Article pubs.acs.org/biochemistry

Conformational Disorganization within the Active Site of a Recently Evolved Organophosphate Hydrolase Limits Its Catalytic Efficiency Peter D. Mabbitt,†,# Galen J. Correy,†,# Tamara Meirelles,† Nicholas J. Fraser,† Michelle L. Coote,†,‡ and Colin J. Jackson*,† †

Research School of Chemistry, Australian National University, Acton 2601, Canberra, Australia ARC Centre of Excellence for Electromaterials Science

‡

S Supporting Information *

ABSTRACT: The evolution of new enzymatic activity is rarely observed outside of the laboratory. In the agricultural pest Lucilia cuprina, a naturally occurring mutation (Gly137Asp) in α-esterase 7 (LcαE7) results in acquisition of organophosphate hydrolase activity and confers resistance to organophosphate insecticides. Here, we present an X-ray crystal structure of LcαE7:Gly137Asp that, along with kinetic data, suggests that Asp137 acts as a general base in the new catalytic mechanism. Unexpectedly, the conformation of Asp137 observed in the crystal structure obstructs the active site and is not catalytically productive. Molecular dynamics simulations reveal that alternative, catalytically competent conformers of Asp137 are sampled on the nanosecond time scale, although these states are less populated. Thus, although the mutation introduces the new reactive group responsible for organophosphate detoxification, the catalytic efficiency appears to be limited by conformational disorganization: the frequent sampling of low-energy nonproductive states. This result is consistent with a model of molecular evolution in which initial function-changing mutations can result in enzymes that display only a fraction of their catalytic potential due to conformational disorganization.

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A century of research has resulted in an extraordinarily detailed understanding of enzyme function. In contrast, the molecular principles that underlie the evolution of new enzyme function have not been fully elucidated.9 Conformational sampling and protein dynamics are essential to protein function,10,11 and modulation of protein dynamics through mutation may allow enzymes to adapt to new substrates.12,13 However, the impact of protein dynamics is not always beneficial: recent work has highlighted the negative role that excessive flexibility can have on protein function and evolvability.14,15 Laboratory (directed) evolution has provided unique insights into the pathways and constraints on enzyme evolution,12 although it is by necessity a simplification of natural evolution, in which selection pressure is seldom constant and organismal fitness is not a function of a single gene.16 Thus, the rare examples of enzymes evolving new functions within the last century complement the larger number of directed evolution studies. The evolution of LcαE7-Gly137Asp7 is of particular interest as it occurred extremely rapidly in a eukaryotic system. Here, we present an X-ray crystal structure of the Gly137Asp variant of LcαE7, molecular dynamics (MD) simulations and comparative kinetic analysis of the pH dependence, phosphor-

rganophosphate (OP) insecticides are covalent inhibitors of acetylcholinesterase (AChE), the enzyme that terminates cholinergic neurotransmission in higher eukaryotes.1,2 They were widely used for insect control, which created a selective pressure that led to the evolution of OP detoxification systems in some insect species.3 In the sheep blowfly Lucilia cuprina, resistance has arisen primarily due to the conversion of α-esterase 7 (LcαE7, EC 3.1.1.1) to an OP hydrolase via a single amino acid substitution in the active site (Gly137Asp).4 LcαE7, like other carboxyl/cholinesterases, catalyzes hydrolysis of its native fatty acid methyl ester substrate in a two-step mechanism involving a catalytic triad (Ser218, Glu351, and His471) and an oxyanion hole (formed by the amide nitrogens of Gly136, Gly137, and Ala219).5 Ester hydrolysis proceeds via acylation of the catalytic serine in the first step, followed by deacylation initiated by a water molecule in the second step, with the catalytic histidine acting as a general base. Organophosphate insecticides phosphorylate the catalytic serine in a manner similar to the first step of ester hydrolysis, but the second step (dephosphorylation) is significantly slower.4,6 The Gly137Asp mutation results in a significant increase in the rate of dephosphorylation and confers a commensurate increase in OP-resistance to the insect.4,7 The protective benefit of this mutation, coupled with strong selective pressure in the form of OP insecticide use, has resulted in the prevalence of the Gly137Asp mutation reaching greater than 95% in some wild populations of L. cuprina.7,8 © 2016 American Chemical Society

Received: December 8, 2015 Revised: January 19, 2016 Published: February 16, 2016 1408

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ylation (burst) and dephosphorylation (steady-state) steps of the reaction for the mutant and WT enzymes. These results highlight the conformational disorganization within the newly formed active site and the negative impact that this has on its catalytic efficiency. Interestingly, and in contrast to many recent laboratory directed evolution experiments,17−19 we also observe a large activity trade-off between the ancestral and evolved function, which reflects the disruptive effects that new catalytic groups can have on specialized and preorganized active sites.

k3

k5

E + S ⇌ ES → EPQ → E + P + Q k2

(1)

where E, S, Q are the enzyme, substrate, and fluorescent leaving group, respectively. k1 and k2 are the rate constants governing the formation of the Michaelis complex (ES), k3 is the rate constant for the formation of the phosphorylated intermediate (EP), and k5 is the rate constant for dephosphorylation. Purified LcαE7 was exchanged into ATM buffer (50 mM acetic acid, 100 mM Tris, 50 mM MES, 100 mM NaCl), which allows activity assays to be performed at a range of pH values with constant ionic strength.28 The hydrolysis of DEUP was monitored fluorometrically (excitation at 330 nm and emission at 450 nm) with a Cary Eclipse Fluorescence spectrophotometer (Agilent Technologies, USA). All experiments were carried out at 25 °C and pH 7.5 unless otherwise stated. Product formation was quantified using a standard curve of 4methylumbelliferone (Sigma-Aldrich, USA) in the assay buffer. To determine the rate constant describing the pre-steady state burst, solutions containing DEUP serially diluted 1:2 from 24 to 1.5 μM, or 0.75 μM LcαE7, were mixed in a 1:1 ratio using a RX2000 rapid mixing stopped-flow unit fitted with a RX/DA pneumatic drive (Applied Photophysics, UK). The first five seconds of the stopped-flow fluorescence trace were fitted via nonlinear regression to a single exponential function (eq 2). All curve fitting was achieved using GraphPad Prism 6.0 (GraphPad Software, USA).

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MATERIALS AND METHODS Protein Expression and Purification. The Gly137Asp mutation was introduced into the previously described variant of LcαE7 whose X-ray crystal structure was solved previously (LcαE7-4).5 Wild-type and Gly137Asp LcαE7 were expressed in BL21(DE3) Esherichia coli at 26 °C for 18 h with autoinduction. Cells were collected by centrifugation and resuspended in Buffer A (50 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 20 mM imidazole) and lysed by sonication. Cell debris was pelleted by centrifugation, and the soluble fraction was applied to a HisTrap-FF Ni-Sepharose column (GE Healthcare, USA). Protein was eluted in Buffer B (50 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 300 mM imidazole). Fractions containing LcαE7 were concentrated with a 30 kDa molecular mass cutoff centrifuge concentrator (Amicon, USA) and applied to a HiLoad 26/60 Superdex-200 size-exclusion column (GE Healthcare, USA), pre-equilibrated in Buffer C (50 mM HEPES-NaOH pH 7.5, 200 mM NaCl). Protein elution was monitored at 280 nm, and fractions containing the monomeric protein were pooled and concentrated for crystallography or enzyme assays. Protein concentration was determined by measuring absorbance at 280 nm with an extinction coefficient calculated using the Protparam online tool.20 Crystallization and Structure Determination. Crystals of the Gly137Asp mutant were grown at 290 K using the hanging-drop vapor diffusion method, with reservoir solutions of 100 mM sodium-acetate (pH 4.6) and 20% PEG 2K MME. For cryoprotection the PEG 2K MME concentration was increased to 35%, and crystals were vitrified at 100 K in a stream of nitrogen. Diffraction data were collected at beamline ID 23-2 of the European Synchrotron Radiation Facility with wavelength of 0.8726 Å. Data were indexed, integrated, and scaled using the XDS package.21 The resolution limits of the data were assessed on the basis of the significance of the CC1/2 at the P = 0.001 level.22,23 Phases were obtained by molecular replacement with the previously determined structure of LcαE7 (PDB: 4FNG) using the PhaserMR program implemented in the CCP4 software package.24 The Gly137Asp model was improved through iterative cycles of model building in Coot25 and refinement in Refmac5.26 The coordinates were submitted to the Protein Data Bank (PDB: 5C8V). Organophosphate Hydrolase Activity. The proposed scheme for the hydrolysis of diethylumbelliferyl phosphate (DEUP; Sigma-Aldrich, USA) by LcαE7 is outlined in eq 1. Importantly, the phosphorylated enzyme produced through reaction with DEUP is identical to that produced through reaction with the diethyl-substituted OP-insecticide diazinon. Diazinon is currently used for blowfly control and L. cuprina bearing the LcαE7 Gly137Asp mutation are resistant to diazinon.27

Fobs = A(1 − e−kobst ) + C

(2)

where Fobs is the observed fluorescence, A is the amplitude, kobs is the observed rate of the pre-steady state burst, t is time, and C is an amplitude-offset. The values for KM and k3 were determined by nonlinear regression to the quadratic equation for tight binding substrates (eq 3).29 This model was used because it eliminates the assumption that the ligand concentration remains constant over the course of an enzyme-catalyzed reaction (e.g., that [ET] ≪ [S]).29 kobs = k 3{([E T] + [DEUP] + KM) −

([E T] + [DEUP] + KM)2 − 4[E T][DEUP] }

/{2[E T]}

(3)

where k3 is the rate constant for the formation of the covalent enzyme−substrate intermediate, kobs is the observed rate of the pre-steady state burst, [ET] is the total concentration of enzyme, and KM is the Michaelis constant. For the characterization of the pH−activity profiles, the turnover rate of DEUP was determined under steady state conditions at a range of pH values from 5.9 to 8.5 by monitoring product formation for 180 s (excluding the initial 30 s for the pre-steady state burst). The values of kcat as a function of pH were fitted via nonlinear regression to eq 4.30 (kcat)H =

k max − k min 1 + eb(pH − c)

(4)

where b is a slope constant and c is the pKA of the species upon which the dephosphorylation reaction depends. Carboxylesterase Activity. Hydrolysis of 4-nitrophenyl butyrate (4-NPB; Sigma-Aldrich, USA) was monitored at 405 nm (pH 7.5, 25 °C) using an Epoch microplate spectrophotometer (BioTeK Instruments, USA). The concentration of 1409

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oxygen atom closest to the phosphorus was selected for distance and orientation measurements.

enzyme used ensured that less than 10% of the total amount of substrate was consumed during the monitored reaction. Seven concentrations of 4-NPB were used, serially diluted 1:2 from 500 to 7.81 μM. Velocities were obtained from the initial linear portion of the reaction progress curves, and product concentration was determined with the extinction coefficient calculated from a standard curve of 4-nitrophenol (SigmaAldrich, USA). KM and kcat were determined by nonlinear regression of the initial velocities to the Michaelis−Menten equation. Molecular Dynamics Simulations. Coordinates of the WT enzyme (PDB: 4FNG), phosphorylated WT (PDB: 4FNM), and the Gly137Asp mutant (PDB: 5C8V) were used as a starting model for the MD simulations within the GROMACS suite.31 A model for phosphorylated Gly137Asp intermediate was built by manually replacing the catalytic serine with the diethylphosphoserine from the structure of the phosphorylated WT enzyme (PDB: 4FNM). The ligand was parametrized using the Automated Topology Builder,32 and the GROMOS 53a7 force-field33 was updated to include the modified residue. Simulations were performed using periodic boundary conditions. Short-range nonbonded interactions were evaluated via a twin-range cutoff scheme with the Coulombic and van der Waals terms truncated at 0.9 and 1.4 nm, respectively. Long-range electrostatic interactions were calculated with the fast smooth particle-mesh Ewald (PME) method.34 The LINCS35 algorithm was used to constrain covalent bond lengths, allowing a time step of 2 fs. Temperature and pressure coupling was achieved with the Berendsen weak coupling method36 utilizing coupling constants of 0.1 ps to 300 K and 0.2 ps to 1 bar. To prepare the simulation system, atomic coordinates were solvated in a cubic box of SPC water37 with minimum distance of 0.8 nm maintained between the protein and the box wall. Charge neutrality was achieved by replacing the appropriate number of solvent molecules with counterions. The resulting system was energy minimized via the steepest descent to the limit of machine precision. Three replicate systems were constructed with variation generated by assigning initial velocities at different temperatures (296, 300, and 304 K) followed by 100 ps of position-restrained simulation (1000 kJ/ mol nm2) coupled to the temperature used to generate initial velocities. All systems were equilibrated for a further 6 ns with temperature coupling at 300 K. To eliminate backbone conformational flexibility influencing the investigation of the accessible conformations of the Asp137 side chain, production simulations (120 ns) were conducted with position restraints applied to backbone atoms. Velocities and coordinates were written out every 10 ps. The hydrogen bonding of the catalytic triad for both systems was analyzed by extracting the distances between the relevant atoms. The flexibility of binding pocket residues was assessed by extracting χ1 and χ2 dihedral angles with g_angle and plotted as two-dimensional histograms. To analyze the two conformations of Asp137 observed in the simulation of the phosphorylated enzyme, a python script utilizing the pymol module was written to identify water molecules that could potentially act as nucleophiles for the dephosphorylation reaction. This was accomplished using the following criteria for each of the 36 000 structural snapshots: waters were identified with a hydrogen within 2.5 Å of the Asp137 carboxylate oxygens, and subsequently the water with an

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RESULTS X-ray Crystallography. To investigate the structural consequences of the introduction of Asp137 into the oxyanion hole of LcαE7, we solved the crystal structure of the Gly137Asp variant under conditions identical to those used to crystallize the WT enzyme (Figure S1, Table 1).5 No large changes in Table 1. Data Processing and Refinement Data for the X-ray Crystal Structure of the Gly137Asp Variant of Lucilia cuprina Alpha-carboxylesterase 7 Lucilia cuprina αE7:Gly137Asp (PDB 5C8V) Data Processing space group C2221 cell dimensions (Å) a, b, c 49.67, 102.58, 225.48 resolution range (Å) 44.7- 2.01 (2.06−2.01)a total number of reflections 146001(9661) number of unique reflections 37150 (2490) multiplicity 3.9 (3.9) completeness (%) 95.4 (97.8) mean I/σ(I) 7.7(1.55) Wilson B factor (Å2) 24.6 CC1/2b 0.992 (0.581) Rmerge 0.132 (0.906) Refinement Rwork/Rfree 0.185/0.245 (0.302/0.333) total number of atoms 4795 number of macromolecules 1 number of waters 208 number of protein residues 566 RMSD for bonds (Å) 0.0149 RMSD for angles (deg) 1.666 Ramachandran favored (%) 96.6 Ramachandran outliers (%) 0 Clashscore 2.09 average B factor (Å2) 27.0 a

Values in parentheses are for the highest resolution shell. bPearson’s correlation coefficient calculated from two half-sets of the data.22,23

backbone conformation were observed between the WT and the Gly137Asp variant (Cα r.m.s.d. 0.26 Å). Alignment of the substrate binding pockets of WT, the Gly137Asp variant and phosphorylated WT reveals that the positions of the atoms of the catalytic triad and the oxyanion hole are essentially identical in all three structures (Figure 1). In the structure of the Gly137Asp variant, the carboxylate of Asp137 protrudes into the binding pocket and is approximately 4 Å from the Ser218 Oγ. The opening of the binding pocket is reduced from 8.3 to 5.5 Å upon introduction of the Asp137 side chain, and hence the Asp137 rotamer observed in the crystal structure would prevent productive binding of a diethyl OP substrate into the active site (Figures 1 and S2). Indeed, when the Gly137Asp variant was aligned with the phosphorylated WT structure, there was steric overlap between the carboxyl group of Asp137 and the phospho-adduct of the serine, suggesting that the orientation of the carboxylate is incompatible with phosphorylation (Figure 1). There were two other notable changes within the substrate-binding pocket of the Gly137Asp variant relative to both the apo WT and the phosphorylated WT structures. First, the χ1 dihedral angle of Phe309 has rotated by 1410

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Figure 1. Gly137Asp mutation does not significantly alter the geometry of the LcαE7 catalytic triad or oxyanion hole. (A) Close up of the LcαE7:Gly137Asp active-site with the residues making up the catalytic triad (Ser218, His471, Glu351), oxyanion hole (Gly136, Asp137, Ala219) and binding pocket (Met308, Phe309, Phe354, Phe355, Tyr420, Phe421, Tyr457) shown as white sticks. The 2|FO − FC| map centered on the aforementioned residues is shown as a blue mesh contoured at 1.5 σ. The hydrogen bonds between catalytic residues are shown as dashed black lines. (B) Alignment of the binding pocket residues of the Gly137Asp variant (white sticks) and apo WT LcαE7 (blue sticks). (C) Alignment of the binding pocket residues of the Gly137Asp variant (white sticks) and the phosphorylated WT LcαE7 (pink sticks) with the covalently bound diethyl OP shown as white spheres.

Figure 2. Rapid phosphorylation of LcαE7 and the slow dephosphorylation of an organophosphate compound by LcαE7 were monitored via stopped-flow fluorescence. Only the basic side of the pH profile could be precisely determined because LcαE7 was unstable at pH values below 6.0. (A) Representative progress curves of diethylumbelliferyl phosphate hydrolysis by WT (green 3 μM substrate, black 24 μM substrate) and Gly137Asp LcαE7 (red 3 μM substrate, blue 24 μM substrate) at pH 7.5. Points indicate the concentration of the product 7-hydroxy-4-methyl coumarin (HMC). (B) Diethylumbelliferyl phosphate hydrolase activity of WT (▲) and Gly137Asp (●) LcαE7 as a function of pH. Values of k5 are the mean ± standard deviation for four replicates. The apparent pKA of the diethylumbelliferyl phosphate hydrolase activity of LcαE7-Gly137Asp was 6.4 ± 0.4 (mean ± standard deviation).

30°, thereby increasing the distance to the Ser218 Oγ by 2.3 Å relative to the WT structure. Second, the conformation of Met308 is shifted in tandem with Phe309, maintaining the 3.5 Å separation between the two side chains (Figure 1). Kinetic Analysis. To investigate the trade-off between carboxylesterase and OP-hydrolase activity that has occurred in LcαE7 evolution, we characterized the catalytic efficiencies of the WT and the Gly137Asp variant. In contrast to previous studies where activity was measured in insect cell lysates,4,38 in this study both proteins were purified through affinity chromatography followed by size-exclusion chromatography and only pure monomeric fractions were assayed. The presteady state and steady state kinetic course of OP hydrolysis was monitored using stopped-flow with fluorescence detection of the product (Figure 2). The pre-steady state burst corresponds to phosphorylation of the catalytic serine and release of the fluorescent umbelliferone leaving group. While the rates of phosphorylation were similar for the two enzymes, there was a 6-fold decrease in OP affinity, as estimated from the concentration dependence of the pre-steady state burst (Figure 2, Table 2). In contrast to the similar phosphorylation kinetics, the steady state rate, corresponding to the rate-determining dephosphorylation step, increased 14-fold from the WT enzyme to the Gly137Asp variant (Figure 2, Table 2). We

Table 2. Kinetic Parameters for the Hydrolysis of the Model Organophosphate (Diethylumbelliferyl Phosphate) and the Model Fatty Acid Methyl Ester (4-Nitrophenyl Butyrate) at pH 7.5 by WT and Gly137Asp LcαE7a Diethylumbelliferyl Phosphate k3 (s−1) wild type Gly137Asp

wild type Gly137Asp

1.8 ± 0.2 1.3 ± 0.1

KM (μM)

k5 (s−1) × 10−4

70 160 ± 30

KM (μM)

kcat (s−1)

kcat/KM (M−1·s−1)

44 ± 6 130 ± 20

31 ± 2.0 5.6 ± 0.5

6.9 ± 1 × 105 4.5 ± 0.8 × 104

Values are the mean ± standard error (diethylumbelliferyl phosphate, n = 8; 4-nitrophenyl butyrate, n = 3).

a

next characterized the enzyme with a carboxylester substrate. The native substrate of LcαE7 has not been identified but is most likely a fatty acid methyl ester; in this work we have used 4-nitrophenyl butyrate (4-NPB) as a model ester substrate, as has been the case in studies of other carboxylesterases.39−41 We observed a 5-fold reduction in the kcat of the Gly137Asp variant, compared with the WT LcαE7 enzyme (Table 2). Additionally, we observed a 3-fold increase in KM for this reaction. Thus, the 1411

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catalytic triad. Furthermore, substantial backbone motions are unlikely to occur within the tightly packed active site over the time scale of side chain fluctuations.44 Measurement of interatomic distances between residues of the catalytic triad and oxyanion hole suggested that in simulations of both systems, hydrogen bonds were formed between the key residues of catalytic triad and the oxyanion hole (Figure S3). We assessed the conformational flexibility of the substratebinding pocket in both systems by analyzing the distribution of χ1 and χ2-dihedral angles across the 360 ns of simulation (Figure S4). Most residues adopted generally homogeneous conformations that were similar to those in the crystal structure, which confirms that our MD approach is able to reproduce the crystallographic conformations of key binding pocket residues. Interestingly, several side-chains, including Asp137, were observed to sample additional conformations. In the LcαE7 Gly137Asp:apo simulation, we observe that, although some residues (e.g., Trp251) are conformationally homogeneous, several residues that together comprise the substrate-binding pocket sample a number of alternative conformations in addition to their conformations in the crystal structure (Figure S4). Most notably, Asp137 samples two closely related conformations: the crystallographic (C1) conformation, which obstructs OP binding, was the most populated (Figure 3), but an alternative conformation (C2), with the carboxylate of Asp137 rotated ∼90° relative to the crystallographic conformation, was sampled with significant frequency. In the LcαE7 Gly137Asp:OP simulation, the substrate binding pocket was significantly more homogeneous:

14-fold increase in the rate of OP hydrolysis coincided with a 15-fold decrease in the catalytic specificity for the native substrate. Mechanism. To probe the mechanism by which Asp137 confers catalytic activity to LcαE7, we investigated the pHdependence of the rate-determining dephosphorylation step of the reaction. For the WT enzyme, there was no detectable change in the rate of dephosphorylation with changing pH (Figure 2). This suggests that this rate-limiting step is independent of any ionizable groups with pKA values within the pH range tested. In contrast, the rate of OP hydrolysis by the Gly137Asp variant was strongly dependent on the pH of the reaction (Figure 2). Although we could not measure activity at pH values below 6 due to the instability of LcαE7, we could accurately determine the basic region of the pH−activity profile, revealing decreasing activity with increasing pH. This was consistent with efficient dephosphorylation requiring protonation of at least one ionizable group. The pKA value for the group responsible for the basic region of the curve was calculated to be 6.4, which is consistent with a buried histidine residue requiring protonation for full enzymatic activity.42 The only histidine residue in the vicinity of the phosphorylated serine is the histidine of the catalytic triad, His471 (Figure 1). The observed pH dependence suggests that His471 acts as a general acid in the dephosphorylation reaction, protonating Ser218 as the phosphodiester departs. The only available general base to activate a water molecule for nucleophilic attack at the phosphorylated serine is the newly introduced Asp137. The Asp137 side-chain is located opposite the phosphoserine bond, which potentially satisfies the stereochemical constraints on the dephosphorylation reaction which require a water molecule to approach with a trajectory colinear to the phosphoserine bond.43 Molecular Dynamics Simulations. Molecular dynamics simulations at a physiologically relevant temperature (300 K) were conducted in order to investigate whether conformational change in LcαE7 Gly137Asp could allow Asp137 to act as a general base and provide sufficient space for substrate binding. Although the resolution of the LcαE7 Gly137Asp crystal structure was reasonably high (2.01 Å), it is possible that not all alternative conformations of the protein will be visible without ultrahigh resolution data. Additionally, some sparsely populated states might have been “frozen-out” during the snap-freezing process to allow cryoprotection.10 Room temperature data collection was not feasible with these crystals owing to loss of resolution. Thus, we used atomistic molecular dynamics simulations at a physiologically relevant temperature (300 K) to investigate whether conformational change in LcαE7 Gly137Asp could allow this residue to act as a general base and provide sufficient space for substrate binding. To investigate the conformational flexibility of Asp137, we performed simulations on the apoenzyme crystal structure and modeled the covalent diethyl OP adduct into the crystal structure as the phosphorylated intermediate of LcαE7 Gly137Asp could not be captured crystallographically. Production runs (postequilibration) of 120 ns were carried out in triplicate for each system. Simulations were performed with the backbone heavy-atoms (N, Cα, C) restrained to the crystallographic coordinates via a 1000 kJ/mol nm2 force constant as described in Materials and Methods. Although this could bias sampling of the side-chain conformational landscape, as large-scale backbone motions would not be accessible, restraints were necessary to obtain plausible arrangement of the

Figure 3. MD simulations reveal multiple conformations of the Asp137 side-chain in the Gly137Asp variant of LcαE7. (A) Histograms of χ1 and χ2 dihedral angles of 36 000 snapshots from each simulation show the distribution of Asp137 conformers. Because the aspartate carboxylate has a 2-fold axis of symmetry, χ2 dihedral angles greater than 180° were adjusted to the symmetry related conformation (e.g., 270° = 90°). The crystallographic conformation (C1) is shown as an open circle. (B) Representative snapshots from the major populations shown in (A). The catalytic serine (Ser218) and oxyanion hole residues (Gly136, Asp137) are shown as white sticks. The covalently bound diethyl OP is shown as white sticks with transparent white spheres. 1412

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transition states for OP hydrolysis (∼3.9−4.2 Å).45 In contrast to the similarity in distance to the electrophilic phosphorus, the two conformations differ in the orientation of the carboxylate with respect to the incoming water molecules (Figure 4). The carboxylate orientation is an important factor in the ability of aspartate to act as a general base: model studies examining hydrogen bonding to carboxylates indicate that bonding is most favorable when a water hydrogen (HW) is in the plane of the carboxylate and least favorable when HW is at right angles to the plane of the carboxylate.46 Thus, the two Asp137 conformations identified in this work can be differentiated based on the position of a potential water nucleophile relative to the plane of the carboxylate (Oδ−Cγ−Oδ). Analysis of the relative orientations of the carboxylate indicates that the HW of water molecules approaching the phosphorus would be positioned approximately in the plane of the carboxylate when Asp137 is in the C2 conformation, whereas in the C3 conformation HW would be at right angles to the carboxylate plane (Figure 4). These results suggest that the C2 conformation is more appropriately configured to activate a water molecule for attack at the phosphorus. A Dynamic Conformational Network in the LcαE7 Substrate-Binding Pocket. We next investigated the structural features of the LcαE7 binding pocket that allow the nonproductive C1 and C3 conformations of Asp137 to be populated. The conformational change between the C1 and C2 states in the apoenzyme is relatively subtle, and no obvious network was apparent, although, as observed in the LcαE7 Gly137Asp-OP structure, the local environment of this residue can constrain the number of conformers that it can sample. With respect to the C3 conformation, the MD simulations revealed that, in LcαE7 Gly137Asp:OP, the only other residues that sampled alternative conformations with significant frequency were Met308 and Phe309, which are adjacent to Asp137 (Figure 1 and Figure S3). These residues were in different conformations in the structure of the Gly137Asp variant relative to the WT enzyme (Figure 1). The alternate Phe309 conformation involves a 115° rotation of the χ1 dihedral angle, resulting in burial of the aromatic side-chain in a hydrophobic pocket away from Asp137, thereby removing steric interactions between the two side-chains. In the 360 ns simulation, Phe309 frequently (∼270 times) transitions between the crystallographic conformation and the buried conformation. When the snapshots from the MD trajectory were sorted by whether Phe309 was in the crystallographic or buried conformation, conformational coupling was observed with both the Asp137 and Met308 side-chains (Figure 5). Sampling of the C3 conformation is dependent on Phe309 occupying the buried conformation; Asp137 is restricted to sampling the C2 conformation when Phe309 is in the crystallographic conformation due to the steric overlap between the crystallographic Phe309 conformation and the C3 Asp137 conformation. In a similar manner, Met308 is restricted to a single conformation when Phe309 is in the crystal conformation. However, the Met308 side-chain samples several discrete conformations when the Phe309 side-chain is buried (Figure 5). Therefore, the conformation of Asp137 is determined by interaction with the Phe309 side-chain; the alternative, buried, conformation of Phe309 removes a steric constraint on the conformation of the Asp137 side-chain and enables it to sample conformations that are not catalytically competent. In simulations of the apo and phosphorylated WT LcαE7, the Phe309 side-chain is largely restricted to the

a single conformation (or two very similar conformations) was observed for the Tyr148, Trp251, Phe354, Phe355, Tyr420, Phe421, and Tyr457 side-chains, with the distribution of dihedral angles centered on or near the crystallographic conformation (Figure S4). However, in addition to the crystallographic conformations, we observed significant sampling of alternative conformations for the residues Asp137, Met308, and Phe309. Unsurprisingly, the crystallographic (C1) conformation of Asp137, which clashes with the bound OP, was not sampled (Figure 3). Instead, the C2 conformation was the dominant state. A third conformation (C3) of Asp137 is also observed. This conformation results from a 100° rotation in the χ1 dihedral angle, bringing the carboxylate into a significantly different orientation compared to the C1 or C2 conformations. Despite the different geometry, the Oδ position is largely identical to that of C2, although the orientation of the carboxylate headgroup is significantly different (Figure 3). Given that both Asp137 conformations were similar, we examined the positions of water molecules hydrogen bonding to the carboxylate in the MD simulations to determine whether either geometry was better suited to Asp137 acting as a general base (Figure 4). Water molecules can approach within ∼3.8−6 Å of the phosphorus in either conformation, which is consistent with distances calculated through quantum chemical simulations for near-attack conformations immediately preceding

Figure 4. C2 Asp137 conformation is more appropriately configured to activate a water nucleophile in the dephosphorylation reaction. (A) Histograms describing the distribution of the water oxygen (OW) to phosphorus (P1) distances versus the position of the water hydrogen (HW) relative to the plane of the Asp137 carboxylate (dihedral Oδ−Cγ−Oδ−HW). For each of the 36 000 structural snapshots, only the water closest to the phosphorus with a hydrogen bond to the Asp137 carboxylate was considered (see Materials and Methods). (B) A representative water molecule satisfying the geometry of the major population in (A) was mapped onto representative snapshots of the C2 and C3 conformations. The hydrogen bond and distance between the water oxygen (OW) and the phosphorus (P1) are shown as black dashed lines. The oxyanion hole residues (Asp137, Gly136, and Ala219) are shown as white sticks, and the covalently bound diethyl OP is shown as white sticks with transparent white spheres. 1413

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Figure 5. Conformation of Phe309 determines the flexibility of the key catalytic residue Asp137 and the adjacent residue Met308. (A) Surface representation of the LcαE7:Gly137Asp binding pocket showing the crystallographic (crystal) and alternate (buried) conformations of Phe309. Met308, Phe309, Asp137, and the covalently bound diethyl OP are shown as blue sticks. (B) The 36 000 snapshots from the Gly137Asp:OP simulation were sorted by Phe309 conformation. Histograms of the χ1 and χ2 dihedral angles of residues Asp137 and Met308 reveal that the C3 conformation of Asp137 is only sampled when Phe309 rotates to the buried conformation. (C) Conformations from (B) mapped onto the LcαE7:Gly137Asp crystal structure. The residues of interest (Met308, Phe309, Asp137, Ser218, Ala219) and the covalently bound diethyl OP are shown as white sticks. Transparent spheres are shown for Met308 (pink), Phe309 (white), and Asp137 (blue).

crystallographic conformation, although the buried conformation is accessible (Figure S5). The adjacent Met308 side-chain is highly flexible, sampling several discrete conformations.

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DISCUSSION

The data presented here suggest that, under selective pressure to break down and detoxify OPs, LcαE7 has evolved a novel catalytic mechanism. Whereas the first step in the reaction was already efficient (phosphorylation; Table 2), the second step (dephosphorylation) was very slow in the wild-type enzyme. Interestingly, the 14-fold increase in the OP-hydrolase activity is countered by a 15-fold reduction in the native carboxylesterase activity, which is not unexpected given the major disruption to the active site. This result is notable and suggests that the natural evolution of OP-hydrolase activity in LcαE7 appears to be a counter-example to recent studies that have suggested promiscuous activities can often be enriched without significant disruption to native activities.17,18 The pH−activity profiles presented in this work provide experimental evidence to support the previous proposal that Asp137 increases the rate of dephosphorylation by acting as a general base, activating a water molecule for nucleophilic attack with His471 acting as a general acid to reprotonate Ser2184 (Figure 6). This mechanism is also supported by the observation that Asp137 can adopt an alternative conformation that allows it to act as a general base to a water molecule positioned for in-line nucleophilic displacement (Figure 4). Indeed, an equivalent mechanism has been suggested for the Gly117His variant of BuChE.30,47,48 However, crystal structures of the Gly117His variant of BuChE indicate that some structural rearrangement would be required to accommodate the attacking water.49 This led to the proposal of an alternative mechanism in which steric strain imposed by His117 acts to

Figure 6. Proposed catalytic mechanism for the dephosphorylation reaction of LcαE7:Gly137Asp. Asp137 acts as a general base to activate a water molecule for nucleophilic substitution at the phosphorus center. The catalytic histidine (His471) acts as a general acid to reprotonate Ser218.

destabilize the trigonal bipyramidal dephosphorylation transition state.49 This has been supported by Quantum Mechanics/Molecular Mechanics simulations (QM/MM) of Gly117His BuChE, in which nucleophilic attack originates from a position adjacent the active site serine, potentially involving Glu197.50 Given the results presented here show that in LcαE7 rearrangement of Asp137 can occur to allow accommodation of a nucleophilic water molecule (Figure 4), and the previous observation that a Glu217Met mutation (equivalent to BuChE:Glu197Met) did not affect the rate of dephosphorylation of LcαE7,6 we favor a general base role for Asp137. QM/MM simulations of LcαE7 could provide additional insight into alternate mechanisms. 1414

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Biochemistry The evolution of OP-hydrolase activity in L. cuprina is a rare example of a new catalytic activity naturally evolving in a eukaryotic organism during the past 50−100 years.4 The catalytic efficiency (kcat/KM) for the Gly137Asp variant is less than that of typical naturally evolved enzymes (102 versus 104− 107),51 which is not unexpected given that LcαE7 Gly137Asp has had only a few decades to evolve versus millions of years for other natural enzymes. However, it provides a valuable model system: by contrasting this recently evolved, inefficient, enzyme with highly evolved efficient enzymes, we can gain insight into what happens between the initial and late stages of the evolution of catalytic efficiency. One of the defining features of highly evolved and proficient enzymes is the TS stabilization afforded by the electrostatic preorganization of their polar active sites.52−56 In contrast, we observe the key catalytic residue in LcαE7 Gly137Asp, Asp137, is highly mobile, requires conformational change from the apoenzyme state, and frequently adopts nonproductive conformations. The impact of this conformational disorganization is particularly apparent in the increased KM in the Gly137Asp variant relative to WT: the loss of OP binding affinity can be explained by the partial occlusion of the binding pocket entrance by the Asp137 sidechain, and the adjustment of substrate-binding site residues Phe309 and Met308 in the dominant, crystal structure, conformation (Figure 1 and Figure S2). Similarly, the low kcat can be at least partially explained by its frequent sampling of nonproductive conformations (C3; Figure 3), and the significant reorganization of the active site that would be required to attain a TS-like conformation. This is evident through the comparison with the naturally evolved bacterial phosphotriesterase (kcat/KM ≈ 104-fold higher than LcαE7 Gly137Asp), in which the crystallographic capture of enzyme:substrate complexes reveals very little conformational change from the apoenzyme to a Michaelis complex that is a very close approximation of the transition state.11,57 In highly evolved enzymes, the inner shell active-site residues are often stabilized in their optimal geometries through interactions with second and third shell residues that have evolved over thousands of generations of evolutionary selection. This suggests that future improvement of LcαE7 Gly137Asp could be achieved through the introduction of remote mutations that reduce the sampling of nonproductive rotamers. Indeed, this is consistent with the frequently observed pattern in directed evolution experiments of mutations predominantly being located in second- and third-shell positions in later generations.18 In summary, we have performed structural, kinetic, and dynamic characterization of a recently evolved OP hydrolase from a eukaryotic organism. These results have revealed that, rather than modifying the substrate range, the Gly137Asp mutation has resulted in a qualitatively new catalytic mechanism. Additionally, the trade-off between the new and ancestral activities (OP hydrolase and carboxylesterase) is strong-negative, which is unlike many recent observations of weak-negative trade-offs in laboratory evolution or bacterial systems.17,18,58 This recently evolved enzyme exhibits relatively low catalytic efficiency that can be at least partially explained by the level of disorder within the active site and the poor electrostatic preorganization of the active site, exemplified by the key catalytic residue frequently sampling nonproductive conformations. The observation that this enzyme can provide protection against OP intoxication in insects and has extremely high affinity (low KM) for OPs suggests that it could be a valid

candidate for development as a catalytic bioscavenger to protect against OP intoxication as a prophylactic or in the treatment of poisoning. However, with respect to the continued evolution of insecticide resistance, the observed imperfections in the enzyme suggests that additional mutations could lead to substantial improvement by reducing the sampling of nonproductive conformations. Thus, the Gly137Asp mutation could be the first of many mutational steps toward efficient insecticide detoxification and resistance.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b01322. Difference density map illustrating changes to the LcαE7:Gly137Asp active-site relative to the LcαE7:WT active-site (Figure S1). Surface representation of the phosphorylated WT LcαE7 and the Gly137Asp variant binding pockets (Figure S2). Key distances between catalytic residues in the Gly137Asp:apo simulation and Gly137Asp:OP simulation (Figure S3). Histograms of the χ1, χ2 dihedral angles sampled in the Gly137Asp:apo simulation and Gly137Asp:OP simulation (Figure S4). Histograms of the χ1 and χ2 dihedral angles sampled by Met308 and Phe309 in MD simulations of apo and phosphorylated LcαE7 (Figure S5) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions #

P.D.M. and G.J.C. contributed equally to this work.

Funding

This work was supported by a Defense Threat Reduction Agency Contract HDTRA1-11-C-0047. C.J.J. and M.L.C. were supported by Australian Research Council Future Fellowships. P.D.M. was the recipient of a John Stocker Postdoctoral Fellowship from the Science and Industry Endowment Fund (Australia). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS DEUP, diethylumbelliferyl phosphate; 4-NPB, 4-nitrophenyl butyrate; LcαE7, Lucilia cuprina alpha-carboxylesterase 7; MD, molecular dynamics; OP, organophosphate

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REFERENCES

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