Exploring the challenges of computational design by rebuilding the of a dehalogenase

Garima Jindala,1, Katerina Slanskab, Veselin Koleva, Jiri Damborskyb,c,d, Zbynek Prokopb,c,d, and Arieh Warshela,2

aDepartment of Chemistry, University of Southern California, Los Angeles, CA 90089; bLoschmidt Laboratories, Department of Experimental Biology, Masaryk University, 625 00 Brno, Czech Republic; cResearch Centre for Toxic Compounds in the Environment, Masaryk University, 625 00 Brno, Czech Republic; and dInternational Clinical Research Center, St. Anne’s University Hospital, 656 91 Brno, Czech Republic

Contributed by Arieh Warshel, April 4, 2018 (sent for review March 22, 2018; reviewed by Shina Caroline Lynn Kamerlin and Vicent Moliner)

Rational enzyme design presents a major challenge that has not close to that of the wild-type (WT) enzyme. Thus, we should be able been overcome by computational approaches. One of the key to assess the effectiveness of some crucial steps in a given compu- challenges is the difficulty in assessing the magnitude of the tational design approach. As a model system, we take the enzyme maximum possible catalytic activity. In an attempt to overcome haloalkane dehalogenase DhlA, which catalyzes the conversion of this challenge, we introduce a strategy that takes an active toxic haloalkanes to alcohols (11). DhlA from bacterium Xantho- bacter autotrophicus enzyme (assuming that its activity is close to the maximum GJ10 converts 1,2-dichloroethane (DCE) to chloroethanol via a series of steps that involve an S 2 reaction, possible activity), design mutations that reduce the catalytic − N activity, and then try to restore that by mutating other hydrolysis, isomerization, and a Cl ion departure (12). The SN2 residues. Here we take as a test case the enzyme haloalkane step involves the attack of the nucleophilic D124 on DCE, and the dehalogenase (DhlA), with a 1,2-dichloroethane . We formation of an intermediate, which subsequently undergoes hydrolysis and leads to the formation of an alcohol (Fig. 1). The start by demonstrating our ability to reproduce the results of − final step involves the departure of Cl ion from the active site, single mutations. Next, we design mutations that reduce the whichisbelievedtobeprecededbyanisomerization. enzyme activity and finally design double mutations that are The , which is commonly found in α/β-, aimed at restoring the activity. Using the computational predic- is composed of D124, H289, and D260. Other residues, that have tions as a guide, we conduct an experimental study that confirms been proven important using mutation studies, include W125, our prediction in one case and leads to inconclusive results in W175, F172, and V226 as shown in Fig. 2A. It has been found, BIOPHYSICS AND another case with 1,2-dichloroethane as substrate. Interestingly, using both experimental and computational studies, that W125 COMPUTATIONAL BIOLOGY − one of our predicted double mutants catalyzes dehalogenation of and W175 help in stabilizing the departing Cl ion by forming H 1,2-dibromoethane more efficiently than the wild-type enzyme. bonds, while F172 has been shown to interact with the DCE substrate. V226 does not interact with the substrate directly, al- enzyme design | EVB | transient kinetics | dehalogenase | nucleophilic though it forms van der Waals interactions with W125 and F172. substitution The mutants V226A (13) and F172W (14) are interesting, as they lead to a decrease in the rate of the S 2 step but to an increase in − N he need to progress with enzyme design is a major practical the rate of the Cl ion release. The hydrolysis step becomes the Tand fundamental challenge (1, 2). Unfortunately, despite rate-limiting step in the V226A, F172W, and W175Y mutants. interesting progress (3–7), the main advances have been made by Apart from these, other residues that line the active site cavity directed evolution and not by computational design (7, 8). This include E56, F128, F164, F222, L262, and L263. The backbone point can be concluded by examining the references mentioned in NH group of W125 and E56 form H bonds with the oxygen of the ref. 7 or, for example, in ref. 9. Furthermore, at present, there are major difficulties in using directed evolution to obtain the catalytic Significance effects that approach those of naturally evolved . Apparently, reasonable attempts to use charge−charge inter- The goal of rational computer-aided enzyme design is ham- actions with a high dielectric, to predict the effect of distanced pered by the lack of knowledge of the maximum possible rate ionized groups on the reactivity of the substrate (e.g., ref. 1), are enhancement. We address this problem by considering the not likely to lead to a large catalytic effect. Thus, it seems that enzyme DhlA, which is naturally adapted for the degradation one must focus on groups at a close and medium distance to the of dihalogenated ethanes. Using empirical valence bond cal- substrate (although directed evolution appears to also use distant culations, we determine the effect of finding mutations that groups). In the case of groups in proximity to the substrate, it reduce the catalysis and then introducing mutations that re- seems that approximations such as the linear response approxi- store catalysis. One of our predicted cycles is confirmed ex- mation (LRA) are not likely to give sufficiently reliable results, perimentally, while the other attempt remains inconclusive. and one should probably move to the more expensive empirical We believe that the proposed strategy provides a very pow- valence bond (EVB) calculations. However, even with the EVB erful way of validating and refining approaches for computa- method, it is not clear what it takes to make a reliable prediction. For example, we recently encountered (10) a major stumbling tional enzyme design. block in the study of the directed evolution of Kemp eliminases. Author contributions: G.J., J.D., Z.P., and A.W. designed research; G.J., K.S., V.K., and A.W. We found that the ability to evaluate the catalytic power of each performed research; G.J., K.S., J.D., Z.P., and A.W. analyzed data; and G.J., J.D., Z.P., and step in the evolution process is not sufficient for constructing a A.W. wrote the paper. good catalyst. We have found that, apart from the knowledge of Reviewers: S.C.L.K., Uppsala University; and V.M., Universitat Jaume I. the key catalytic residues in the active site, we also need to figure out what are the other residues that will lead to the correct Conflict of interest statement: G.J. is an employee of Syngene International Ltd. preorganization of the catalytic residues. Published under the PNAS license. In view of the difficulties of obtaining reliable prediction of the 1Present address: Medicinal Chemistry Department, Biocon Bristol-Myers Squibb R&D sequence needed for optimal catalysis, we explore here a part of Center (BBRC), Syngene International Ltd, Bangalore 560100, India. the problem. That is, we try here to computationally mutate 2To whom correspondence should be addressed. Email: [email protected]. residues that are known to contribute to catalysis and then at- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tempt to find mutations that restore the activity. Such a strategy 1073/pnas.1804979115/-/DCSupplemental. is useful since we know the maximum possible catalysis: probably Published online December 26, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1804979115 PNAS | January 8, 2019 | vol. 116 | no. 2 | 389–394 Downloaded by guest on September 26, 2021 Fig. 1. The SN2 step and the hydrolysis steps in the haloalkane dehaloge- − nase DhlA catalyzed conversion of DCE to chloroethanol and a Cl ion.

carboxylate of D124. These two amide groups form an , which is similar to that in other hydrolases. The current design study used the X-ray structure of DhlA [Protein Data Bank (PDB) ID code 2DHC (12)] as a starting point for the EVB simulations. We started by reproducing the observed effects of single mutations and then explored the effort needed for computer-aided restoration of the WT activity after computational mutations of key catalytic residues. This was fol- Fig. 3. Comparison of observed and calculated activation free energies lowed by experimental determination of the effects of the pre- (kilocalories per mole). The error in estimating the experimental rate con- dicted mutations on the individual catalytic steps, with focus on stants is around 10 to 20%, which would correspond to an error of ∼0.1 kcal/ the rate of the SN2 reaction step. Our computational catalytic mol in the free energy. The assigned error for the reference water reaction is restoration is confirmed in one test case, while another case discussed in the caption of Table 1. remains inconclusive.

Results and Discussion Calculated pKa using the H++ web server was again found to be Computational Studies. We started by using the EVB approach to around 7. Another interesting residue is F128, which is replaced by Ala calculate the activation free energy of the SN2 step in the WT enzyme and several known mutants. The excellent agreement in the LinB and DhaA enzymes (16, 17), and it has been pos- between the calculated and experimental values (Fig. 3 and tulated that the presence of this residue enables the catalysis of Table 1) encouraged us to try to predict mutants that would DCE, which is otherwise not a good substrate for the other two reduce the catalytic activity and then to look for other mutants enzymes (18). It presumably does so by rendering compactness that would enhance the catalytic activity of the enzyme. to the active site. Thus, it would be interesting to see the effect of Before addressing the above challenge, we explored the role of mutating phenylalanine to alanine in the current system as well. the E56 residue and its ionization state. This residue forms a part In the mutant F128A, the active site cavity might enlarge and can of the oxyanion hole, and its backbone NH forms an H bond with help in the catalysis of larger and bulkier haloalkanes. Addi- the D124 nucleophile, as shown in Fig. 2B. Thus, one might tionally, residues F164 and F222 were considered important in assume that the presence of E56 is crucial for the dehalogenase previous studies (19). We also found it interesting to explore the activity. The pKa of E56 was found to be 9.58, by using the mutations of A149 and A227 to charged residues (based on Protein Dipole Langevin Dipole method within its semi- calculating the electrostatic contribution to catalysis of mutants macroscopic LRA (15). Even the increase of the consistent of these residues). The results of the computationally tested protein dielectric from 4 to 6 kept the pKa above 7. This suggests mutants that were inspired by the above consideration are given that it should be protonated during the reaction. In fact, if E56 is in Table 2. Some of the studied mutants led to a significant re- assumed to be ionized, the activation free energy becomes ∼3 duction in catalysis and thus could serve as candidates for the kcal/mol higher than when it is unionized. The E56Q mutation second step of our study, i.e., experimental verification. led to only a 0.5 kcal/mol increase in the activation free energy In looking for a way to restore the activity of the W125F and relative to that of the WT (where E56 is in the unionized form). W175F/Y mutants (Table 1), we noted that the stabilization Thus, it is very likely that E56 is not ionized and offers an at- tractive target for our mutational study, which is explored below. Table 1. Calculated and observed activation free energies (in kilocalories per mole) for different mutants with DCE as substrate ‡ ‡ System ΔG 2,obs ΔG 2,cal

Water (cage) 23.6 25.4* † WT 15.3 14.5 W125F 17.6§ 16.7 F172Y 17.3§ 15.7 † F172W 16.8 17.3 W175F 18.3{ 17.9 W175Y 18.3§ 18.2 † V226A 16.0 14.0

*In contrast to the regular EVB procedure, we did not insist on calibrating the water reaction on the observed value, since we are more interested in the effects of mutations. The value for water (cage) is taken from ref. 28. † Based on k2; values for WT, F172W, and V226A are taken from refs. 14 Fig. 2. (A) The active site of DhlA with the substrate DCE and the residues and 13. § that have been proven to be important, using both experiments and com- Based on kcat; values for W125F, F172Y, and W175Y are taken from refs. 29, putations. (B) Residues lining the active site that have been studied in the 14, and 30, respectively. current study. The dashed lines show the important H-bonding interactions. {Based on specific activity of the W175Y mutant (30).

390 | www.pnas.org/cgi/doi/10.1073/pnas.1804979115 Jindal et al. Downloaded by guest on September 26, 2021 Table 2. Activation free energies (kilocalories per mole) All mutations were successfully introduced into the dhlA gene. calculated for different mutants with DCE as substrate The correct sequence was confirmed by sequencing of both ‡ strands. Variants E56Q, F164A, W175Y/E56N, and W125F/ System ΔG 2,cal V226Q were expressed in Escherichia coli BL21(DE3) and pu- WT (E56 ionized) 18.2 rified to homogeneity using the metalloaffinity chromatography. WT (E56 unionized) 14.5 Expression, purity, and size of purified proteins were checked by E56Q 15.0 SDS/PAGE. Purification yields of E56Q, F164A, and W175Y/ ∼ · −1 · −1 A149D 13.0 E56N mutants were 35 mg L to 60 mg L of culture. The W125/V226 double mutant was purified with a significantly A227D 15.4 · −1 F128A 14.4 lower yield, 5 mg L of culture, and SDS/PAGE showed that most of the protein remained in the insoluble phase after the F164A 12.9 cell lysis (SI Appendix, Fig. S2). Proper folding and secondary F222A 15.0 structure of the newly constructed variants were verified by far- W125F/V226N 15.9/21.9* UV CD spectroscopy (SI Appendix). Similar to DhlA WT, the W125F/V226Q 13.9 mutant enzymes exhibited CD spectra with one positive peak at W175Y/V226N 21.8 about 195 nm and two negative maxima at about 208 nm to W175Y/V226T 17.9 210 nm and at 221 nm, characteristic for alpha-helical content. W175Y/E56N 15.8 W175Y/E56N retains a very similar structure to the WT enzyme, W175Y/A149D 18.8 while, for the remaining variants, small changes in intensity and † 1BEE.pdb (W175Y mutant) 18.2 (18.3) shape of the spectra in comparison with the WT are visible. 1BEE.pdb+Y175W (conf1)§ 18.3 Observed variations reflect a partial loss of alpha-helical structure 1BEE.pdb+Y175W (conf2) 14.1 caused by introduced mutations, implying slight conformational changes of the enzymes. Information obtained using SDS/PAGE *EVB calculations starting from different initial geometries gave different and far-UV CD spectroscopy for the W125/V226 variant suggests results. The lower activation energy is obtained for cases when the NH2 − that the protein is not properly folded and has limited solubility. group of 226Q mutant forms an H bond with the departing Cl ion. † Therefore, this variant was not studied by the pre-steady-state The experimental activation free energy is given in parentheses. §A back-mutation of Y to W, where 1BEE.pdb serves as the starting point for kinetics. the simulation of the W175Y mutant. The rapid mixing of DhlA WT and its variants with DCE was

associated with kinetic quenching of fluorescence intensity of en- BIOPHYSICS AND dogenous (Fig. 6). The initial kinetic phase of fluores- − k COMPUTATIONAL BIOLOGY offered by the tryptophan residues to the departing Cl ion is lost cence traces was fitted to a single exponential to provide obs and amplitudes for each substrate concentration (Fig. 7). Fitting the in these mutants (resulting in an increase in the activation free 1 energy). We therefore decided to mutate an additional residue dependence of observed rate on DCE concentration by Eq. pro- − that can stabilize the Cl ion. Reasonable candidates were V226 vided estimates for rate constants (Table 3) of corresponding min- and E56 that were found to be at a suitable position to interact with imal reaction pathway for DhlA WT and W175Y/E56N (Scheme 1). − the Cl ion (Fig. 4). Thus, these residues were replaced with N/Q, The dependence of amplitude on the concentration of DCE was − used to calculate an estimate of apparent binding constant K for where the NH side chain can form H bonds with the Cl ion. s,app 2 E56Q (Eq. 2). By using K as a fixed parameter, the estimates for In deciding which mutations to explore, we propagate molecular s,app kinetic parameters were obtained for E56Q by fitting the de- dynamics (MD) trajectories with different mutational options. Our pendence of observed rate on DCE concentration by using Eq. 1. analysis of the trajectories of the W175Y/V226Q mutant reveals Kinetic fluorescence quenching has not been detected for that, although the NH2 group of the Q226 side chain interacts with − − F164A. This result indicates that the SN2 reaction became the the Cl ion, the existing H bond between W125 and the Cl ion is rate-limiting step for the conversion of DCE by this variant, and lost, thereby resulting in an increase in the activation free energy the reaction does not provide any observable accumulation of alkyl- (Fig. 4). In the W175Y/E56N mutant, the NH2 group of N56 enzyme intermediate. Since the first chemical step determines the forms H bonds with the OH group of Y175, and thus should be useful for lowering the activation free energy. It should also be noted that, in the enzymes LinB and DhaA, the role of W175 is played by asparagine residue. Thus, we explored the effect of the E56N mutation in DhlA, to see whether it can play the stabilizing role offered by W175. The EVB calculations of the double mu- tants, W125F/V226N and W125F/V226Q, were carried out using different starting structures. For the W125F/V226N mutant, a total of seven profiles starting from seven different trajectories were studied. Out of these seven simulations, four showed a de- crease in the activation free energy with respect to the W175Y mutant, whereas the others showed an increase in the activation free energy. Further analysis revealed that, for the cases where the − H bond between the mutated N226 and Cl ion is maintained, a decrease in activation free energy is obtained (15.9 kcal/mol). The W125F/V226Q mutant also showed a decrease in the activation free energy (13.9 kcal/mol) compared with the W125F mutant (16.7 kcal/mol) (Fig. 5).

Experimental Studies. Based on the above computational design, we have constructed and characterized experimentally four mu- tants: E56Q, F164A, W175Y/E56N, and W125F/V226Q. The first step involved the construction of these variants followed by CD spectroscopy, specific activity measurements, and transient Fig. 4. Attempt to restore the catalytic activity of the W175Y and W125F kinetic analysis. The specific activity was evaluated for both DCE mutants. The mutated residues are highlighted in blue. Distances are given and 1,2-dibromoethane (DBE). in angstroms.

Jindal et al. PNAS | January 8, 2019 | vol. 116 | no. 2 | 391 Downloaded by guest on September 26, 2021 Fig. 5. Calculated activation free energies (kilocalories per mole) for different mutants in the restoration cycle. The observed values are provided in pa- rentheses. Distances are given in angstroms.

steady-state turnover, the steady-state equilibrium constant Km to the values estimated for WT enzyme. This result suggests that closely indicates substrate binding equilibrium Ks.The5.2± 0.9 mM the binding affinity of DCE has not been changed for the F164A value for the equilibrium binding of the DCE to F164A is similar variant.

Fig. 6. Fluorescent traces recorded by multiple- turnover stopped-flow analysis upon rapid mixing of (A) 6.8 μM DhlA WT, (B)6.8μM E56Q, (C)6.2μM F164A, and (D)6.7μM W175Y/E56N with different DCE concentrations. Each trace shown is the average of 5 to 10 individual experiments.

392 | www.pnas.org/cgi/doi/10.1073/pnas.1804979115 Jindal et al. Downloaded by guest on September 26, 2021 Table 4. Calculated and observed activation free energies (kilocalories per mole) ‡ ‡ ‡ System ΔG 2,cal ΔG 2,obs, ΔG obs

DhlA WT 14.5 14.4* E56Q 15.0 15.9* F164A 12.9 19.4† W175Y/E56N 15.8 15.6* † W175Y 18.2 18.3 W125F/V226Q 13.9 n.a.§

*The observed value is taken from k . Experiments are done at 37 °C in Fig. 7. Dependence of the (A) observed rate and (B) amplitude on the 2 100 mM glycine buffer at pH 8.6. Please note that the EVB calculations are concentration of DCE for reactions with DhlA WT (●), E56Q (○), and W175Y/ done at the standard temperature of 300 K. E56N (☐). † The observed value is calculated from kcat of the whole reaction. For W175Y mutant, the observed value is taken from ref. 30. §No activity. The comparison between the computationally predicted and experimental activation free energies is shown in Table 4. While, for E56Q and W175Y/E56N, the results agree with the compu- Conclusions tations, F164A shows a variation. E56Q mutation shows that the This work explored the issue of enzyme design, focusing on one E56 residue is not very important, and its role can be played by of the key difficulties in validating the successes of the design other residues as well. For W175Y/E56N, which includes two process. That is, in trying to design an effective enzyme, it is not mutations, we were able to restore the catalytic activity. That is, clear what the limit of maximum possible improvement is. Thus, although, the activity is lower than that of the WT (15.6 kcal/mol we “modified” the challenge and address the problem of taking a for W175Y/E56N and 14.4 kcal/mol for the WT), it shows an known enzyme, reducing the catalytic residues, and trying to improvement over the W175Y mutant (18.3 kcal/mol). These restore catalysis. In this way, we know what the likely maximum results are encouraging and may provide a guide for the design of possible catalytic effect is, and have a relatively well-defined dehalogenases. The probable reason behind the low activity of problem. In fact, even if there is a possible better enzyme than BIOPHYSICS AND F164A might be a change in other steps of the reaction. In this the WT enzyme, reaching the WT activation free energy is a COMPUTATIONAL BIOLOGY respect, we note that our EVB calculations only reflect the en- well-defined challenge that can provide a powerful guide for the ergetics of the SN2 step and that there are no available values for successes of enzyme design strategies. the separate rate constants. F164A mutation might be affecting In the present case, we chose DhlA, noting several specific − k the hydrolysis or Cl release step, resulting in a low cat. For the challenges. The reaction catalyzed by DhlA is multistep, and W125F/V226Q mutant, the mutations might cause destabilization therefore a single mutation can change the identity of the rate- of the enzyme. In fact, Rossetta (20) estimated an 8 kcal/mol limiting step. Thus, while a new mutant might lower the free energy destabilization in this double mutant in comparison with the WT SI Appendix for the SN2 step, it might have a negative effect on other steps. For enzyme ( ). Although this result might be a significant instance, the V226A mutation (13) decreases the activation free overestimate, the fact that no activity is detected suggests that the − energy for the Cl release while simultaneously increasing the free active site region changes significantly. Interestingly, mutants energy for the SN2 step, thereby resulting in a very small increase in E56Q and W175Y/E56Q show stabilization compared with the −1 −1 SI the overall kcat (3.3 s forWTversus3.8s for V226A mutant). WT, whereas F164A and W125F/V226Q show destabilization ( The success reported in Fig. 3 indicates that our approach Appendix). Of course, we can try to use more reliable and time- allows one to determine the catalytic power of a given mutant consuming ways to assess the change in stability upon mutations whose structure is approximately known. However, as we found (e.g., free energy perturbation methods), but such studies are recently (10), the challenge is to design an optimal enzyme, outside of the scope of the present work. The specific activities of DhlA WT and its variants toward DCE where the active site groups are preorganized in the correct way and DBE were assayed at 37 °C (Table 5). For both substrates, by other residues. The agreement between the computationally W125F/V226Q does not show any activity, while E56Q shows an predicted activation energies and experiments for mutants E56Q improved activity with DCE. Interestingly, all mutants show en- and W175Y/E56N seems remarkable. Additionally, the higher hanced activity with DBE as the substrate. The difference in the specific activities with DBE as the substrate are noteworthy. effect of mutations on two closely related substrates is remarkable. Thus, it is encouraging to see that we obtained some success in For DBE, the kcat of the SN2 step, calculated using EVB simula- tions, remains largely unaffected in comparison with the WT. Thus, Table 5. Specific activities of studied mutants toward DCE the enhanced activity of the mutants might be due to change in the − and DBE hydrolysis or Cl ion release. The activities of E56Q and W175Y/ − − E56N toward DBE are significantly improved over the WT. Specific activity, μmol·min 1·mg 1

Enzyme DCE DBE Table 3. Estimates of kinetic constants for the conversion of DhlA WT 2.0 ± 0.2* 2.9 ± 0.1 DCE by DhlA WT and its mutants E56Q 2.5 ± 0.1 5.1 ± 0.3 Constants DhlA WT E56Q F164A W175Y/E56N F164A 0.022 ± 0.002 3.1 ± 0.1 W175Y/E56N 0.6 ± 0.2 4.2 ± 0.2 Ks,mM 3.7± 0.5 5.4 ± 1.2* n.d. 30 ± 12 −1 W125F/V226Q n.a. n.a. k2,s 201 ± 916± 9 n.d. 25 ± 10 −1 kx,s 23 ± 326± 4 n.d. 10 ± 2 Experiments were done at 37 °C in 100 mM glycine buffer at pH 8.6.

Km,mM 0.5± 0.1 1.2 ± 0.1 5.2 ± 0.9 1.3 ± 0.1 Concentrations of substrates were 12.4 mM and 11.3 mM for DCE and −1 DBE, respectively. Enzyme concentrations varied from 52 nM to 282 nM, kcat,s 2.2 ± 0.1 1.1 ± 0.1 0.05 ± 0.01 0.31 ± 0.01 depending on the catalytic activity; n.a., no activity detected.

*Ks,app calculated based on Eq. 2; n.d., not detected. *Values are given with SDs calculated from three independent runs.

Jindal et al. PNAS | January 8, 2019 | vol. 116 | no. 2 | 393 Downloaded by guest on September 26, 2021 active site restoration in the current study. Of course, this idea k2. ½S kobs = + kx [1] should be extended in the future to more than two mutations. ½S + KS

Methods ½S. A A = lim . [2] Computational Methods. The starting structure for the EVB calculations (21– KS,app + ½S 23) was taken from the X-ray crystal structure of DhlA [PDB ID 2DHC (12)]. In this structure, the substrate is bound to the protein, which circumvents the The kinetic Eq. 1 was derived for fast initial phase of the reaction from ki- docking approach. Additionally, previous computational studies involving netic pathway (Scheme 1) (27). dehalogenase have also considered this PDB structure for the simulations. Ks k2 k3 k4 The EVB calculations were carried out using the MOLARIS package with the E + S ⇌ ES → El → EP → E + P , ENZYMIX force field. The ESP (electrostatic potential) charges for the two diabatic states that represent the reactant and were calculated us- Scheme 1. ing the B3LYP level of theory at the 6-311+G** basis set using Gaussian − software (24). The EVB region consists of the acetate group of Asp124 and where S and P are a substrate and a product, respectively, ES is an enzyme DCE. The FEP/US (free energy perturbation/umbrella sampling) approach substrate complex, EI is a covalent alkyl-enzyme intermediate, EP is an was used to calculate the activation free energies. The center of the reactive enzyme-product complex, KS is an equilibrium dissociation constant for − species was taken as the center of the system which was immersed in a water enzyme substrate complex, and ki is an individual rate constant of ith step. Further details of the methods are provided in SI Appendix. Analytical pro- sphere of 18 Å and solvated using the surface constrained all-atom cedure has been used for data analysis: (i) The initial part of fluorescence model (25). The long-range effects were treated using the local reaction traces was fitted to a single exponential to provide observed rates and am- field method (26). The system was first relaxed for at least 100 ps using a step plitudes, (ii)Eq.1 describing the dependence of observed rate on substrate size of 1 fs, and then three different starting structures were generated. For concentration was derived from minimal reaction pathway (Scheme 1) in the FEP mapping, the simulation was divided into 51 frames, and each frame simplified form, where k represents a net rate constant including contribution was simulated for 30 ps with a step size of 1 fs, resulting in a total simulation x of k and k ,and(iii) fitting the kinetic data (the dependence of observed rate time of over 1.5 ns. The specific EVB parameters used in the current calcu- 3 4 on DCE concentration derived from exponential fitting of fluorescence traces lations are given in SI Appendix. in step i)byEq.1 provided estimates for the rate parameters. The fitting of the dependence of amplitude (A) on the concentration of DCE by hyperbolic Experimental Methods. model Eq. 2,whereA is the upper limit for the observed amplitude, was Stopped-flow analysis. lim Multiple-turnover stopped-flow fluorescence technique used to calculate an estimate of the apparent equilibrium constant for disso- was used to study the DCE conversion catalyzed by DhlA WT, E56Q, F164A, and ciation of enzyme−substrate complex Ks,app. W175Y/E56N. The experiments were performed by using an SFM-300 stopped- flow instrument combined with an MOS-200 spectrometer (BioLogic). Fluores- ACKNOWLEDGMENTS. We thank Dr. Tana Koudelakova (Masaryk Univer- cence emission from W residues was observed through a 320-nm cutoff filter sity) for collaboration on construction and purification of the mutant pro- upon excitation at 295 nm. All reactions were performed at 37 °C in 100 mM teins. G.J. and A.W. thank Mr. Dibyendu Mondal for helpful discussions. We glycine buffer and pH 8.6. The relaxations of active site Trp fluorescence upon also thank the University of Southern California’s High Performance Com- starting the reaction were recorded stepwise at different substrate concentra- puting for computer time. We are grateful for generous computing time ’ tions. from Extreme Science and Engineering Discovery Environment s Comet fa- cility at the San Diego Supercomputing Center. This work was supported by Data analysis. The fluorescence traces were fitted to a single or double ex- National Institutes of Health Grants GM-24492 and R35GM-122472, the ponential using KinTek Explorer (KinTek Corporation). The dependence of Grant Agency of the Czech Republic Grant GA16-07965S, and the Ministry observed rate and amplitude on DCE concentration was fitted by Eqs. 1 and 2, of Education, Youth, and Sports of the Czech Republic Grants LQ1605, respectively, by nonlinear regression using Origin 8.0 (OriginLab corporation). LO1214, LM2015051, LM2015047, and LM2015055.

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