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Electrostatic Control of Chemistry in Terpene Cyclases Dan T. Major ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b01328 • Publication Date (Web): 06 Jul 2017 Downloaded from http://pubs.acs.org on July 6, 2017
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Electrostatic Control of Chemistry in Terpene Cyclases Dan T. Major* Department of Chemistry and the Lise Meitner-Minerva Center of Computational Quantum Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel ABSTRACT: Electrostatic interactions play a major role in stabilizing transition states in enzymes. A crucial question is how general this electrostatic stabilization principle is. To address this point, we study a key common C-C bond formation step in a family of enzymes that is responsible for the biosynthesis of 60% of all natural products. In these terpene cyclases, we have previously shown that the enzymes gain chemical control by raising the energy of initial carbocation intermediates along the reaction coordinate to bypass the formation of unwanted side-products. Here we employ hybrid quantum mechanics-molecular mechanics free energy simulations to show that this energy tuning is achieved by modulation of electrostatic interactions. The tempering of electrostatic interactions allows enzymatically directed chemical control that slows down the reaction temporarily by introducing thermodynamic and activation barriers. We show that this electrostatic control in terpene cyclases is achieved by a unique binary active site architecture with a highly charged region flanked by a hydrophobic region. In the charged region, negatively and positively charged moieties are arranged in approximately a layered manner relative to the carbocation binding pocket, with alternating negative and positive layers. We suggest that this active site architecture can be utilized for rational design. KEYWORDS: Electrostatic interactions, Enzyme catalysis, Lyase, Chemical control, QM/MM simulations
INTRODUCTION Enzymes accelerate chemical reactions by providing a carefully carved out binding site,1 precise positioning of active site residues and cofactors,2-3 and preferentially stabilizing the transition state (TS).4-5 The latter effect of preorganized active sites has been suggested by Warshel and co-workers to be responsible for most of the rate enhancement when compared to a well-defined reference reaction.4-5 This understanding of bio-catalysis has facilitated in-silico design of so-called theozymes that subsequently have been successfully tested in wet-lab settings.6 However, this traditional view of rate enhancement by electrostatic preorganization does not exclude other roles of electrostatics, such as allosteric functions, as suggested by Warshel and co-workers.7 One family of enzymes that does not conform to the traditional view of catalysis is terpene synthases (also termed terpene cyclases),8-9 which are responsible for the synthesis of approximately 60% of all natural compounds. A remarkable feature of these enzymes is that they all employ the same family of acyclic precursors, to produce a great variety of carbon-based designs with intricate stereo- and regiochemistry.8, 10-11 The terpene precursors are composed of an acyclic hydrocarbon chain and a pyrophosphate moiety (PP), such as geranyl diphosphate (GPP; C10), farnesyl diphosphate (FPP; C15) and higherorder hydrocarbons. Each terpene synthase contains a unique active site architecture that pre-folds the acyclic substrate in a conformation analogous to its final target.8,
12
In terpene cyclases, catalysis, in its traditional meaning of rate enhancement, occurs during the initial C-O bond cleavage step (Scheme 1).13-15 However, the subsequent steps involving complex chemical transformations via a sequence of electrophilic cycloadditions and rearrangements of carbocations, arguably present the main enzymatic challenge.16 These intermediates must be guided in the correct reaction direction, as well as be protected from nucleophilic attacks and premature quenching in the active site, thereby minimizing formation of sideproducts. Recently, we suggested that terpene cyclases adopt a counterintuitive strategy of destabilizing reaction intermediates along the productive reaction pathway to achieve chemical control.17-20 A crucial question is how such a general strategy is implemented in enzymes, that traditionally are believed to have evolved to stabilize, rather than destabilize bound states. Clearly, electrostatics play a pivotal role in enzyme catalysis, and this has also been proposed in the context of terpene cyclases. Previous work has suggested that the PP moiety plays an important role both in ligand binding and in forming a controlled electrostatic environment.17-26 Interestingly, the active site contour in terpene cyclases is composed of a highly polar pyrophosphate binding region flanked by a hydrophobic carbocation binding pocket.8 However, little is known about the specific role of the pyrophosphate or other active site residues in the chemical regulation in these enzymes.
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In the current work, we revisit the role of electrostatics in enzyme catalysis by focusing on the large terpene synthase family of enzymes. We study a key common step in the cyclization cascade of many mono- and sesquiterpene cyclases (Scheme 1) using multiscale free energy simulations. Specifically, we employ a charge deletion strategy27-30 to pinpoint the role of electrostatic forces in catalysis. Below, we will show that electrostatics introduce thermodynamic and activation barriers along the reaction pathway, contrary to what is observed in most enzymes. We further show that this is due to the unique active site architecture in these enzymes. OPP-3 4
OPP-3
2 6
OPP-4
1
geranyl diphosphate
(3R)-linalyl diphosphate
linalyl cation
BPPS OPP-4
OPP-3
OPP-4 =
O O P P O O O O O
(+)-bornyl diphosphate
(4R)-α-terpinyl cation
-4
P OP
H
-OPP-4 trichodiene
TDS
(6R)-bisabolyl cation
H
H
6
H
OPP-3
3
10 1
OPP-4
OPP farnesyl diphosphate
(3R)-nerolidyl diphosphate
nerolidyl cation
Scheme 1. Formation of terpenes by mono- and sesquiterpene synthases.
We compute the free-energy for the terpene synthase reactions using a well-established simulation protocol developed in our group, which accounts for all the essential physics of the system (see Computational Details). The effect of electrostatics of the charged residues in the first solvation shell on the reaction rate is accounted for by deleting their partial atomic charges27 and hence neutralizing their effect during the simulations. In the current work, the monoterpene synthase family is represented by bornyl diphosphate synthase (BPPS), while trichodiene synthase (TDS) represents the sesquiterpene synthases.
COMPUTATIONAL DETAILS We adopt a computational protocol like that employed in our multiscale modeling study of the monoterpene system BPPS and sesquiterpene synthase TDS.17-20 Specifically, we employ a combined quantum mechanicsmolecular mechanics (QM/MM) potential to model the first C-C bond formation step of the cyclization cascade to
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form terpinyl cation (in BPPS) and bisabolyl cation (in TDS) (Scheme 1).31-32 Similarly to our previous studies,18 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.33 We term this QM region as large (QML). The QM region is treated by density functional theory (DFT), using the M06-2X functional.17-19, 34 The three-point charge TIP3P model is used for water.35 Free energy molecular dynamics (MD) simulations were performed as previously described, to obtain the potential of mean force for the chemical step of interest.19 To probe the Coulombic effect of selected residues on the thermodynamics and kinetics of the first cyclization step in the mono- and sesquiterpene cyclases, we adopt a charge deletion strategy.27-30 We annul the partial Coulombic charges of selected residues to probe their effect on chemistry. During these simulations only the hydrocarbon framework is treated as QM, while the metalpyrophosphate cluster PP-(Mg2+)3 is treated as MM. We term this QM region as small (QMS). We test the validity of using a small QM region by comparing the free energy profiles obtained using QML and QMS (Table S1, Fig. S2, S3). Complete free energy simulations were conducted for each charge deletion state. To assure structural integrity during the MD simulations of the charge deleted states we employed a series of Nuclear Overhauser Effects (NOE) restraints in the PP and Asp-rich binding region.36 Such restraints assure that key ionic interactions remain intact during the simulations, without directly affecting the energetics of the chemical step studied.
RESULTS We first explore the effect of electrostatics in the catalysis of the first carbocation step of the biosynthesis of the monoterpene BPP (Scheme 1, Fig. 1a). The chargeintact enzyme system has a free energy barrier of 2.8 kcal/mol, and a reaction free energy of -8.3 kcal/mol (Fig. 2a and Table 1). Applying charge deletion to the PP moiety, causes a radical change in the free energy profile, underscoring the great effect of electrostatics. The free energy barrier disappears, and the reaction becomes highly exergonic (-17.2 kcal/mol). Interestingly, this exergonicity is nearly identical to the one observed in the absence of the enzyme for this step (-15.2 kcal/mol).17-18 Consequently, the cofactor, which is tightly bound to the enzyme (Fig, 1), slows down the reaction due to electrostatic effects during this early mechanistic step. The reason for this dramatic effect is the migration of the cation charge from the C1 position to C6, which is located deep inside the hydrophobic binding pocket (Fig. 1, S1 and Movie S1), resulting in loss of electrostatic interactions. Deleting the charges of the side-chain of Asp351 results in a similar effect, although not as pronounced, whereas the same operation on Asp496 hardly changes the free energy profile. This is in line with Asp351 being highly conserved in terpene cyclases and likely plays an important electrostatic role, whereas Asp496 is more variable among terpene
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cyclases. Hence, Asp351 also plays a role of slowing down chemistry, and this role may be evolutionary preserved. Deleting the charges of the three Mg2+ ions introduces a free energy barrier (12.9 kcal/mol), yet also yields a more exergonic reaction (-20.5 kcal/mol). This suggests that the Mg2+ ions play a charge balancing role by removing an activation barrier, yet introducing a thermodynamic barrier. Finally, we delete the charges of a cluster containing all the charge elements investigated for BPPS (i.e. PP, 3Mg2+, Asp351, and Asp496). This combined effect reproduces the result of simply deleting the charges of the PP residue alone, suggesting that the carbocation senses a similar electrostatic field from the combined cluster of residues and from PP alone.
the reaction barrier, and gives a highly exergonic reaction (-11.4 kcal/mol). In comparison, the value for this step is 18.0 kcal/mol in the absence of the enzyme.20 Deleting the charges of the side-chain of Asp100 slightly reduces the free energy barrier, and results in an exergonic reaction (3.6 kcal/mol). These effects are rather like those of the PP moiety, although not quite as marked. Again, this is in line with Asp100 being highly conserved in terpene cyclases, and this residue likely plays an important electrostatic role. Thus, also in the case of the sesquiterpene, the electrostatics originating from the PP cofactor and the conserved Asp100 serve to slow down chemistry during this enzymatic C-C bond formation step (Fig. 1, S1 and Movie S2). a)
a)
40
BPPS
Asp496
OPP-4
PP,3Mg,D351,D496 PP
30
3Mg ∆ G [kcalmol-1]
PP C6 C1
Asp351
D351 D496
20
OPP-4 10
b)
0
Asn225
0.0
1.0
b)
2.0 R (C1-C6) [Å]
3.0
4.0
C6 20
H
TDS
C1
PP,3Mg,D100 PP
15
OPP-4
3Mg
Asp100 Figure 1. Active Site of a) Bornyl Diphosphate Synthase with Linalyl Carbocation Intermediate b) Trichodiene Synthase with Nerolidyl Carbocation Intermediate. Color code: H – white, C – green, N – blue, O – red, Mg – cyan, P – orange. The black dotted line marks the C1-C6 bond formation coordinate.
Subsequently, we performed a similar analysis of the effect of electrostatics in the catalysis of the first carbocation step of the biosynthesis of the sesquiterpene TDS (Scheme 1, Fig. 1b). The activation and reaction free energies are 3.1 and 2.0 kcal/mol, respectively, for the charge intact enzyme (Fig. 2b and Table 1). Similar to the monoterpene case, also here we observe that deleting the PP moiety charges causes a drastic change in the free energy profile. Applying the deletion scheme to PP removes
∆G [kcalmol-1]
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D100 10
H
5 OPP-4
0
-5 0.0
1.0
2.0
3.0
4.0
R (C1-C6) [Å]
Figure 2. Free Energy Profiles for a) Terpinyl Cation Formation Step in Bornyl Diphosphate Synthase b) Bisabolyl Cation Formation Step in Trichodiene Synthase for the Fully Charged Enzyme and Selectively Charge Deleted Enzymes.
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Table 1. Thermodynamic Results (Kcal/Mol) for the Formation Of Terpinyl Cation In Bornyl Diphosphate Synthase a (BPPS) and Bisabolyl Cation In Trichodiene Synthase (TDS). BPPS Charge deleted b residues
∆G
No charge deletion ܲܲ, 3݃ܯ, ߙܦ, ߚܦ
‡
TDS
∆Gr
∆G
2.8
-8.3
3.1
0.0
-17.2
‡
∆Gr
2.0
charge separation occurs during the reaction, and the generated cation becomes more distal to the highly charged region of the active site pocket (Fig. 1, Movies S1, S2), while entering the more hydrophobic parts of the active site (Fig. S1).
a) 8 Positive charges Radial Distribution Function
Deleting the charges of the three Mg2+ ions introduces a more noticeable free energy barrier (7.5 kcal/mol), while also resulting in a more exergonic reaction (-6.2 kcal/mol), similarly to the effect for the monoterpene synthase. Finally, we delete the charges of a cluster containing all the charged moieties investigated for TDS (i.e. PP, 3Mg2+, and Asp100). This combined effect results in free energy profile that bears some reminiscence of the charge-intact enzyme.
Negative charges Mg2+,Arg314,Arg493
Asp351
6
PP
Asp496
4
Asp355,Glu504 Glu429,Asp497, Arg316, Asp509,Asp576 Lys512
2
0
4.5
0
-0.5
5
10
15
Distance (Å)
ܲܲ
0.0
-17.6
0.0
-11.4 b)
3݃ܯ
12.9
-20.5
7.5
8
-6.2
ߙܦ
1.7
-14.1
2.7
-3.6
ߚܦ
3.3
-9.7
NA
NA
a
The thermodynamic data were obtained from multiscale QM/MM simulations where the QM region was composed of the carbocation moiety. The QM region employed the M062X/6-31G(d) level of theory, while the MM region was treated b by the CHARMM forcefield. In the case of BPPS, Dα=D351, Dβ=D496. In the case of TDS, Dα=D100, Dβ=ø (not defined). PP is the pyrophosphate cofactor and 3Mg represents the 3 2+ Mg ions.
In conclusion, the electrostatic field due to the charged active site elements in these enzymes is responsible for the introduction of activation and thermodynamic barriers during an early mechanistic stage, and thus slows down the reaction, contrary to what is common in most enzyme families. The physical basis for this is twofold: First, the active sites in terpene cyclases have a unique binary architecture with a highly charged region flanked by a hydrophobic region (Fig. S1).8 Further insight into the active site design in terpene cyclases is obtained by inspecting the radial distribution function for the charged residues in the highly polar side of the binding pocket. In the charged region, negatively and positively charged moieties are arranged in approximately a layered manner relative to the carbocation binding pocket, with alternating negative and positive layers (Fig. 3). Second,
Positive charges Radial Distribution Function
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Negative charges 6 Asp100 4
Mg2+,Arg182,Arg304 PP Asp226,Glu233 Lys232 Asp101, Asp239
2
0 0
5
10
15
Distance (Å)
Figure 3. Radial Distribution Function for the C7 cation (defined as the origin) in a) Terpinyl Cation in Bornyl Diphosphate Synthase b) Bisabolyl Cation in Trichodiene Synthase for the Fully Charged Enzyme. All marked residues are located in the highly charged active site region region.
An important question is how one can probe these effects experimentally. The electrostatic effect of the Asp residues is readily probed by mutational analysis.37 To understand the effect of the PP moiety and the Mg2+ ions one could use synthetic PP analogues or alternative metal ions, as has been done extensively in the literature.37-38 In the Supporting Information we show that such modifications can have rather radical effect on the electrostatic potential exerted by these cofactors (Table S2, Fig. S4). We suggest that such modifications can change the
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chemistry of terpene cyclases due in part to electrostatic effects.
the reaction temporarily by introducing thermodynamic and activation barriers.
DISCUSSION
ASSOCIATED CONTENT
A crucial aspect in our understanding of enzyme’s function is the role of various catalytic elements, such as amino acid residues and organic and inorganic cofactors. It is widely assumed that enzymes enhance chemical rates by providing a pre-organized active site that has a charge distribution that is more complimentary to the transition state than to the ground state.4-5 In this regard terpene cyclases are an exception, in that they appear to control chemistry rather than enhance chemical rates. We have recently shown that mono- and sesquiterpene- cyclases raise the energy of key carbocation intermediates during early stages of the reaction to avoid longlived, highly reactive intermediates.18-20 What is not clear is how these enzymes implement such a chemical control strategy. In the current study, we reinvestigate the role of electrostatics in enzyme reactions. Specifically, we approach this question by applying a charge deletion scheme to charged first and second solvation shell components of the active site, which include Asp residues, PP, and Mg2+ ions. By selectively neutralizing the electrostatic effect of these residues, we managed to delineate their effect on catalysis, something that is not readily attained from experiments.
Supporting Information Available
It has been postulated that the PP unit is crucial for terpene cyclases and that it functions via Coulomb forces. 17-24 Yet, the role of these forces has not been explored. It is well established that terpene cyclases have a unique binary active site architecture with a highly charged region flanked by a hydrophobic region.8 In the current work, we find that in the charged region, negatively and positively charged moieties are arranged in approximately a layered manner relative to the carbocation binding pocket, with alternating negative and positive layers. Moreover, we discover that the most striking influence on chemistry is that exerted by the PP moiety, which is in the first solvation shell (Fig. 3). The effect of the pyrophosphate is to slow down the carbocation reaction in its early steps, where intermediates are more prone to side reactions.18-20 Rather, the release of energy due to repeated bond formations, is deferred to later stages of the reaction where low-barrier19, 39 side-product formation is less likely. We suggest that one might be able to modulate the Coulomb forces of the active site by mutagenesis, varying the metal ions, or using synthetic pyrophosphate variants.
CONCLUSIONS We have previously shown that terpene cyclases gain chemical control by raising the energy of initial intermediates along the reaction coordinate to bypass the formation of unwanted side-products. In this work, we explain that this is achieved by modulation of electrostatic interactions. The tempering of Coulomb forces allows enzymatically directed chemical control that slows down
The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. (1) Comparison of free energy profiles using small and large QM regions. (2) Partial atomic charges for various model pyrophosphate-metal ion complexes. (3) Molecular electrostatic potentials for various model pyrophosphate-metal ion complexes. (4) Figure showing highly charged and hydrophobic regions of active site (PDF).
AUTHOR INFORMATION Corresponding Author *
[email protected] Author Contributions D.T.M designed the research, performed the studies, and wrote the paper.
ACKNOWLEDGMENT This work has been supported by the Israel Science Foundation (Grant # 2146/15). The author thanks Prof. Lynn Kamerlin for helpful comments to the manuscript, and Dr. Anil Mhashal with help in preparing figure 1.
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