An Activator–Blocker Pair Provides a Controllable On–Off Switch for a

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An Activator−Blocker Pair Provides a Controllable On−Off Switch for a Ketosteroid Isomerase Active Site Mutant Vandana Lamba,† Filip Yabukarski,† and Daniel Herschlag*,†,‡,§,∥ †

Department of Biochemistry, ‡Department of Chemistry, §Department of Chemical Engineering, and ∥Stanford ChEM-H, Stanford University, Stanford, California 94305, United States S Supporting Information *

ABSTRACT: Control of enzyme activity is fundamental to biology and represents a long-term goal in bioengineering and precision therapeutics. While several powerful molecular strategies have been developed, limitations remain in their generalizability and dynamic range. We demonstrate a control mechanism via separate small molecules that turn on the enzyme (activator) and turn off the activation (blocker). We show that a pocket created near the active site base of the enzyme ketosteriod isomerase (KSI) allows efficient and saturable base rescue when the enzyme’s natural general base is removed. Binding a small molecule with similar properties but lacking general-base capability in this pocket shuts off rescue. The ability of small molecules to directly participate in and directly block catalysis may afford a broad controllable dynamic range. This approach may be amenable to numerous enzymes and to engineering and screening approaches to identify activators and blockers with strong, specific binding for engineering and therapeutic applications.

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INTRODUCTION As our understanding of the mechanisms that enzymes use for catalysis and the means used by nature to control enzymatic activity has matured over the past decades, there has been growing interest in engineering enzymes for industrial and medical applications.1−3 Indeed, there has been enormous progress in these areas, including the de novo design of enzymes and the ability to introduce allosteric control.4−7 Nevertheless, engineered enzymes have low activity, relative to naturally occurring ones,4,5 and engineered allosteric control typically involves fusing a functional protein domain with a domain known to respond to a particular ligand.7,8 While these fusions often result in allostery, the on−off switch is a secondary response that potentially limits the dynamic range, and highly predictive engineering algorithms may be difficult to effectuate. Similarly, engineered light-induced control of protein activity using azobenzene moieties and LOV domain photoswitches has been successful but faces challenges in efficiently coupling the photoswitch to the target protein.9−11 Chemical rescue provides another path for enzyme engineering. There are many examples of enzymes that, upon removal of a catalytic residue by site-directed mutagenesis, can be “rescued” by addition of a small molecule containing the ablated functional group.12−19 In some of these instances, saturated binding could be observed, indicating that the region surrounding the ablated side chain remained “imprinted” to recognize similar molecules.17−19 Similarly, proteins with ablated interior side chains have been stabilized by binding of small molecules in the resultant cavities.20−23 Karanicolas and co-workers have extended this approach by removing a side chain near the active site, such that the active site is disrupted © 2017 American Chemical Society

and activity is rescued by addition of a small molecule mimic of the ablated side chain to restore the catalytic conformation of the active site.24,25 The creation of a new binding site within an existing protein scaffold, while more highly constrained, may be a simpler task than efficient de novo enzyme design. From such a starting point; designing, screening, or selecting for enhanced binding affinity and specificity may be more tractable than de novo design, may allow for simple and systematic tests of predictions to further the development of computational and design algorithms, and may provide a broader response window than approaches involving fusion of additional subunits.26,27 One of the goals of bioengineering and systems biology is to create signal transducers that can integrate information from multiple inputs, and there are similar goals for targeted and precision therapeutic strategies.28−31 In this context, chemical rescue can be considered as one-dimensional rheostat where the concentration of the rescuing molecule can be varied to tune activity up and down. A useful elaboration from this onedimensional rheostat would be the ability to turn on the activity with one input molecule and turn off the activity via a distinct input. A particularly elegant example of such an on/off switch is an aptamer against coagulation Factor IXa that inhibits thrombin generation and blood clotting but has its inhibition rapidly reversed by addition of an oligonucleotide complementary to the aptamer (see ref 32 and references therein). Such drug-antidote control may be particularly beneficial during a surgical procedure when clotting needs to be inhibited during Received: April 13, 2017 Published: July 18, 2017 11089

DOI: 10.1021/jacs.7b03547 J. Am. Chem. Soc. 2017, 139, 11089−11095

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Figure 1. KSI reaction mechanism and rate constants. (A) KSI utilizes general base D38 to carry out a double bond isomerization through formation of a dienolate intermediate that is stabilized by oxyanion hole hydrogen-bond donors Y14 and (protonated) D99. Residues are numbered for the enzyme from Comamonas testosteroni (tKSI), and experiments were also carried out with KSI from Pseudomonas putida (pKSI). (B) The second- and first-order rescue rate constants obtained in general base mutants with exogenous base and saturating steroid substrate (S).

cavity at the site of the ablated F54 side chain (Figure 2D), whereas the RMSD of 0.35 Å between the Cα coordinates of this structural model and that for an enzyme with F54 intact provided strong evidence for the lack of any major structural changes beyond the cavity (Figure S4C,D). These results were generalized to another KSI variant, that from Pseudomonas putida (pKSI; Figures S5 and S6 and Table S3), where analogous enhanced rescue and saturation binding of longer-chain carboxylates was observed upon mutation of F56, the residue homologous to F54 in tKSI. Finally, the inhibition of base rescue by t-butanol (t-BuOH) specifically for the F54A tKSI and F56A pKSI general base variants described below provides additional strong evidence for the creation of a saturation binding site. Having established the creation of a binding pocket in F54A tKSI and F56A pKSI general base mutants for larger alkyl chain exogenous bases, we next evaluated the catalytic efficiency of these rescued reactions. We compared the reactions of WT tKSI and pKSI (kcat) with the enzymes with the general base mutation (D38G or D40G) and the F54A or F56A mutation, respectively, each with bound exogenous base (kcat,EB, defined in Figure 1B; in all cases with saturating steroid substrate). The rescued reaction rates were within about an order of magnitude of the WT reactions (Figure 3).34,36 For comparison, we show the rate for general base mutants, D38E tKSI and D40E pKSI. The D38E tKSI mutation was previously shown to compromise positioning of the carboxylate general base,37 so the observation that the rescued reactions are 1−2 orders of magnitude faster than reactions of the D38E tKSI and D40E pKSI mutants suggests that the noncovalently bound rescuing general base is better positioned for catalysis than a carboxylate that is covalently tethered at the correct residue but has one methylene added relative to the wild-type carboxylate. Measurements of effective molarities (EM) have been valuable in many mechanistic studies.16,19,38−41 Here, the rescue by TMA corresponds to an EM of ∼1 M for tKSI and pKSI, based on comparisons of reactions of these general base and F54/F56-ablated mutant enzymes with exogenous base to the reactions of WT tKSI and pKSI, and the same values are obtained, within error, whether reactions with saturating or subsaturating steroid substrate (S) are compared (Table S4). We previously observed much higher EMs of ∼103−104 M for general base rescue with active site residues other than F54/ F56 mutated.33 The lower EMs observed with TMA and F54 or F56 ablated result from faster rescue reactions with exogenous bases that can bind. This conclusion is strongly supported by

treatment but rapidly restored at the end of the procedure to limit bleeding and reduce associated morbidity.32 Here, we have built upon the approach of chemical rescue, creating a pocket directly adjacent to the active site that can bind a small-molecule general base to provide rescue and also bind a noncatalytic competitive inhibitor. Thus, we have created a primitive controllable enzyme with distinct on/off channels. Future elaborations with stronger and more specific ligands may provide a practical and powerful approach to bioengineering and may yield therapeutic applications.

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RESULTS Bacterial ketosteroid isomerase (KSI) catalyzes double bond isomerization in β,γ-unsaturated keto system to give a more stable α,β-conjugated unsaturated product (Figure 1A). In the first step, Asp38 acts as a general base to abstract a proton α to a carbonyl group and generates a dienolate intermediate. Subsequently, the protonated Asp38 acts as a general acid and reacts with the intermediate to generate the conjugated product. We have previously shown that the KSI reaction can be efficiently rescued in general base-ablated (e.g., tKSI D38G) mutants by the addition of exogenous carboxylate bases.33 With an additional mutation of F54, a residue that helps position the general base by anion−aromatic interactions,34 to alanine; significantly larger second-order rate constants (kbase, defined in Figure 1B) were observed with larger alkyl chain bases, whereas the rate constant for formate rescue remained within 2-fold for the two mutants (Figure 2A and Table S1). The simplest model for this rate enhancement is the creation of a binding pocket for the larger alkyl chain of rescuing bases upon removal of the F54 aromatic side chain, and the following observations test and provide strong support for this model. First, extension of rescue to higher carboxylate base concentrations shows saturation behavior for trimethyl acetate (TMA) and valerate but only in the mutant with the F54 side chain ablation (Figure 2B and Table S1). Similar enhanced and saturable rescue (kcat,EB, defined in Figure 1B) was also observed for other longer-chain carboxylate compounds, again only with the F54A mutation (Figure S1 and Table S1). In addition to the above demonstration that the F54A mutation is sufficient for the enhanced rescue by longer-chain carboxylates, we also showed that the F54A mutation was necessary for this enhanced rescue in several mutant backgrounds (Figure 2C; Figures S1 and S2 and Table S1). In addition, the 1.8 Å crystal structure of the D38G/F54A mutant (Table S2) revealed a 11090

DOI: 10.1021/jacs.7b03547 J. Am. Chem. Soc. 2017, 139, 11089−11095

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Figure 2. Exogenous base rescue in a KSI binding pocket. (A) The second-order rescue rate constants for trimethyl acetate (TMA; ●) and formate (■) in general base mutant D38G with (blue) and without Phe54 present (red, F54A) (Figure 1B). Data reproduced from ref 33. (B) Saturation binding is observed for exogenous bases valerate (○) and trimethyl acetate (●) with D38G/F54A tKSI. (C) The second-order rescue rate constants for a series of carboxylate exogenous bases with tKSI general base mutants D38G, D38G/P39G/V40G, and D38G/A114 each with F54 (blue) or with F54A (red). Arrows represent upper limits. (Rate constants were obtained from least-squares fit of the data to a linear equation and are given in Figure S2 and Table S1; data for mutants with F54 present and for D38G/F54A tKSI are from ref 33.) (D) Left: Crystal structure of D38N tKSI (surface representation in green) with bound equilenin (sticks shown in orange and blue represent different orientations of equilenin as observed in two crystallographically independent protein chains in the asymmetric unit of D38N crystal) (PDB 1QJG).35 Right: Crystal structure of D38G/F54A (surface representation in green) (PDB 5UGI; Table S2) with bound equilenin (sticks shown in purple for two alternative orientations of equilenin modeled in the same molecule); the position of F54 in D38N tKSI is shown in red (PDB 1QJG) after alignment with the D38G/F54A structure and is used to illustrate the space opened upon its removal in the mutant with F54A. The side chains lining the D38G/F54A cavity are shown explicitly in Figure S3. D38N tKSI was chosen for this comparison because it contains a bound equilenin transition state analog. Additional comparisons of these structures are provided in Figure S4.

binding in the pocket created by F54/F56 ablation, to within about an order of magnitude of WT KSI (Figure 3), was also unexpected, especially given the established role of the ablated phenylalanine in positioning of the native general base.34 The F54/F56 ablation rescue results bolster the perspective that the high EM of WT KSI may arise from factors in addition to simple positioning, as providing highly precise positioning through a fortuitous binding pocket is unlikely. It is possible that juxtaposing a general base and carbon acid removes a solvation barrier for proton abstraction from a carbon acid and allows a substantial rate increase in the absence of highly precise positioning.33,43,44 Additional studies will be required to further elucidate the underlying mechanisms.

the observed saturation behavior (Figures S1 and S5) but is in general difficult to diagnose, underscoring the importance of making multiple comparisons, as we did in the previous study via a series of KSI mutants and a series of carboxylates of varying steric properties to control for binding or steric interactions with the rescuing base that can help or hinder the rescue reaction.33 The prior high EM of 103−104 M for comparing the WT positioned general base to reaction with a rescuing base lacking favorable or unfavorable active site interactions was unexpected, given the need to abstract and donate protons at different positions in differ reaction steps and with different steroid substrates.42 The efficient rescue observed from fortuitous 11091

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similarity of the observed and predicted rate constants for each step (Table S5) demonstrates our ability to exert quantitative control over this system.

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DISCUSSION We have built on the many observations of chemical rescue of enzymes with catalytic groups removed12−19 and observations of cavities and binding site formation in proteins with truncated side chains20−23 to create an enzyme that binds to and responds to a small molecule catalytic activator and also binds to a second small molecule that lacks the catalytic functional group and thus blocks activation. This approach adds to the array of approaches to introduce control into enzymes6,7 and may have several advantages over current approaches, as briefly outlined below. Small molecule activators that directly participate in catalysis may be able to provide a larger dynamic activation range than most indirect modulators. In KSI, the addition of saturating TMA provides a 1300-fold rate increase (Table S7). More generally, mutations of active site residues directly involved in chemical catalysis are most deleterious12,45,46 and thus can provide low background activity and the potential for substantial activation, that is, a large dynamic range. There has been much excitement about the prospect of engineering a variety of logical functions into cellular systems.28−30 Nevertheless, putting such ideas into practice is challenging, so generating new molecular controls may be an important step in developing systems that can be robustly controlled on demand. The application of distinct small molecules that act as activators and blockers allows greater control relative to using an activator alone, as the activator and blocker concentrations can be independently varied (e.g., Figure 4D). It may also be possible to expand the approach of Karanicolas and co-workers24,25 of removing a side chain near the active site, such that one small molecule might bind and restore the catalytic conformation while another molecule binds but does not restore the catalytic conformation and thereby blocks the activator. The Factor IXa aptamer example described in the Introduction provides a powerful therapeutic example of the benefits from higher dimensionality that allows distinct control for turning on and turning off a process, and many more therapeutic applications that require temporally or spatially targeted activation and inhibition can be envisioned.47−49 Similarly, engineering of organisms or cell-free systems to produce drugs and other beneficial products can require careful and complex metabolic engineering and control.50 A final advantage of the approach demonstrated herein is the potential ease of improving binding affinity and specificity. Our activator and inhibitor bind with high millimolar affinities, and multiple hydrophobic compounds can bind to the site we uncovered, so that this system would not be amenable for specific cellular applications. However, enhanced sensitivity of these on/off switches may be achievable via empirical screens and selections, which can help optimize binding and catalysis, analogous to the value of these approaches in improving designed enzymes and analogous to standard structure−activity approaches used in drug development.5,51,52 We anticipate that it may be easier to apply design to optimize binding and specificity for rescuing and blocking compounds than it has been to design molecular switches into enzyme de novo. As noted above, there are numerous examples of chemical rescue of enzymatic reactions. Nevertheless, it is not clear what

Figure 3. Catalytic efficiency of trimethyl acetate (TMA) rescued reactions. First-order rescue rate constants (kcat,EB) with saturating TMA in tKSI and pKSI general base mutants that contain the F54A and F56A binding pockets, respectively, as compared to WT and general base D → E mutant tKSI and pKSI. Values for WT tKSI and D38E are from ref 37, WT pKSI values are from ref 36, and the other values were obtained herein (Tables S1 and S3).

Given that we had created a binding pocket in place of a phenylalanine residue and that no saturation behavior was observed for formate, even at supra-molar concentrations (data not shown), we hypothesized that there might be little or no binding interactions with the carboxylate portion of the rescuing bases. Thus, the F54A and F56A KSI general base mutants might be able to bind other water-soluble but hydrophobic small molecules. We looked for a molecule with properties similar to those of the activator molecule but without its general base functional group. We chose t-butanol to match the carbon skeleton of TMA, and thereby retain binding interactions, while replacing the carboxylate group that functions as the general base with a hydroxyl group (Figure 4A). We reasoned that the hydroxyl group would maintain solubility while eliminating effective proton abstraction and thereby potentially provide an off-switch. As predicted, t-butanol inhibited the TMA-rescued reaction, giving an inhibition constant of 63 ± 4 mM in the presence of subsaturating TMA (Figure 4B), similar to KM,base of 68 ± 10 mM for TMA. This similarity is consistent with the model that these molecules exploit the same or similar binding interactions. With rescuing TMA concentrations increased to 75 and 250 mM (above its KM value), higher concentrations of t-butanol were required for inhibition, as predicted for competitive binding (Figure 4C), and the observed inhibition constants were consistent with simple competitive inhibition (Table S6). As expected for competitive binding to the F54A binding site, t-butanol had a negligible effect on the WT tKSI reaction or water reaction with F54 or F54A present (Figure S8). Analogous activation and blocking results were obtained for pKSI and its F56A general base mutant (Figure S9). Given the stimulation of activity induced by TMA and the inhibition thereof observed with t-butanol, we reasoned that we should be able to turn D38G/F54A tKSI on and off and back on by successive additions of the activator and inhibitor small molecules. Figure 4D shows an example of one such experiment, where enzyme was turned on and off over multiple rounds by successive additions of TMA and t-BuOH. The 11092

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Figure 4. On and off switch from trimethyl acetate base rescue and t-butanol (t-BuOH) inhibition of base rescue. (A) Structures of TMA and tBuOH used for base rescue and inhibition of base rescue, respectively. (B) t-BuOH inhibition with subsaturating TMA (2 mM) in D38G/F54A tKSI. A Ki of 63 ± 4 mM was obtained from least-squares fit of the data. (C) At 75 and 250 mM TMA, Ki values of 140 ± 20 and 270 ± 30 mM were obtained for t-BuOH, respectively, corresponding to t-BuOH affinities of 67 and 58 mM and consistent with the value of 63 mM obtained with subsaturating TMA in part (B). Values were obtained from a competitive binding model, using the observed KM,base value for TMA of 68 mM (Table S1). (D) Turning the activity on and off in D38G/F54A tKSI by successive additions of activator (TMA) and inhibitor (t-BuOH). The starting absorbance of each step was normalized with respect to the end absorbance of the previous step to facilitate the comparisons of rates for the successive rounds. The raw data are presented in Figure S7, and Table S5 compares the observed rates to those predicted on the basis of kinetic parameters obtained for catalysis and inhibition.

functional needs, the two-component activator/blocker control introduced in this work provides a valuable addition to the current arsenal, and we look forward to further developments and to applications of this and other methods to control and engineer enzyme and metabolic function.

fraction of enzymatic systems will be amenable to rescue, and saturation binding has not been observed in most instances. For example, while we found enhanced rescue and saturation binding upon removal of the F54 side chain in tKSI (and homologous F56 side chain in pKSI), removal of F116 or W120 that sit on the other side of the aspartate general base in tKSI and pKSI, respectively, does not lead to such enhanced rescue by longer-chain carboxylate compounds.33 Matthews and co-workers observed in their extensive and systematic studies of T4 lysozyme that side chain truncations led to creation of binding sites of varying size, dependent on the degree of rearrangement of neighboring residues.53 Thus, it would be of value to determine what structural features promote cavity stability versus rearrangement. This information may be valuable toward the ultimate goal of predicting binding pockets and engineering enhanced affinity and specificity for small molecule activator and inhibitor of choice, as well as a stepping stone toward highly effective engineering of full protein systems. While it is likely that no single approach to developing multidimensional, controllable enzymes will serve all future

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EXPERIMENTAL SECTION

Materials. All materials were of highest purity available. 5(10)Estrene-3,17-dione (5(10)-EST)) was purchased from Steraloids. Sodium formate and sodium acetate were purchased from J.T. Baker. Sodium propionate was purchased from Alfa Aesar. Sodium butyrate, valeric acid, and trimethyl acetic acid were purchased from SigmaAldrich. Expression and Purification of KSI Mutants. Proteins were expressed and purified as previously described.33 Briefly, BL21 cells transformed with plasmid carrying the desired KSI construct were grown at 37 °C to an OD of 0.5−0.6 in Luria Broth media containing 50 μg/mL carbenicillin, and then protein expression was induced with 0.5 mM isopropyl-β-D-1-thiogalactopyranoside. After induction, cultures were grown for another 6−10 h at 37 °C. Cells were harvested by centrifugation at 4000g for 20 min at 4 °C and lysed using an emulsiflex. Lysed cells were centrifuged at 48 000g for 20 min 11093

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Journal of the American Chemical Society at 4 °C. Mutants were purified either from soluble fraction or the inclusion body pellets after refolding, first using an affinity column (deoxycholate resin) followed by a size exclusion chromatography column (SEC) Superose 12, as previously described.36 Freshly folded and purified proteins were used for measurements. Prior to the purification of each construct; affinity column, FPLC loops, and SEC column were washed with 40 mM sodium phosphate (NaPi), 6 M guanidinium, pH 7.2 buffer, and then equilibrated with 40 mM NaPi, 1 mM sodium EDTA, pH 7.2 buffer. Purity of the mutants was >97% determined by SDS gel electrophoresis based on Coomassie blue staining. KSI Mutants Nonbase Rescued Reactions. Michaelis−Menten parameters for nonbase rescued reactions were obtained by monitoring the production formation (extinction coefficient 14 800 M−1 cm−1) for 5(10)-EST as described earlier.33 Reactions were measured in a continuous fashion at 248 nm in a PerkinElmer Lambda 25 spectrophotometer at 25 °C in 4 mM sodium MOPS, pH 7.2 buffer, with 2% DMSO added for substrate solubility. Values of kw (maximal rate constant without added rescuing base, defined in Scheme S1) and KM,S were determined by fitting the initial rates as a function of substrate concentration to the Michaelis−Menten equation. Typically, eight substrate concentrations were used, varying from 2 to 600 μM. The kw and KM,S values and associated errors were obtained from least-squares fits to the data averaged from two independent experiments using different enzyme concentrations that varied over 2−10-fold (D38G/P39G/V40G/F54A and D38G/F54A/ A114G, 2.5 and 7.5 μM; D40G/F56A, 0.5 and 5 μM; and D40G/ F56A/W120A, 2 and 5 μM) and are given in Table S8. KSI Mutants Base Rescued Reactions. Base rescued reactions were performed in 20 mM sodium MOPS, pH 7.2 buffer, with 2% DMSO, ionic strength 1 M (NaCl) at 25 °C by monitoring the product formation for 5(10)-EST as described previously.33 The base concentration was varied from 0 to 0.25 M for valerate and trimethyl acetate and to 0.75 M for rest of the bases while keeping the substrate concentration constant at 300 μM. At the substrate concentration of 300 μM, KSI mutants were 80% or more saturated based on the KM,S values obtained from the nonrescued enzymatic reactions (Table S8). Substrate concentrations higher than 300 μM caused precipitation. For D38G/F54A tKSI, second-order acetate rate constants within 10% were observed at substrate concentrations of 100 and 300 μM, both above KM,S but with different calculated saturation levels (84% and 94%). Given this similarity, we did not correct the obtained values to theoretical saturation (Figure S10). To obtain second-order rate constants for base rescued reactions (kbase, defined in Figure 1B), the initial rates of product formation were divided by enzyme concentration and fitted to a linear equation as a function of base concentration. Intercepts agreed with measured kw values obtained in the absence of base. The first-order rate constant kcat,EB and KM,base (defined in Scheme S2) were determined by dividing the initial rates by enzyme concentration and then fitting to a Michaelis−Menten equation as a function of exogenous base concentration. The values of rate constants and errors associated with them were obtained from least-squares fit of the averaged data obtained from 2−3 independent experiments using enzyme concentrations varied over a range of 0.25−1.5 μM. For second-order rate constants, the linear fits to the averaged data are given in Figures S2 (tKSI) and S6 (pKSI), and values and associated errors are given in Tables S1 (tKSI) and S3 (pKSI). For first-order rate constants and KM,base values, the nonlinear fits to the averaged data are given in Figures S1 (tKSI) and S5 (pKSI), and values and associated errors are given in Tables S1 (tKSI) and S3 (pKSI). Inhibition of TMA Rescued Reaction with t-BuOH. Inhibition of TMA rescued reaction in KSI general mutants was observed by monitoring the 5(10)-EST reaction in 20 mM sodium MOPS pH 7.2 buffer, 2% DMSO, at ionic strength 1 M (NaCl) and at 25 °C as described above. The substrate concentration was kept at 300 μM. For each TMA concentration (2, 75, and 250 mM), eight different concentrations of t-BuOH varying from 0 to 250 mM were used. To obtain the observed inhibition constant Kiobsd, the initial rates of product formation were fit to eq 1.

rate/[E] = (ratemax /[E])/(1 + [I]/K i obsd)

(1)

The observed inhibition constants and associated errors were obtained from least-squares fit of the data and are reported in Figure 4. Turning KSI On and Off. The experiment in Figure 4D was performed in nine steps, and data were collected in 20 mM sodium MOPS, pH 7.2, 2% DMSO at 25 °C with a saturating concentration of substrate (∼300 μM). In step 1, the nonbase rescued reaction of 2 μM KSI mutant D38G/F54A was followed. After 17 min, TMA was added to a final concentration of 2.5 mM to turn on the enzyme, and data were collected for ∼6 min, at which time the enzyme was turned off by addition of t-BuOH to a final concentration of 250 mM. After an additional ∼12 min, the enzyme was turned on again by adding 25 mM TMA and data were collected for ∼4 min. After that, the reaction mixture was diluted 10-fold with MOPS buffer to a final enzyme concentration of 0.2 μM enzyme, and additional substrate was added to a final concentration of 300 μM. Data were collected for ∼13 min, at which time enzyme activity was ramped up by the addition of 25 mM TMA. After ∼8 min, enzyme was shut off for ∼13 min by adding 250 mM t-BuOH and then turned on again by the addition of 100 mM TMA. In the last step, the reaction mixture was diluted by 1.5-fold while turning off enzyme activity with a final mixture of 1 M t-BuOH, 125 mM TMA, and 0.13 μM enzyme. In Figure 4D, the absorbance of each step was offset to match to the end point of the previous step for ease of visualization. The raw absorbance traces are given in Figure S7. The observed and predicted rates for each step are given in Table S5. X-ray Crystallography. A solution containing 1.4 mM tKSI D38G/F54A mutant and 3 mM equilenin was prepared prior to crystallization. 1 μL of this solution was mixed with 1 μL of mother liquor (1.0 M ammonium sulfate, 0.5 M TMA, 40 mM potassium phosphate, pH 7.2) in a vapor diffusion hanging drop setup. Crystals were obtained in about 3 weeks. Prior to data collection, crystals were cryoprotected by briefly soaking them in the mother liquor solution supplemented with 10% glycerol. Single-crystal diffraction data were collected at SSRL, beamline BL11-1, using a wavelength of 0.979 and at 100 K. Data reduction was done using the XDS package,54 and data scaling and merging was done using Aimless.55 Initial phases were derived by molecular replacement using Phaser56 and using as a model the PDB entry 3NXJ in which residues 38−43 were removed. Model building was carried out with the program BUCCANEER57 and manually in Coot.58 The model was refined manually after visual inspection with Coot and using phenix.refine.59 Torsion-angle simulated annealing and averaged kicked maps (as implemented in phenix.refine) were used during the initial stages of refinement and in combination with conventional maps. Ligand restraints were generated using ELBOW,60 and ligand geometry was checked using the ValLigURL server. Model quality was checked by MolProbity61 and gave an overall score of 1.45. In the D38G/F54A tKSI electron density map, we observed additional density close to the region occupied by the general base D38 in WT. This electron density can be fit with a glycerol molecule (which was present in the cryo-protection solution) with reasonable geometry (Figure 11A) or to a TMA molecule (Figure 11B). Because of this ambiguity, this electron density was not modeled in the deposited structure.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b03547. X-ray crystallographic data (CIF) X-ray crystallographic data (CIF) Figures S1−S11, Tables S1−S8, and Schemes S1 and S2 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*[email protected] 11094

DOI: 10.1021/jacs.7b03547 J. Am. Chem. Soc. 2017, 139, 11089−11095

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Vandana Lamba: 0000-0002-8608-6292 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by National Science Foundation grant MCB-1121778 to D.H. F.Y. was supported by a postdoctoral fellowship provided by Human Frontiers Science Program. Diffraction data were obtained at Stanford Synchrotron Radiation Laboratory, a facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. We thank Irimpan Mathews for help with diffraction data collection. We also thank members of the Herschlag group for insightful discussions and suggestions.

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DOI: 10.1021/jacs.7b03547 J. Am. Chem. Soc. 2017, 139, 11089−11095