profiles for the generation of cation D directly site architecture of terpene synthases, wherein a highly polar from C (black curve) or via cation F (gray curve). Profiles were region is flanked by a hydrophobic pocket, and such a binary generated from free energy simulations using QM(M06-2X)/MM and active site cavity facilitates charge migration con- − Feynman path integrals. trol.6,24 27,32,50,51 The polar region in TXS is arranged in an approximately layered manner relative to the cation binding The reaction free energy for the C → D formation is −15.8 pocket (Figure 4), similar to that observed for BPPS and kcal/mol, which is significantly lower than the value of 1.8 kcal/ trichodiene synthase.27 Since the pyrophosphate group mol obtained in the gas phase. The barrier for the direct C → D composes the inner layer, the charge migration control is proton transfer is 4.0 kcal/mol, which is also lower than the largely dictated by this moiety. On the other hand, π−cation value of 11.2 kcal/mol in the absence of the enzyme. Hence, interactions with Trp753 are seemingly weakened during the enzyme significantly changes the free energy profile and formation of either cation D or F (Table S1 in the Supporting importantly reduces the free energy barrier. The indirect proton Information), and such interactions are unlikely to control the transfer, C → F → D, is associated with a sequential downhill proton transfer step. We note that such electrostatic steering

7655 DOI: 10.1021/acscatal.7b02824 ACS Catal. 2017, 7, 7653−7657 ACS Catalysis Research Article

that terpene synthases actively control chemistry via traditional catalytic elements. A crucial such element is electrostatic interactions of intermediate carbocations with the pyrophosphate cofactor and amino acid residues. Such a realization is likely to be important in attempts to design terpenes in nonenzymatic 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 low-barrier proton transfer in the terpene synthase taxadiene synthase dictates a two-step proton transfer as the most favorable pathway. This raises the question as to 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 Figure 4. Radial distribution function for the C7 cation (defined as the address the nature of the facile intramolecular proton transfer in origin) of D in taxadiene synthase. All marked residues are located in TXS, and we find that in the enzyme the direct proton transfer the highly charged active site region region. is slightly preferred over the indirect two-step pathway. This suggests that the enzyme might have evolved to favor a simpler, has been suggested by Peters and co-workers and is likely a direct mechanistic pathway, reducing its promiscuity. Under- general feature in terpene synthases.50,51 The current view of standing the underpinnings of the chemical control in enzymes the chemistry in TXS, where the enzyme plays an active role in is crucial when attempting to design natural products in controlling chemistry, is in contrast with that of earlier studies − nonenzymatic environments. where the enzyme was assigned a more passive role.18 20 Additionally, premature quenching of either of the C6 ■ ASSOCIATED CONTENT protons or C7 exocyclic protons is avoided, as neither are in *S Supporting Information proton abstraction distance from the pyrophosphate, active site The Supporting Information is available free of charge on the water molecule, or other alternative bases. On the other hand, ACS Publications website at DOI: 10.1021/acscatal.7b02824. the indirect pathway, which entails an intermediate with a Free energy profiles for proton transfer steps and details carbocation at the C3 position, may be deprotonated at the C3 exocylic position, by the PP moiety, or at the C4 position by an of taxadiene synthase modeling (PDF) active site water molecule. This may explain the observed verticillene side product in TXS.16,20 Indeed, the only slight ■ AUTHOR INFORMATION preference for the direct C → D pathway suggests that the Corresponding Author indirect pathway may compete with the preferred pathway. *E-mail for D.T.M.: [email protected]. ORCID ■ DISCUSSION Dan Thomas Major: 0000-0002-9231-0676 The current TXS simulation studies indicate that the direct Author Contributions proton transfer mechanism is slightly preferred in the enzyme 18,19 D.T.M. designed the research and Y.F., T.A., and D.T.M. over a previously proposed indirect mechanism. Hence, performed the studies and wrote the paper. evolution might have selected the direct proton transfer Notes pathway over an indirect one, to reduce the likelihood of side fi product formation, although this evolutionary pressure is The authors declare no competing nancial interest. seemingly only moderate, as in the plant world some promiscuity is tolerated. We note that the free energy barrier ■ ACKNOWLEDGMENTS reductions reported here due to the enzyme environment have This work has been supported by the Israel Science Foundation no implication for the overall rate of the catalytic cascade, as the (Grant No. 2146/15). rate-limiting step in class I terpene synthases is typically the initial C−O bond cleavage or product release.26 Therefore, the ■ REFERENCES effect of the enzyme on the reaction cascade that is responsible (1) Warshel, A.; Sharma, P. K.; Kato, M.; Xiang, Y.; Liu, H.; Olsson, for some of the most intricate molecular architectures known is M. H. M. Chem. 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7656 DOI: 10.1021/acscatal.7b02824 ACS Catal. 2017, 7, 7653−7657 ACS Catalysis Research Article

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7657 DOI: 10.1021/acscatal.7b02824 ACS Catal. 2017, 7, 7653−7657