Active Site Desolvation and Thermostability Trade-Offs in the Evolution

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Article

Active site desolvation and thermostability tradeoffs in the evolution of catalytically diverse triazine hydrolases Elena Sugrue, Paul D Carr, Colin Scott, and Colin J. Jackson Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00731 • Publication Date (Web): 21 Oct 2016 Downloaded from http://pubs.acs.org on October 23, 2016

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Active site desolvation and thermostability tradeoffs in the evolution of catalytically diverse triazine hydrolases Elena Sugrue,† Paul D Carr,† Colin Scott,‡ Colin J Jackson*† †Research School of Chemistry, Australian National University, Canberra, Australia ‡CSIRO Land & Water, Canberra, Australia

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Abstract

The desolvation of ionizable residues in the active sites of enzymes and the subsequent effects on catalysis and thermostability have been studied in model systems, yet little is known regarding how enzymes can naturally evolve active sites with highly reactive and desolvated charges. Variants of triazine hydrolase (TrzN) with significant differences in their active sites have been isolated from different bacterial strains: TrzN from Nocardioides sp. strain MTD22 contains a catalytic glutamate residue (Glu241) that is surrounded by hydrophobic and aromatic second-shell residues (Pro214, Tyr215), whereas TrzN from Nocardioides sp. strain AN3 has a non-catalytic glutamine residue (Gln241) at an equivalent position, surrounded by hydrophilic residues (Thr214/His215). In order to understand how and why these variants have evolved, a series of TrzN mutants were generated and characterized. These results show that desolvation by second-shell residues raises the pKa of Glu241, enabling it to act as a general acid at neutral pH. However, significant thermostability trade-offs are required to incorporate the ionizable Glu241 in the active site and to then enclose it in a hydrophobic microenvironment. Analysis of high-resolution crystal structures shows that there are almost no structural changes to the overall configuration of the active site due to these mutations, suggesting that the changes in activity and thermostability are purely based on the altered electrostatics. The natural evolution of these enzyme isoforms provides a unique system in which to study the fundamental process of charged-residue desolvation in enzyme catalysis and its relative contribution to the creation and evolution of an enzyme active site.


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Many enzyme reactions involve ionizable residues in nucleophilic, electrophilic or general acid-base catalysis.1 The propensity of a residue to be protonated or deprotonated and thereby perform a catalytic role in the protein interior is not just dependent on its solution pKa; the lowered dielectric constant of the interior of an enzyme, the local microenvironment and conformational changes significantly alter pKa values of amino acid side chains.2-4 For instance, hydrophobic shielding of certain residues by their neighbouring residues (the microenvironment) can significantly increase the pKa of a sidechain.5-8 The process of removing water molecules from active site residues as they fold into a hydrophobic protein interior is inherently destabilizing, in accordance with fundamental thermodynamics, and is often compensated for by stabilizing effects elsewhere in the structure.9-13 These concepts have been studied separately,14-16 and in artificial systems,8, 17-19 but analyzing their relative contributions to the creation and efficiency of an enzyme active site using naturally occurring enzyme variants would advance our understanding. These effects are examples of groundstate destabilization,20,

21

which is generally considered to contribute less to catalysis by

highly efficient enzymes than transition state stabilization.22, 23 This is of course a simplified description, as many other factors can contribute to enzyme efficiency, and enzyme efficiency is not the only criteria required and selected for by nature.24 However, the selection pressure for xenobiotic-degrading enzymes is generally directed towards high turnover rates,25-27 as the efficient breakdown of these compounds can directly impact organism survival.28 Accordingly, the natural evolution of such enzymes is often rapid and inefficient ancestral sequences are often inherently transient. This difficulty in isolating short-lived evolutionary intermediates restricts our understanding of enzyme catalysis to extant (and often very efficient) enzymes. Triazine hydrolases (TrzNs) have been identified in Arthrobacter aurescens strain TC1, and Nocardioides sp. strains C190, MTD22 and AN3, and have been shown to catalyze 3 ACS Paragon Plus Environment

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aromatic nucleophilic substitution reactions on triazine-based herbicides atrazine (2-choloro4-isopropylamino-1,3,5-triazine) and ametryn (2-thiomethyl-4-isopylamino-6-ethylamino1,3,5-triazine) (Figure 1).29-32 In the likely catalytic mechanism, a nucleophilic hydroxide is stabilized by His274, Thr325 and a coordinated zinc ion.31 A glutamatic acid, Glu241, has an atypical role in the hydrolysis of ametryn, acting as a general acid and protonating the triazine ring at the N-1 position, which enhances reactivity at the C-2 position where nucleophilic substitution occurs (Figure 1). Although rare, given the proportion of buried glutamate residues found in proteins,33 glutamate has been shown to act as a general acid in other enzyme scaffolds.34, 35 Glu241 is required to act as a proton donor for catalysis of ametryn and related methylthiotriazine herbicides, which have electron donating leaving groups, but the catalysis of atrazine with its electron withdrawing chloride leaving group is sufficiently labile to proceed without a proton donor.31, 32 Previous work has identified strain-dependent differences in the proton donor and second shell residues surrounding it in TrzN. TrzN isolated from Nocardioides sp. strain MTD22 has Glu241 flanked by hydrophobic and aromatic residues (Pro214, Tyr215) that shield this residue, and catalyzes hydrolysis of both atrazine and ametryn.32 In contrast, TrzN isolated from Nocardioides sp. strain AN3 has Gln241 at an equivalent position, flanked by polar residues (Thr214, His215), it can catalyze hydrolysis the of atrazine, but not ametryn (Figure 1).31, 32 These differences in the active sites, and substrate ranges of these enzymes may be related to the presence or absence of the respective herbicides in the environment where these strains grow; this could lead to different selection pressures on the various strains and the catalytic diversity in these isoforms.36 In the present study, we have generated a series of variants that encapsulate the natural diversity in Nocardioides sp. derived TrzN, and investigated the fundamental concepts and trade-offs associated with charged residue desolvation. The pH-dependent activity analysis 4 ACS Paragon Plus Environment

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has revealed that the hydrophobic and aromatic second shell residues do contribute to the raised pKa of Glu241 and thus catalysis. Screening of all variants for activity and stability has revealed a trade-off in the evolution of TrzN, not explained by compensatory structural changes. This naturally occurring enzyme family has provided a unique system with which we can investigate fundamental concepts in enzymology and a unique example where the thermodynamic basis of the increased activity and reduced stability can be clearly defined as being due to one specific effect: charged residue desolvation. MATERIALS AND METHODS Mutagenesis. Solubility optimized trzNG337 was used as template DNA for subsequent mutagenesis. Site-directed mutagenesis of trzNG3 and variants was performed using QuikChange mutagenesis primers (Stratagene)38 which were used with T7 primers to generate fragments that were subsequently cloned into pET-MCSIII39 using Gibson Assembly.40 Amplified PCR products were purified and sequenced on an AB 3730xl DNA Analyzer (at the ACRF Biomolecular Resource Facility, The John Curtin School of Medical Research, Australian National University) following the manufacturer's protocol (Applied Biosystems 2002). For protein solubility analysis, wild type trzN from Arthrobacter aurescens strain TC1 was also cloned into pET-MCSIII39 using Gibson Assembly and the Glu241Gln mutant was generated using the previously described methodology.40 Protein expression, purification and solubility analysis. trzNG3 and variants were transformed into BL21 λDE3 cells. Variants were grown in LB media with 3 % (v/v) ethanol41 and 500 µM ZnSO4 for 42 h at 25 ºC. Cells were pelleted by centrifugation and lysed, the lysate was then sedimented before filtering and resuspension in buffer A (50 mM NaH2PO4, 100 mM NaCl, 8 mM imidazole, 10 % (v/v) glycerol, pH 7.5), loading onto a NiNTA column (Qiagen) and elution using buffer B (50 mM NaH2PO4, 100 mM NaCl, 300 mM imidazole, 10 % (v/v) glycerol, pH 7.5). Concentrated enzyme was then purified further 5 ACS Paragon Plus Environment

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using a Superdex 200 Hiload 26/6 column (GE Healthcare) equilibrated with buffer C (50 mM imidazole, 400 mM NaCl, 10 % (v/v) glycerol, pH 7.5). Prior to kinetic analysis protein samples were desalted into imidazole free-buffer D (50 mM NaH2PO4, 400 mM NaCl, 10 % (v/v) glycerol, pH 7.5) using a PD-10 column. For solubility analysis trzN and variants were transformed into BL21 λDE3 cells and grown in LB media at 30 ºC for 20 h Cells were lysed with BugBuster (Merck Millipore), the soluble fraction was loaded as clarified lysate on an SDS-PAGE gel, the resultant insoluble pellet was resuspended in lysis buffer and loaded as the insoluble fraction. The amount of sample loaded on the gel was determined by normalisation of the OD600. The intensity of the bands corresponding to TrzN were quantified using ImageJ42 and solubility was calculated as Is/(Is + II) × 100, where Is is the intensity of the soluble band and II is the intensity of the insoluble band.43 Standard error values for solubility were calculated from two independent replicates. Enzyme assays. Kinetic parameters for atrazine and ametryn hydrolysis, as catalyzed by the variants, were quantified as previously described by monitoring the disappearance of substrate at 264 nm using an Epoch microplate spectrophotometer.30 The reaction buffer contained 50 mM NaH2PO4, 400 mM NaCl, 10 % (v/v) glycerol at pH 7.5 and reactions were tested at 25 ºC. Reactions were carried out in triplicate and catalytic parameters calculated using molar absorbance values of ε = 3.5 mM-1cm-1 for atrazine and ε = 5 mM-1cm-1 for ametryn.30 The pH dependence of kcat for the enzymes tested was determined using a buffer of constant ionic strength (50 mM acetic acid, 50 mM MES, 100 mM Tris)44 at pH points between 5.5 and 8.5 using atrazine as a substrate. The apparent pKa values were determined using a previously described equation (Eqn 1)45, 46 =

10 + 10 10 10


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The apparent activity (y) is log(kcat) and pKa1and pKa2 are the apparent pKa values for the acidic and basic groups, respectively Protein stability measurements. Enzymes were incubated for 20 min at 16 temperatures between 38.7 and 70.0 ºC, after incubation the mixture was returned to 25 ºC. Kinetic analysis was performed at 25 ºC in triplicate for each temperature, the activity with atrazine was then plotted as a function of incubation temperature; this generates a sigmoidal curve which represents the transition between folded and unfolded protein. The inflection point or Tm50 can then be calculated using a Boltzmann function (Eqn 2): = +

− − 1+

The maximum and minimum activity values are ymax and ymin respectively, T is the incubation temperature, Tm the melting temperature and λ the gradient within Tm. Thermal shift fluorescence assay The thermal shift of all TrzN mutants was measured using the 7900HT Fast Real-Time PCR System (Applied Biosciences) in MicroAmp EnduraPlate Optical 384 well plates (Life Technologies).47 Samples had a final volume of 20 µL per well, containing 2 mg.mL-1 of protein in Buffer C, and 5000 X SYPRO Orange dye stock diluted to a final 5 X concentration. The samples were mixed and covered with an optical seal before denaturation, with triplicate samples tested per protein. Thermal denaturation was measured between 20 °C to 90 °C at a rate of 0.017 °C/s, and the plate was measured with wavelengths of excitation at 470 nm and emission at 580 nm. The Tm was calculated with the Bolzmann sigmoidal equation in R.48 Crystallization,

data

Glu241/Thr214/His215,

collection

and

Gln241/Pro214/His215

structure and

determination.

Gln241/Thr214/His215

Purified were

concentrated to 16 mg-1mL-1, 20 mg-1mL-1 and 25 mg-1mL-1 in buffer C, respectively. 7 ACS Paragon Plus Environment

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Conditions for crystal screens were optimized from previously published work.31, 37 Crystals of all variants were obtained several days after mixing the protein solution with precipitant solution (100 mM Bis-Tris [2,2-bis(hydroxymethyl)-2,2’,2”-nitrilotriethanol], pH 6.5 100 mM ammonium acetate, 16% (w/v) polyethylene glycol [PEG] 3350) in hanging droplets (2 µL reservoir: 1 µL protein) over a reservoir consisting of the precipitant solution. The concentration of PEG 3350 was increased to 35% for use as a cryoprotectant when crystals were flash-cooled to 100K under a stream of nitrogen gas. Diffraction data for Glu241/Thr214/His215 were collected at the MX2 beamline of the Australian Synchrotron at a wavelength of 0.9537 Å, data for Gln241/Pro214/His215 was collected at the MX1 beamline at a wavelength of 0.9537 Å and Gln241/Thr214/His215 data were collected at the MX1 beamline at a wavelength of 0.9501 Å.49 The diffraction data were indexed and integrated in XDS for Glu241/Thr214/His215 and Gln241/Pro214/His215 crystals50 the MOSFLM suite for Gln241/Thr214/His215 crystals,51 BLEND in the CCP4 suite was used to combine datasets collected on different parts of the Glu241/Thr214/His215 crystal.52, 53 The respective data for all variants were scaled in AIMLESS.54 4LH8 was used as an initial model for all variants in phenix.refine and iterative model building was performed in COOT.37, 55, 56 Calculation of pKa using the DEPTH server Chains with the highest structural similarity were used for pKa analysis. A TrzN variant with ametryn bound (3LSB)31 was superimposed to Chain B of 4LH837 (99% sequence similarity, 0.45 C-α RMSD) and chain A of 5HME (99% sequence similarity, 0.45 C-α RMSD) to best replicate the substrate-bound form in pKa calculations. Waters that directly overlapped with the ligand were removed from both structures, and structures with and without a water molecule near to Glu241 were prepared separately. The resultant PDB files were prepared in the 2016-1 Schrodinger Suite release using the Protein Prep Wizard.57 Epik was used to generate the most likely ionization states for the ligand and metal ion at pH 7, the 8 ACS Paragon Plus Environment

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most favorable state with the lowest score was selected for each.58 Hydrogen bonds were added to the structure and a minimization was performed using an OPLS3 force field.59 PRIME was used for minimization, with a VSGB solvation model and OPLS3 force field.59 The minimized structures were assessed separately using the DEPTH server.60

RESULTS The contribution of second shell residues to the pKa of Glu241 To determine whether the local microenvironment facilitates the high pKa of Glu241, (previously determined to be ~8.5),31 TrzN:Glu241/Pro214/Tyr215 was mutated in a combinatorial fashion to Glu241/Thr214/His215 (Table 1), and the pKa of Glu241 determined

using

pH-activity

curves,

with

a

pH-independent

variant

(TrzN:Gln241/Pro214/His215) also tested as a control (Figure 2, Table 1). The pKa of Glu241 flanked by Pro214 and Tyr215 (TrzN:Glu241/Pro214/Tyr215) was determined to be 8.2 ± 0.1, decreasing to 8.0 ± 0.1 in Glu241/Thr214/Tyr215, 6.8 ± 0.2 in Glu241/Pro214/His215, and to 7.6 in Glu241/Thr214/His215 (Figure 2, Table 1). The modest decrease in the pKa of Glu241 upon Thr214 (although there is a clear reduction in activity at basic pH in the Pro214Thr variant; Figure 2) contrasts with the much larger reduction due to the Tyr215His mutation (Table 1, Figure 2). The structural basis for this is discussed below. The significantly higher pKa of Glu241 when it is in a more hydrophobic microenvironment is shown to improve the catalysis (kcat) of ametryn by >4-fold at physiological pH (Table 2, Figure 3). When the ionizable glutamic acid was replaced by glutamine in the TrzN:Gln241/Pro214/His215

variant,

the

activity

was

pH-independent.

However,

Glu241/Pro214/His215 TrzN exhibited a narrowed pH–activity curve (Table 1, Figure 2), indicative of greater sensitivity to changes in pH, similar to what has previously been observed in the activity of a cyclodextrin glucotransferase (CGTase) variant.61 In that work, 9 ACS Paragon Plus Environment

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His233, which does not directly hydrogen bond to the substrate but is thought to be involved in transition state stabilization, was modified to Asn233.62, 63 This His233Asn mutation was thought to change the manner of substrate recognition by changing the hydrogen bonding network of water molecules in the active site and the size of the sidechain, increasing the distance to the substrate and the strength of the pH-dependence on the activity.63 Given that the modification of Tyr215 to His215 results in a reduction in the size of the size-chain and will also change the hydrogen bonding network of water molecules in the active site, a similar mechanism to that seen in CGTase could underlie the narrowing of the pH-activity profile in this variant. To complement the pH-activity analyses, we solved an X-ray crystal structure of the TrzN:Glu241/Thr214/His215 variant at 2.15 Å resolution (Table 3), to compare with the previously solved structure of the TrzN:Glu241/Pro214/Tyr215 variant.37 To test whether the changes in pKa that we observe kinetically can be reproduced computationally using experimentally determined structures, the DEPTH algorithm, which incorporates the distance away from bulk solvent, as well the local microenvironment, in calculating the pKa values of ionizable residues was used.60 The pKa of Glu241 in both variants was determined after positioning

of

ametryn

and

energy

minimization.

The

predicted

pKa

of

TrzN:Glu241/Pro214/Tyr215 was 8.2 (8.2 experimentally) the predicted pKa of TrzN:Glu241/Thr214/His215

was

7.7

(7.6

experimentally).

Given

the

increased

hydrophilicity of the active site pocket in TrzN:Glu241/Thr214/His215, it appears that there is enhanced potential for this water molecules to be located in the active site, which would contribute to the lowered pKa of Glu241 in this variant. These results show that the decrease in the pKa of the key ionizable general acid in the reaction quantitatively correlates with the decrease in the pKa of Glu241, further supporting the idea that Glu241 is indeed the catalytic


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general acid, and that hydrophobic shielding by the surrounding residues can increase its pKa and thus its reactivity. Reduced thermostability of ametryn-hydrolyzing TrzN variants To investigate why the TrzN:Gln241/Thr214/His215 variant is found in Nocardioides sp. strain AN3 TrzN, despite significantly reduced activity with triazine compounds,32 these residues were mutated in a combinatorial fashion to the TrzN:Glu241/Pro214/Tyr215 motif observed in Nocardioides sp. strain MTD22 TrzN and characterized using activity and thermostability assays. Modifying Gln241 to Glu241 and the second shell hydrophilic residues to their hydrophobic counterparts results in substantial reductions in the melting temperature (thermostability, Tm), which contrasts with significant increases in the catalytic activity (kcat); (Figure 4, Table 4). The magnitude of the destabilization due to the Gln241Glu mutation was significant as determined by catalytic (8.8 ºC) and differential scanning fluorimetry methods (4.2 ºC), which suggests that introducing this charged group into the active site places it in a high-energy state (Figure 4-5, , Table 4). The increased thermostability of Gln241-containing variants in TrzN compared to Glu241-containing variants is consistent with previously published stability increases upon mutating an interior charged residue to a neutral amino acid in different enzyme scaffolds.64,

65

Modifying the

hydrophilic residues flanking Glu241 to their hydrophobic counterparts also decreases stability (Figure 4, Table 4). Glu241/Pro214/Tyr215 TrzN was found to be 2.9 ºC to 1.5 ºC (catalytic Tm, structural Tm) less stable than Glu241/Thr214/His215 TrzN (Figure 4, Table 4), which is again consistent the pH-activity profiles that suggest Glu241 becomes destabilized and exists predominantly in a reactive, protonated state. Changes to the Km of TrzN with atrazine or ametryn between all variants are not particularly significant, the most significant changes are observed in the kcat values (Table 2). Catalysis of atrazine is improved two-fold at neutral pH through the addition of Glu241 and 11 ACS Paragon Plus Environment

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flanking hydrophobic residues (Table 2). The ability to hydrolyze ametryn is gained when Glu241 is present, and improved three-fold when the second-shell residues are made more hydrophobic

(Figure

3,

Table

2).

Mutation

of

TrzN:Gln241/Thr214/His215

to

TrzN:Gln241/Pro214/His215 does not significantly impact stability or activity, but in the TrzN:Glu241/Pro214/Tyr215 background, modification of Tyr215 to His215 significantly decreases activity with both substrates, potentially due to the smaller sidechain of His214 impacting substrate recognition, as previously described. The solubility of TrzN variants was also assessed to determine whether protein solubility correlated with the previously determined thermostability differences. There is greater soluble expression of Gln241-containing TrzN (28% ± 2%) compared to Glu241-containing TrzN (22% ± 2%) in Escherichia coli (Figure 4). Enzymes with higher thermostability are often observed to have higher soluble expression, as less stable proteins have shorter lifetimes and do not accumulate in the cytosol to high levels.66, 67 The relatively small difference in solubility in this instance, may be due to poor stability of apo-TrzN in E. coli, which is a bottleneck in the the accumulation of soluble holo-TrzN.37 This is thought to be partly due to differences in codon usage, metabolism, and the chaperones present in E. coli and the native host (Arthobacter and Nocardioides).68, 69 The increased soluble expression of Gln241-TrzN, while small, does represent a physiologically relevant trade-off between a poorer catalyst able to reach a higher effective concentration in the cell (Gln241-TrzN) and a catalytically superior TrzN with a lower effective concentration (Glu241-TrzN).66, 70-73 Structural characterization of TrzN variants In addition to the previously reported structure of TrzN:Glu241/Pro214/Tyr215 (4LH8)37 and the structure of TrzN:Glu241/Thr214/His215 (5HME), discussed above, we solved structures of the TrzN:Gln241/Pro214/His215 (2.10 Å) and Gln241/Thr214/His215 (1.84 Å) to investigate whether any large-scale structural changes were associated with the mutation of 12 ACS Paragon Plus Environment

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these residues or whether the observed changes in activity and stability were due to loop movements or significant rearrangements of the active site. All four structures were essentially identical, with the exception of the mutated residues, with C-α RMSD values of 0.32

Å,

0.33

Å

and

0.30

Å,

TrzN:Glu241/Thr214/His215,

vs

TrzN:Glu241/Pro214/Tyr215

for

TrzN:Gln241/Pro214/His215

the and

TrzN:Gln241/Thr214/His215 structures, respectively.23 TM-align (template modelling alignment) scores of 0.99 between all structures vs each other also indicates the very high structural similarity between variants.74 There were no significant changes observed in loop conformations, bonding interactions or residue conformations when the structures were visually inspected in a graphics terminal. The only substantial difference between structures was the presence of many more water molecules

in

the

active

site

of

TrzN:Gln241/Thr214/His215

compared

to

TrzN:Glu241/Pro214/Tyr215 TrzN (Figure 6). There are 19 water molecules in the substrate tunnel and active site of TrzN:Gln241/Thr214/His215, with 17 potential bonds with the protein, compared to the 12 water molecules observed in TrzN:Glu241/Pro214/Tyr215 with 10 potential bonds (Figure 6). It is likely that the more hydrophilic interior of TrzN:Gln241/Thr214/His215 is more conducive to the presence of extra water molecules, as the structures are so similar that the same interactions between the additional water molecules and the protein backbone could be formed in TrzN:Glu241/Pro214/Tyr215. The removal of the bulky tyrosine residue could also facilitate the entry of more water molecules into the active site, as Tyr215 lies at the narrowest point in the substrate tunnel of TrzN:Glu241/Pro214/Tyr215. In the absence of structural changes, the changes in activity can thus be ascribed to changes in the electrostatic character of otherwise identical active sites.


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A water molecule coordinated to Glu241/Gln241 is observed in every structure solved in this work, it is also positioned for hydrogen bonding between Glu241 and Tyr215 in a previously solved structure (Figure 6).37 However, this water-mediated hydrogen bond is displaced in the crystal structure of a TrzN variant with ametryn bound (Figure 6),31 resulting in Glu241 and Tyr215 being shielded from solvent, contributing to the raised pKa of Glu241 in catalysis.

DISCUSSION Determining the impact of the local microenvironment on the pKa of catalytic ionizable residues is often complex, as many factors contribute to the energetics of ionization of a buried group.75,

76

The relative contribution of a microenvironment with increased

hydrophobicity, created through the modification of second shell residues (Thr214/His215 to Pro214/Tyr215), on the raised pKa of Glu241 in TrzN was investigated. The experimentally and computationally determined differences in the pKa of Glu241between these TrzN variants indicates that the hydrophobic wall formed by Pro214/Tyr215 contributes to the raised pKa of Glu241 by shielding it from solvent. The increased pKa of Glu241 effectively destabilizes it, or raises its reactivity, so that it can exist in a protonated state at pH values where it would normally be deprotonated (pKa of 8.2 vs 4.5 in solution). The ability to remain protonated improves the catalytic efficiency of TrzN and allows it to catalyze the hydrolysis of ametryn, which it would otherwise be unable to do. It is likely that the remaining disparity between the pKa of glutamate in water (4.5) and the experimentally determined pKa of Glu241 with flanking hydrophilic residues (7.6) is due to it being buried ~10 Å in the protein interior; again, the low dielectric constant of the protein contributes to this increase in pKa.60, 77, 78


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The ionizable Glu241 residue is required for the efficient catalysis of atrazine and ametryn by TrzN, but there is an energetic cost associated with the desolvation of charged residues upon protein folding.12 The energy cost associated with the desolvation of charged residues when they are folded into the interior of a protein where the dielectric constant is lower, unless new stabilizing interactions are formed, has been well documented.12, 79, 80 The watermediated hydrogen bond between Glu241 and Tyr215 is a stabilizing interaction, but this interaction is observed to be lost upon ametryn binding.31 The lack of any structural differences between the variant TrzN crystal structures indicates that no other compensatory interactions have been formed (Figure 6), and it has previously been shown that stable enzyme scaffolds can withstand the destabilizing addition of an interior charged residue without extensive compensatory mutations.15,

81

In fact, in this case, the lack of any

compensatory interactions to neutralize the charge is essential to the driving force behind this mutation, which is to make the glutamic acid more reactive. This is a clear example of the role of electrostatics in enzyme catalysis because the only differences between the three TrzN variant structures that we solved were the mutations themselves; there were no secondary effects on the structure. The observation of many more water molecules in the active site cavity of the most polar active site structure TrzN:Gln241/Thr214/His215

compared

to

TrzN:Glu241/Pro214/Tyr215

gives

some

indication of the thermodynamic basis for the greater stability of this variant: both structures were solved to the same resolution (1.8 Å), and all water molecules are well defined in electron density (Figure 4), yet we observe many more (7) water molecules in the cavity of the TrzN:Gln241/Thr214/His215, consistent with a number of stabilizing interactions and additional H-bonds (7) with the protein. The presence of Glu241 or Gln241 in the active site of TrzN is most likely determined by the different niches the respective bacterial sources occupy.32, 82 Atrazine has been used for 15 ACS Paragon Plus Environment

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longer and far more extensively than methylthiotriazine based herbicides such as ametryn and simetryn.83 Therefore, it is likely that there are locations in which atrazine has been the only triazine-based herbicide applied, where the selective pressure would only be towards the breakdown of atrazine as an additional carbon source for bacteria in these contaminated areas. In this context, whole cell activity, which would correlate closely with the level of soluble expression, is known to be linked to the thermostability of the enzyme (Figure 4),84, 85 would be under strong selective pressure. Thus, the high thermostability but slightly slower rate of atrazine hydrolysis by TrzN:Gln241/Thr214/His215 TrzN could meet the requirements

of

bacteria

in

these

environments

for

enhanced

survival

vs

TrzN:Glu241/Pro214/Tyr215, where the higher hydrolysis rate compensates for the significantly lower thermostability. However, in environments where ametryn or related methylthiotriazines are found, the selective pressure will be directed towards the hydrolysis of these compounds. Loss of TrzN stability due to the burial of the catalytic Glu241, to facilitate a gain in catalytic activity with ametryn, and improved activity with atrazine if it is also present, would result in enhanced survival for bacteria in areas with methylthiotriazines present.36, 86 CONCLUSIONS We have investigated the naturally evolved variation of TrzN isolated from different bacterial strains and characterized a trade-off between variants based on the fundamental concept of active site desolvation. We have shown that the considerably less active Gln241containing TrzN variant is more stable than the Glu241 variant due to a trade-off between the enhanced catalysis of a glutamate proton donor shielded by hydrophobic residues and the thermodynamic cost of this burial. This natural example of these fundamental concepts illustrates the importance of active site desolvation and trade-offs in the creation of an efficient active site and thermostability in natural evolution. Similarly clear examples of 16 ACS Paragon Plus Environment

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ground-state (GS) destabilization in the natural evolution of enzymes are rare,20,

87, 88

and

unlike the majority of naturally evolved enzymes that appear to have evolved to stabilize the transition state (TS).24,

89-91

The catalytic efficiency of TrzN is low, given that “average”

enzymes exhibit kcat values of ~10 s-1 (~1 s-1 in TrzN) and kcat/KM values of ~105 s-1M-1 (~104 s-1 M-1 in TrzN),92 and it remains to be seen whether further evolutionary optimization of TrzN will result in fixation of mutations that better stabilize the TS, rather than destabilize the GS. It is possible that the evolution of GS destabilization is a common early step in enzyme development, introducing catalytic reactivity prior to the evolution of efficient TS stabilization.

ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author *E-mail:[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT We thank Hafna Ahmed for assistance with the energy minimization of the protein structures. This research was undertaken on the MX1 and MX2 beamlines at the Australian Synchrotron, Victoria, Australia. ABBREVIATIONS TrzN, Triazine hydrolase; PDB, Protein Data Bank; CGTase, cyclodextrin 17 ACS Paragon Plus Environment

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glucanotransferase.


Page 19 of 50

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

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(1) Harris, T. K., and Turner, G. J. (2002) Structural basis of perturbed pKa values of catalytic groups in enzyme active sites, IUBMB life 53, 85-98. (2) Fafarman, A. T., Sigala, P. A., Schwans, J. P., Fenn, T. D., Herschlag, D., and Boxer, S. G. (2012) Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase, Proc. Natl. Acad. Sci. U.S.A. 109, E299-E308. (3) Ho, M.-C., Ménétret, J.-F., Tsuruta, H., and Allen, K. N. (2009) The origin of the electrostatic perturbation in acetoacetate decarboxylase, Nature 459, 393-397. (4) Hanoian, P., Liu, C. T., Hammes-Schiffer, S., and Benkovic, S. (2015) Perspectives on Electrostatics and Conformational Motions in Enzyme Catalysis, Acc. Chem. Res. 48, 482489. (5) Schwans, J. P., Sunden, F., Gonzalez, A., Tsai, Y., and Herschlag, D. (2013) Uncovering the Determinants of a Highly Perturbed Tyrosine p K a in the Active Site of Ketosteroid Isomerase, Biochemistry 52, 7840-7855. (6) Czerwinski, R. M., Harris, T. K., Massiah, M. A., Mildvan, A. S., and Whitman, C. P. (2001) The structural basis for the perturbed p K a of the catalytic base in 4-oxalocrotonate tautomerase: Kinetic and structural effects of mutations of Phe-50, Biochemistry 40, 19841995. (7) Yévenes, A., Espinoza, R., Rivas-Pardo, J. A., Villarreal, J. M., González-Nilo, F. D., and Cardemil, E. (2006) Site-directed mutagenesis study of the microenvironment characteristics of Lys 213 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase, Biochimie 88, 663-672.


Biochemistry

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 20 of 50

(8) Isom, D. G., Castañeda, C. A., and Cannon, B. R. (2011) Large shifts in pKa values of lysine residues buried inside a protein, Proc. Natl. Acad. Sci. U.S.A. 108, 5260-5265. (9) Shoichet, B. K., Baase, W. A., Kuroki, R., and Matthews, B. W. (1995) A relationship between protein stability and protein function, Proc. Natl. Acad. Sci. U.S.A. 92, 452-456. (10) Robinson, A. C., Castañeda, C. A., and Schlessman, J. L. (2014) Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein, Proc. Natl. Acad. Sci. U.S.A. 111, 11685-11690. (11) Aghera, N., Dasgupta, I., and Udgaonkar, J. B. (2012) A buried ionizable residue destabilizes the native state and the transition state in the folding of monellin, Biochemistry 51, 9058-9066. (12) Warshel, A., Sussman, F., and Hwang, J.-K. (1988) Evaluation of catalytic free energies in genetically modified proteins, J. Mol. Biol. 201, 139-159. (13) Karp, D. A., Gittis, A. G., Stahley, M. R., Fitch, C. A., and Stites, W. E. (2007) High apparent dielectric constant inside a protein reflects structural reorganization coupled to the ionization of an internal Asp, Biophys. J. 92, 2041-2053. (14) Harms, M. J., Castañeda, C. A., Schlessman, J. L., Sue, G. R., Isom, D. G., and Cannon, B. R. (2009) The pK a values of acidic and basic residues buried at the same internal location in a protein are governed by different factors, J. Mol. Biol. 389, 34-47. (15) Chimenti, M. S., Khangulov, V. S., Robinson, A. C., Heroux, A., Majumdar, A., Schlessman, J. L., and García-Moreno, B. (2012) Structural reorganization triggered by charging of Lys residues in the hydrophobic interior of a protein, Structure 20, 1071-1085.


Page 21 of 50

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

Biochemistry

(16) Karp, D. A., Stahley, M. R., and García-Moreno E, B. (2010) Conformational consequences of ionization of Lys, Asp, and Glu buried at position 66 in staphylococcal nuclease, Biochemistry 49, 4138-4146. (17) Isom, D. G., Cannon, B. R., Castañeda, C. A., and Robinson, A. (2008) High tolerance for ionizable residues in the hydrophobic interior of proteins, Proc. Natl. Acad. Sci. U.S.A. 105, 17784-17788. (18) Pey, A. L., Rodriguez-Larrea, D., Gavira, J. A., Garcia-Moreno, B., and Sanchez-Ruiz, J. M. (2010) Modulation of buried ionizable groups in proteins with engineered surface charge, J. Am. Chem. Soc. 132, 1218-1219. (19) Harms, M. J., Schlessman, J. L., and Sue, G. R. (2011) Arginine residues at internal positions in a protein are always charged, Proc. Natl. Acad. Sci. U.S.A. 108, 18954-18959. (20) Frushicheva, M. P., Cao, J., Chu, Z. T., and Warshel, A. (2010) Exploring challenges in rational enzyme design by simulating the catalysis in artificial kemp eliminase, Proc. Natl. Acad. Sci. U.S.A. 107, 16869-16874. (21) Kraut, D. A., Carroll, K. S., and Herschlag, D. (2003) Challenges in enzyme mechanism and energetics, Annu. Rev. Biochem. 72, 517-571. (22) Benkovic, S. J., and Hammes-Schiffer, S. (2003) A perspective on enzyme catalysis, Science 301, 1196-1202. (23) Warshel, A., and Florián, J. (1998) Computer simulations of enzyme catalysis: finding out what has been optimized by evolution, Proc. Natl. Acad. Sci. U.S.A. 95, 5950-5955. (24) Warshel, A., Sharma, P. K., Kato, M., Xiang, Y., Liu, H., and Olsson, M. H. (2006) Electrostatic basis for enzyme catalysis, Chem. Rev. 106, 3210-3235. 21 ACS Paragon Plus Environment

Biochemistry

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 22 of 50

(25) Jackson, C. J., Foo, J.-L., Tokuriki, N., Afriat, L., Carr, P. D., Kim, H.-K., Schenk, G., Tawfik, D. S., and Ollis, D. (2009) Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase, Proc. Natl. Acad. Sci. U.S.A. 106, 21631-21636. (26) Omburo, G. A., Kuo, J. M., Mullins, L. S., and Raushel, F. (1992) Characterization of the zinc binding site of bacterial phosphotriesterase, J. Biol. Chem. 267, 13278-13283. (27) Seffernick, J. L., de Souza, M. L., Sadowsky, M. J., and Wackett, L. P. (2001) Melamine deaminase and atrazine chlorohydrolase: 98 percent identical but functionally different, J. Bacteriol. 183, 2405-2410. (28) Sugrue, E., Hartley, C. J., Scott, C., and Jackson, C. J. (2016) The Evolution of New Catalytic Mechanisms for Xenobiotic Hydrolysis in Bacterial Metalloenzymes, Aust. J. Chem., -. (29) Topp, E., Mulbry, W. M., Zhu, H., Nour, S. M., and Cuppels, D. (2000) Characterization of s-triazine herbicide metabolism by a Nocardioides sp. isolated from agricultural soils, Appl. Environ. Microbiol. 66, 3134-3141. (30) Shapir, N., Pedersen, C., Gil, O., Strong, L., Seffernick, J., Sadowsky, M. J., and Wackett, L. P. (2006) TrzN from Arthrobacter aurescens TC1 is a zinc amidohydrolase, J. Bacteriol. 188, 5859-5864. (31) Seffernick, J. L., Reynolds, E., Fedorov, A. A., Fedorov, E., Almo, S. C., Sadowsky, M. J., and Wackett, L. P. (2010) X-ray structure and mutational analysis of the atrazine chlorohydrolase TrzN, J. Biol. Chem. 285, 30606-30614.


Page 23 of 50

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

Biochemistry

(32) Yamazaki, K., Fujii, K., Iwasaki, A., Takagi, K., Satsuma, K., Harada, N., and Uchimura, T. (2008) Different substrate specificities of two triazine hydrolases (TrzNs) from Nocardioides species, FEMS Microbiol. Lett. 286, 171-177. (33) Kim, J., Mao, J., and Gunner, M. (2005) Are acidic and basic groups in buried proteins predicted to be ionized?, J. Mol. Biol. 348, 1283-1298. (34) McIntosh, L. P., Hand, G., Johnson, P. E., Joshi, M. D., Körner, M., Plesniak, L. A., Ziser, L., Wakarchuk, W. W., and Withers, S. G. (1996) The p K a of the General Acid/Base Carboxyl Group of a Glycosidase Cycles during Catalysis: A 13C-NMR Study of Bacillus circulans Xylanase, Biochemistry 35, 9958-9966. (35) Bashiri, G., Squire, C. J., Moreland, N. J., and Baker, E. N. (2008) Crystal structures of F420-dependent glucose-6-phosphate dehydrogenase FGD1 involved in the activation of the anti-tuberculosis drug candidate PA-824 reveal the basis of coenzyme and substrate binding, J. Biol. Chem. 283, 17531-17541. (36) Arnold, F. H., Wintrode, P. L., Miyazaki, K., and Gershenson, A. (2001) How enzymes adapt: lessons from directed evolution, Trends Biochem. Sci. 26, 100-106. (37) Jackson, C. J., Coppin, C. W., Carr, P. D., Aleksandrov, A., Wilding, M., Sugrue, E., Ubels, J., Paks, M., Newman, J., and Peat, T. S. (2014) 300-Fold Increase in Production of the Zn2+-Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization, Appl. Environ. Microbiol. 80, 4003-4011. (38) Zheng, L., Baumann, U., and Reymond, J.-L. (2004) An efficient one-step sitedirected and site-saturation mutagenesis protocol, Nucleic Acids Res. 32, e115-e115.


Biochemistry

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 24 of 50

(39) Neylon, C., Brown, S. E., Kralicek, A. V., Miles, C. S., Love, C. A., and Dixon, N. E. (2000) Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance, Biochemistry 39, 11989-11999. (40) Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., and Smith, H. O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases, Nat. Methods 6, 343-345. (41) Oganesyan, N., Ankoudinova, I., Kim, S.-H., and Kim, R. (2007) Effect of osmotic stress and heat shock in recombinant protein overexpression and crystallization, Protein Expression Purif. 52, 280-285. (42) Schneider, C. A., Rasband, W. S., and Eliceiri, K. W. (2012) NIH Image to ImageJ: 25 years of image analysis, Nat. Methods 9, 671-675. (43) Wyganowski, K. T., Kaltenbach, M., and Tokuriki, N. (2013) GroEL/ES buffering and compensatory mutations promote protein evolution by stabilizing folding intermediates, J. Mol. Biol. 425, 3403-3414. (44) Ellis, K. J., and Morrison, J. F. (1981) Buffers of constant ionic strength for studying pH-dependent processes, Methods Enzymol. 87, 405-426. (45) Khurana, J. L., Jackson, C. J., Scott, C., Pandey, G., Horne, I., Russell, R. J., Herlt, A., Easton, C. J., and Oakeshott, J. G. (2009) Characterization of the phenylurea hydrolases A and B: founding members of a novel amidohydrolase subgroup, Biochem. J 418, 431-441. (46) Sugrue, E., Fraser, N. J., Hopkins, D. H., Carr, P. D., Khurana, J. L., Oakeshott, J. G., Scott, C., and Jackson, C. J. (2015) Evolutionary expansion of the amidohydrolase 24 ACS Paragon Plus Environment

Page 25 of 50

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

Biochemistry

superfamily in bacteria in response to the synthetic compounds molinate and diuron, Appl. Environ. Microbiol. 81, 2612-2624. (47) Niesen, F. H., Berglund, H., and Vedadi, M. (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability, Nat. Protoc. 2, 22122221. (48) Team, R. C. (2013) R: A language and environment for statistical computing. (49) McPhillips, T. M., McPhillips, S. E., Chiu, H.-J., Cohen, A. E., Deacon, A. M., Ellis, P. J., Garman, E., Gonzalez, A., Sauter, N. K., and Phizackerley, R. P. (2002) Blu-Ice and the Distributed Control System: software for data acquisition and instrument control at macromolecular crystallography beamlines, Synchrotron Radiation 9, 401-406. (50) Kabsch, W. (2010) Xds, Acta Crystallogr. Sect. D. Biol. Crystallogr. 66, 125-132. (51) Battye, T. G. G., Kontogiannis, L., Johnson, O., Powell, H. R., and Leslie, A. G. (2011) iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM, Acta Crystallogr. Sect. D. Biol. Crystallogr. 67, 271-281. (52) Foadi, J., Aller, P., Alguel, Y., Cameron, A., Axford, D., Owen, R. L., Armour, W., Waterman, D. G., Iwata, S., and Evans, G. (2013) Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography, Acta Crystallogr. Sect. D. Biol. Crystallogr. 69, 1617-1632. (53) Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G., and McCoy, A. (2011) Overview of the CCP4 suite and current developments, Acta Crystallogr. Sect. D. Biol. Crystallogr. 67, 235-242.


Biochemistry

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 26 of 50

(54) Evans, P. R. (2011) An introduction to data reduction: space-group determination, scaling and intensity statistics, Acta Crystallogr. Sect. D. Biol. Crystallogr. 67, 282-292. (55) Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L.-W., Kapral, G. J., and Grosse-Kunstleve, R. W. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution, Acta Crystallogr. Sect. D. Biol. Crystallogr. 66, 213-221. (56) Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics, Acta Crystallogr. Sect. D. Biol. Crystallogr. 60, 2126-2132. (57) Sastry, G. M., Adzhigirey, M., Day, T., Annabhimoju, R., and Sherman, W. (2013) Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments, J. Comput.-Aided Mol. Des. 27, 221-234. (58) Greenwood, J. R., Calkins, D., Sullivan, A. P., and Shelley, J. C. (2010) Towards the comprehensive, rapid, and accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution, J. Comput.-Aided Mol. Des. 24, 591-604. (59) Harder, E., Damm, W., Maple, J., Wu, C., Reboul, M., Xiang, J. Y., Wang, L., Lupyan, D., Dahlgren, M. K., and Knight, J. L. (2015) OPLS3: a force field providing broad coverage of drug-like small molecules and proteins, J. Chem. Theory Comput. 12, 281-296. (60) Tan, K. P., Nguyen, T. B., Patel, S., Varadarajan, R., and Madhusudhan, M. S. (2013) Depth: a web server to compute depth, cavity sizes, detect potential small-molecule ligandbinding cavities and predict the pKa of ionizable residues in proteins, Nucleic Acids Res., gkt503.


Page 27 of 50

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

Biochemistry

(61) Nakamura, A., Haga, K., and Yamane, K. (1993) Three histidine residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011: effects of the replacement on pH dependence and transition-state stabilization, Biochemistry 32, 66246631. (62) Ishii, N., Haga, K., Yamane, K., and Harata, K. (2000) Crystal structure of alkalophilic asparagine 233-replaced cyclodextrin glucanotransferase complexed with an inhibitor, acarbose, at 2.0 Å resolution, J. Biochem. 127, 383-391. (63) Ishii, N., Haga, K., Yamane, K., and Harata, K. (2000) Crystal structure of asparagine 233‐replaced cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 determined at 1.9 Å resolution, J. Mol. Recognit. 13, 35-43. (64) Inoue, M., Yamada, H., Hashimoto, Y., Yasukochi, T., Hamaguchi, K., Miki, T., Horiuchi, T., and Imoto, T. (1992) Stabilization of a protein by removal of unfavorable abnormal pKa: substitution of undissociable residue for glutamic acid-35 in chicken lysozyme, Biochemistry 31, 8816-8821. (65) Mukaiyama, A., Haruki, M., Ota, M., Koga, Y., Takano, K., and Kanaya, S. (2006) A hyperthermophilic protein acquires function at the cost of stability, Biochemistry 45, 1267312679. (66) Socha, R. D., and Tokuriki, N. (2013) Modulating protein stability–directed evolution strategies for improved protein function, FEBS J. 280, 5582-5595. (67) DePristo, M. A., Weinreich, D. M., and Hartl, D. L. (2005) Missense meanderings in sequence space: a biophysical view of protein evolution, Nature Reviews Genetics 6, 678687.


Biochemistry

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 28 of 50

(68) Pal, A., Mondal, U. K., Mukhopadhyay, S., and Bothra, A. K. (2011) Genomic heterogeneity within conserved metabolic pathways of Arthrobacter species-a bioinformatic approach, Bioinformation 5, 446-454. (69) Terpe, K. (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems, Appl. Microbiol. Biotechnol. 72, 211-222. (70) Cabrita, L. D., Gilis, D., Robertson, A. L., Dehouck, Y., Rooman, M., and Bottomley, S. P. (2007) Enhancing the stability and solubility of TEV protease using in silico design, Protein Sci. 16, 2360-2367. (71) Mayer, S., Rüdiger, S., Ang, H. C., Joerger, A. C., and Fersht, A. R. (2007) Correlation of levels of folded recombinant p53 in Escherichia coli with thermodynamic stability in vitro, J. Mol. Biol. 372, 268-276. (72) Randles, L. G., Lappalainen, I., Fowler, S. B., Moore, B., Hamill, S. J., and Clarke, J. (2006) Using model proteins to quantify the effects of pathogenic mutations in Ig-like proteins, J. Biol. Chem. 281, 24216-24226. (73) Tokuriki, N., Stricher, F., Serrano, L., and Tawfik, D. S. (2008) How protein stability and new functions trade off, PLoS Comput Biol 4, e1000002. (74) Zhang, Y., and Skolnick, J. (2005) TM-align: a protein structure alignment algorithm based on the TM-score, Nucleic Acids Res. 33, 2302-2309. (75) Fitch, C. A., Karp, D. A., Lee, K. K., Stites, W. E., Lattman, E. E., and GarcíaMoreno, E. B. (2002) Experimental pK a values of buried residues: analysis with continuum methods and role of water penetration, Biophys. J. 82, 3289-3304. 28 ACS Paragon Plus Environment

Page 29 of 50

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

Biochemistry

(76) Li, H., Robertson, A. D., and Jensen, J. H. (2004) The determinants of carboxyl pKa values in turkey ovomucoid third domain, Proteins: Struct., Funct., Bioinf. 55, 689-704. (77) Matthew, J. B., Gurd, F. R., Garcia-Moreno, B. E., Flanagan, M. A., March, K. L., and Shire, S. J. (1985) pH-dependent processes in protein, CRC Crit. Rev. Biochem. 18, 91-197. (78) Isom, D. G., Castañeda, C. A., Cannon, B. R., and Velu, P. D. (2010) Charges in the hydrophobic interior of proteins, Proc. Natl. Acad. Sci. U.S.A. 107, 16096-16100. (79) Warshel, A., and Russell, S. T. (1984) Calculations of electrostatic interactions in biological systems and in solutions, Q. Rev. Biophys. 17, 283-422. (80) Pace, C. N., Grimsley, G. R., and Scholtz, J. M. (2009) Protein ionizable groups: pK values and their contribution to protein stability and solubility, J. Biol. Chem. 284, 1328513289. (81) Merski, M., and Shoichet, B. K. (2012) Engineering a model protein cavity to catalyze the Kemp elimination, Proc. Natl. Acad. Sci. U.S.A. 109, 16179-16183. (82) Satsuma, K. (2006) Characterisation of new strains of atrazine‐degrading Nocardioides sp. isolated from Japanese riverbed sediment using naturally derived river ecosystem, Pest Manage. Sci. 62, 340-349. (83) LeBaron, H. M., Mc Farland, J., and Burnside, O. (2011) The triazine herbicides, Elsevier. (84) Tokuriki, N., and Tawfik, D. S. (2009) Stability effects of mutations and protein evolvability, Curr. Opin. Struct. Biol. 19, 596-604.


Biochemistry

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(85) Peña, M. I., Davlieva, M., Bennett, M. R., Olson, J. S., and Shamoo, Y. (2010) Evolutionary fates within a microbial population highlight an essential role for protein folding during natural selection, Mol. Syst. Biol. 6, 387. (86) Seffernick, J. L., and Wackett, L. P. (2001) Rapid evolution of bacterial catabolic enzymes: a case study with atrazine chlorohydrolase, Biochemistry 40, 12747-12753. (87) Frushicheva, M. P., Cao, J., and Warshel, A. (2011) Challenges and advances in validating enzyme design proposals: The case of Kemp eliminase catalysis, Biochemistry 50, 3849-3858. (88) Ruben, E. A., Schwans, J. P., Sonnett, M., Natarajan, A., Gonzalez, A., Tsai, Y., and Herschlag, D. (2013) Ground state destabilization from a positioned general base in the ketosteroid isomerase active site, Biochemistry 52, 1074-1081. (89) Warshel, A. (1998) Electrostatic origin of the catalytic power of enzymes and the role of preorganized active sites, J. Biol. Chem. 273, 27035-27038. (90) Warshel, A., Štrajbl, M., Villa, J., and Florián, J. (2000) Remarkable rate enhancement of orotidine 5'-monophosphate decarboxylase is due to transition-state stabilization rather than to ground-state destabilization, Biochemistry 39, 14728-14738. (91) Štrajbl, M., Shurki, A., Kato, M., and Warshel, A. (2003) Apparent NAC effect in chorismate mutase reflects electrostatic transition state stabilization, J. Am. Chem. Soc. 125, 10228-10237. (92) Bar-Even, A., Noor, E., Savir, Y., Liebermeister, W., Davidi, D., Tawfik, D. S., and Milo, R. (2011) The moderately efficient enzyme: evolutionary and physicochemical trends shaping enzyme parameters, Biochemistry 50, 4402-4410. 30 ACS Paragon Plus Environment

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Biochemistry

Table 1. Dissociation constants for the hydrolysis of atrazine by variants of triazine hydrolase (mean ± standard error) Variant

pKe1

pKe2

Glu241/Pro214/Tyr215

5.4 ± 0.1

8.2 ± 0.1

Glu214/Thr214/Tyr215 5.4 ± 0.1

8.0 ± 0.1

Glu241/Pro214/His215

6.4 ± 0.3

6.8 ± 0.2

Glu241/Thr214/His215 5.4 ± 0.0

7.6 ± 0.0


N/A

N/A


Biochemistry

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Table 2. Kinetic parameters for triazine hydrolase variants with atrazine and ametryn Atrazine Variant

Ametryn

kcat

Km

kcat/Km

kcat

Km

kcat/Km

(s-1)

(µM)

(104 M-1 s-1)

(s-1)

(µM)

(104 M-1 s-1)


1.18 ± 0.10

36 ± 7

3.4

0.87± 0.06

67± 1

1.3

Glu214/Thr214/Tyr215

1.49 ± 0.11

51 ± 5

2.9

0.79± 0.08

49± 4

1.6


0.20 ± 0.07

26 ± 6

0.8

0.24± 0.06

40± 12

0.6

Glu241/Thr214/His215

0.55 ± 0.04

54 ± 4

1.0

0.21± 0.02

55± 17

0.4

Gln241/Pro214/Tyr215

0.11 ± 0.01

29 ± 6

0.4

ND

ND

ND


0.40 ± 0.02

51 ± 14

0.8

ND

ND

ND


0.50 ± 0.03

38 ± 8

1.4

ND

ND

ND


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Biochemistry

Table 3. Crystallographic data and refinement statistics for triazine hydrolase variants Glu241/Thr214/His215



P1 21 1

P1 21 1

P1 21 1

a (Å)

56.55

56.15

57.25

b (Å)

100.72

99.42

101.68

c (Å)

78.85

77.18

80.44

β (°)

90, 102.3, 90

90, 101.12,90

90, 104.01, 90

Data collection Space group Unit-cell parameters

Wavelength (Å)

0.9537

0.9537

0.9501

Resolution range (Å)*

35.98-2.15 (2.23-2.15)

19.69-2.10 (2.16-2.10)

55.54-1.84 (1.88-1.84)

No. of unique reflections

46811

47950

72511

Completeness (%)

99.7 (98.5)

98.6 (93.3)

94.0 (87.5)

6.4 (6.4)

5.3 (4.9)

3.3 (2.9)

0.230 (1.043)

0.131 (0.557)

0.078 (0.636)

0.154 (0.686)

0.098 (0.420)

0.072 (0.553)

7.3 (2.3)

11.1 (2.7)

12.8 (1.6)

0.985 (0.860)

0.995 (0.797)

0.983 (0.641)

Reflections used

46686

47914

72488

Resolution range (Å)

35.98-2.15 (2.23-2.15)

19.69-2.10 (2.16-2.10)

55.54-1.84 (1.88-1.84)

0.2018/0.2269 (0.3407/0.3837)

0.1509/0.2132 (0.2104/0.2651)

0.1641/0.1941 (0.2734/0.2908)

Bond Length (Å)

0.015

0.012

0.010

Bond Angles (°)

1.43

1.37

1.25

5HMD

5HMF

Multiplicity Rmerge(I)† Rpimψ Mean CC1/2# Refinement

RWork/RFree‡ R.M.S Deviations

PDB ID 5HME *Values in parenthesis are for the highest-resolution shell

†Rmerge(I) = (Σhkl Σj |Ihkl,j - 〈Ihkl〉|)/(Σhkl Σj Ihkl,j) where 〈Ihkl〉 is the average intensity of j symmetry-related observations of reflections with Miller indices hkl. ψRpim = (Σhkl1/ − 1 Σj |Ihkl,j - 〈Ihkl〉|)/(Σhkl Σj Ihkl,j) #CC1/2 = percentage of correlation between intensities from random half-datasets ‡RWork = Σhkl|F(obs)-F(calc)|/Σhkl|F(obs)|; 5% of the data that were excluded from the refinement were used to calculate RFree.


Biochemistry

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Table 4. Thermostability of triazine hydrolase variants measured using kinetic activity (Tm1) and also structurally using a thermal shift fluorescence assay (Tm2) (mean ± standard error) Variant

Tm(ºC) Tm1

Tm2


52.9±0.7

62.1±0.6

Glu214/Thr214/Tyr215

53.7±1.6

62.4±0.1


55.1±1.7

63.8±0.5

Gln241/Pro214/Tyr215

61.7±0.6

66.2±0.8

Glu241/Thr214/His215

55.8±0.7

63.6±0.5


61.8±0.6

64.9±0.8


60.5±0.8

65.6±0.6


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Biochemistry

Figure 1. (A) The displacement of chlorine from the triazine ring of the herbicide atrazine is catalysed by triazine hydrolase (TrzN), with Glu241 enhancing the reactivity of the substrates via ring protonation. (B) TrzN catalyses nucleophilic ring substitution reactions on the striazine based herbicides: atrazine and ametryn Figure 2. The influence of microenvironment on the pKa of Glu241 in TrzN was determined using the pH dependent activity (kcat) of triazine hydrolase (TrzN) variants on atrazine, from which dissociation constants were generated. (A) Glu241/Pro214/Tyr215 TrzN (black, pKe1 5.4, pKe2 8.2) and Glu241/Thr214/Tyr215 TrzN (grey, pKe1 5.4, pKe2 8.0). (B) Glu241/Thr214/His215 TrzN (grey, pKe1 5.4, pKe2 7.6). (C) Gln241/Pro214/His215 TrzN (grey, pKe1 6.4, pKe2 6.8). (D) Glu241/Pro214/His215 TrzN (grey, pKe1 5.4, pKe2 7.6). Points are an average of three replicates, with error bars representing the standard error of three independent replicates. Figure 3. Stepwise modification of the Gln241/Thr214/His215 TrzN scaffold to the Glu241/Pro214/Tyr215 TrzN scaffold significantly impacts catalysis. Catalysis of atrazine (A) significantly increases in TrzN variants with Glu241 surrounded by the hydrophobic microenvironment created by Tyr215 alone and with Pro214/Tyr215 combined. Hydrolysis of ametryn (B) is seen to significantly increase upon incorporation of Glu241 alone and is further improved via the incorporation of Tyr215 and Pro214/Tyr215. Figure 4 Variants that exhibit faster ametryn turnover are observed to have lower thermostability. The addition of Glu241 significantly decreases thermostability and the incorporation of hydrophobic residues decreases thermostability further, as measured using catalytic activity (A) and a thermal shift fluorescence assay (B). (C) Glu241- containing TrzN has reduced soluble expression compared to Gln241-containing TrzN. Points are an average


Biochemistry

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of three replicates, with error bars representing the standard error of three independent replicates Figure 5. Thermostability of triazine hydrolase variants tested using thermostability assays with atrazine. Variants with Glu241 and any combination of Pro214/Thr214 and Tyr215/His215 were significantly less stable than variants with Gln241 and any combination of Pro214/Thr214 and Tyr215/His215. Thermal denaturation curves for each replicate are shown. Figure 6. Differences in the internal charge between TrzN variants did not cause noticeable compensatory structural changes in the crystal structures. The only substantial difference between the solved structures is the presence of fewer water molecules in the more hydrophobic active site of Glu241/Pro214/Tyr215 TrzN (A, PDB:4LH8)37 compared to the more hydrophilic Gln241/Thr214/His215 TrzN (B, 5HMF). Placement of ametryn in a previously determined binding mode,31 with displacement of the water molecule coordinated Glu241 in the apo-form, results in a calculated pKa of 8.2 for Glu241 (C, 4LH8). Retention of the water molecule hydrogen bonded to Glu241 in the more hydrophilic active site of Glu241/His214/Thr215 TrzN results in a calculated pKa of 7.7 for Glu241 (D, 5HME).31 Electron density (2mFo-DFc) is shown in grey, contoured at 1.0 σ.


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Biochemistry

Figure 1. (A) The displacement of chlorine from the triazine ring of the herbicide atrazine is catalysed by triazine hydrolase (TrzN), with Glu241 enhancing the reactivity of the substrates via ring protonation. (B) TrzN catalyses nucleophilic ring substitution reactions on the striazine based herbicides: atrazine and ametryn.


Biochemistry

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Figure 2. The influence of microenvironment on the pKa of Glu241 in TrzN was determined using the pH dependent activity (kcat) of triazine hydrolase (TrzN) variants on atrazine, from which dissociation constants were generated. (A) Glu241/Pro214/Tyr215 TrzN (black, pKe1 5.4, pKe2 8.2) and Glu241/Thr214/Tyr215 TrzN (grey, pKe1 5.4, pKe2 8.0). (B) Glu241/Thr214/His215 TrzN (grey, pKe1 5.4, pKe2 7.6). (C) Gln241/Pro214/His215 TrzN (grey, pKe1 6.4, pKe2 6.8). (D) Glu241/Pro214/His215 TrzN (grey, pKe1 5.4, pKe2 7.6). Points are an average of three replicates, with error bars representing the standard error of three independent replicates.


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Biochemistry

Figure 3. Stepwise modification of the Gln241/Thr214/His215 TrzN scaffold to the Glu241/Pro214/Tyr215 TrzN scaffold significantly impacts catalysis. Catalysis of atrazine (A) significantly increases in TrzN variants with Glu241 surrounded by the hydrophobic microenvironment created by Tyr215 alone and with Pro214/Tyr215 combined. Hydrolysis of ametryn (B) is seen to significantly increase upon incorporation of Glu241 alone and is further improved via the incorporation of Tyr215 and Pro214/Tyr215.


Biochemistry

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Figure 4 Variants that exhibit faster ametryn turnover are observed to have lower thermostability. The addition of Glu241 significantly decreases thermostability and the incorporation of hydrophobic residues decreases thermostability further, as measured using catalytic activity (A) and a thermal shift fluorescence assay (B). (C) Glu241- containing TrzN has reduced soluble expression compared to Gln241-containing TrzN. Points are an average of three replicates, with error bars representing the standard error of three independent replicates


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Biochemistry

Figure 5. Thermostability of triazine hydrolase variants tested using thermostability assays with atrazine. Variants with Glu241 and any combination of Pro214/Thr214 and Tyr215/His215 were significantly less stable than variants with Gln241 and any combination of Pro214/Thr214 and Tyr215/His215. Thermal denaturation curves for each replicate are shown.


Biochemistry

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Figure 6. Differences in the internal charge between TrzN variants did not cause noticeable compensatory structural changes in the crystal structures. The only substantial difference between the solved structures is the presence of fewer water molecules in the more hydrophobic active site of Glu241/Pro214/Tyr215 TrzN (A, PDB:4LH8)37 compared to the more hydrophilic Glu241/Thr214/His215 TrzN (B, 5HME). Placement of ametryn in a previously determined binding mode,31 with displacement of the water molecule coordinated Glu241 in the apo-form, results in a calculated pKa of 8.2 for Glu241 (C, 4LH8). Retention of the water molecule hydrogen bonded to Glu241 in the more hydrophilic active site of Glu241/His214/Thr215 TrzN results in a calculated pKa of 7.7 for Glu241 (D, 5HME).31 Electron density (2mFo-DFc) is shown in grey, contoured at 1.0 σ.


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Biochemistry

“For Table of Contents Use Only”

Active site desolvation and thermostability tradeoffs in the evolution of catalytically diverse triazine hydrolases Elena Sugrue,† Paul D Carr,† Colin Scott,‡ Colin J Jackson*†


Biochemistry

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Biochemistry

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Active Site Desolvation and Thermostability Trade-Offs in the Evolution

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