A Single Active Site Mutation in the Pikromycin Thioesterase Generates a More Effective Macrocyclization Catalyst

Macrolactonization of natural product analogs presents a significant challenge to both biosynthetic assembly and synthetic chemistry. In the preceding paper, we identified a thioesterase (TE) domain catalytic bottleneck processing unnatural substrates in the pikromycin (Pik) system, preventing the formation of epimerized macrolactones. Here, we perform molecular dynamics simulations showing the epimerized hexaketide was accommodated within the Pik TE active site; however, intrinsic conformational preferences of the substrate resulted in predominately unproductive conformations, in agreement with the observed hydrolysis. Accordingly, we engineered the stereoselective Pik TE to yield a variant (TES148C) with improved reaction kinetics and gain-of-function processing of an unnatural, epimerized hexaketide. Quantum mechanical comparison of model TES148C and TEWT reaction coordinate diagrams revealed a change in mechanism from a stepwise addition–elimination (TEWT) to a lower energy concerted acyl substitution (TES148C), accounting for the gain-of-function and improved reaction kinetics. Finally, we introduced the S148C mutation into a polyketide synthase module (PikAIII-TE) to impart increased substrate flexibility, enabling the production of diastereomeric macrolactones.


Structure Determination
Colorless needles of S2 were grown from a hexanes solution of the compound at -30 deg. C. A crystal of dimensions 0.26 x 0.01 x 0.01 mm was mounted on a Rigaku AFC10K Saturn 944+ CCD-based X-ray diffractometer equipped with a low temperature device and Micromax-007HF Cu-target micro-focus rotating anode (λ = 1.54187 A) operated at 1.2 kW power (40 kV, 30 mA). The X-ray intensities were measured at 85(1) K with the detector placed at a distance 42.00 mm from the crystal. A total of 2633 images were collected with an oscillation width of 1.0° in ω. The exposure times were 15 sec. for the low angle images, 75 sec. for high angle. The integration of the data yielded a total of 98528 reflections to a maximum 2θ value of 136.48° of which 9383 were independent and 8176 were greater than 2σ(I). The final cell constants (Table  1) were based on the xyz centroids 44319 reflections above 10σ(I). Analysis of the data showed negligible decay during data collection; the data were processed with CrystalClear 2.0 and corrected for absorption. The structure was solved and refined with the Bruker SHELXTL (version 2008/4) software package, using the space group P2(1)2(1)2(1) with Z = 4 for the formula C25H35NO7.
There are two crystallographically independent molecules per asymmetric unit. All non-hydrogen atoms were refined anisotropically with the hydrogen atoms placed in idealized positions. Full matrix least-squares refinement based on F2 converged at R1 = 0.0518 and wR2 = 0.1275 [based on I > 2sigma(I)], R1 = 0.0591 and wR2 = 0.1328 for all data. Additional details are presented in Table 1

Kinetic analysis of thioesterase catalysis
Product formation for all kinetic experiments was quantified by linear regression using equations derived from fitting the peak areas of the corresponding standard curves. Standard curves were generated by analyzing product standards in triplicate at a range of concentrations and were linear in all cases. Prior to performing kinetic analysis, the pH dependence for TE macrolactonization was established by running a series of assays at pH 6, 6.5, 7, 7.2, 7.5, 7.8, 8. The reactions consisted of sodium phosphate buffer (50 mM), 1 μM Pik TE (WT or S148C), 1 mM 3 and 10% (v/v) DMSO in a total volume of 50 μL. The reactions were incubated at RT for 1 h and quenched by addition of MeOH (150 μL). All reactions were run in duplicate and following centrifugal clarification (17,000 x g, 15 min, 4˚C) the samples were directly analyzed for product formation, pH 7.2 was found to be optimum.

Macrolactonization of 4:
Macrolactonization of SNAC-MeHK 4 to 6 was monitored via analytical high performance liquid chromatography (HPLC) using an Agilent 1100 Series HPLC and a Zorbax SB-Phenyl (3.5 µm, 4.6 x 150 mm, part number 863953-912) column at a wavelength of 250 nm. Products were separated using a linear gradient via the following method: 3.0 mL/min, solvent A: H 2 O, solvent B: MeCN, 20% B 0-1 min, 20-60% B linear gradient 1-10 min, 100% B 10-11 min, 20% B 11-12 min. A time course for the macrolactonization reaction of each TE at pH 7.2 was obtained by analyzing reaction mixtures containing 400 mM phosphate buffer (pH 7.2), 10% (v/v) DMSO, 0.2 mM 4, and 0.3 μM TE S148C or 1.0 μM TE WT in a total reaction volume of 1500 μL incubated at room temperature for intervals of 5, 10, 20, and 30 min at which point they were quenched and analyzed via HPLC for macrolactone production. Reactions were run in duplicate and the velocities were determined to be linear by linear regression analysis of the data over a period of 20 min ( Figure S6).
Steady-state kinetic analysis was performed by determining the initial velocities for the macrolactonization of 4 at concentrations of 0.25 mM, 0.5 mM, 1 mM, 2mM, 3 mM, 4mM, 6mM, 8mM. The reactions consisted of 400 mM phosphate buffer (pH 7.2), 10% (v/v) DMSO, variable concentrations of 4, and 0.3 μM TE S148C or 1.0 μM TE WT . The reactions were incubated at room temperature for 10 min at which point they were quenched with MeOH (150 μL) and analyzed via HPLC for macrolactone production. All reactions were run in triplicate and the initial velocities at different substrate concentrations were fit to the Michaelis-Menten equation by nonlinear least-squares regression to calculate k cat and K M ( Figure S7). The 6 mM and 8 mM concentration points were omitted from the plots with TE S148C as these concentrations resulted in significant substrate inhibition.

Hydrolysis and macrolactonization of 5:
The hydrolysis (TE WT ) and macrolactonization (TE S148C ) of 5 to either 7 or 9, respectively, was monitored via HPLC using the same method as above for 4. A time course for the hydrolysis and cyclization reaction for each TE was accomplished by incubating assay mixtures containing 400 mM phosphate buffer (pH 7.2), 10% (v/v) DMSO, 0.03 mM (TE S148C ) or 0.125 mM (TE WT ) 5, and 0.3 μM TE S148C or 1.0 μM TE WT in a total reaction volume of 1500 μL at room temperature for intervals of 5, 10, 20, and 30 min, at which point they were quenched and analyzed via HPLC. Reactions were run in duplicate and the velocities were determined to be linear by linear regression analysis of the data over a period of 30 min ( Figure S8).
Steady-state kinetic analysis was performed by determining the initial velocities for the macrolactonization and hydrolysis of 5 at concentrations of 0.25 mM, 0.5 mM, 1 mM, 2mM, 3 mM, 4mM, 6mM, 8mM for TE WT and 0.03 mM, 0.06 mM, 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, 2mM, 3 mM, 4mM, 6mM, 8mM for TE S148C . The reactions consisted of 400 mM phosphate buffer (pH 7.2), 10% (v/v) DMSO, variable concentrations of 5, and 0.5 μM TE S148C or 1.0 μM TE WT . The reactions were incubated at room temperature for 10 min at which point they were quenched and analyzed via HPLC for product formation. All reactions were run in triplicate and the Initial velocities at different substrate concentrations were fit to the Michaelis-Menten Figure S7. Steady state kinetic analysis for the TE catalyzed macrocyclization of 4. Figure S8. Linear velocity determination for the hydrolysis (WT) and macrocylization (S148C) of 5. S13 equation by nonlinear least-squares regression to calculate k cat and K M ( Figure S9). The concentration points not represented were removed from the plot as they resulted in significant substrate inhibition.

Macrolactonization of 3:
Macrolactonization of the native hexaketide 3 was monitored via analytical high performance liquid chromatography (HPLC) using an Agilent 1100 Series HPLC and a Zorbax SB-Phenyl (3.5 µm, 4.6 x 150 mm, part number 863953-912) column at a wavelength of 250 nm. Products were separated using a linear gradient via the following method: 3.0 mL/min, solvent A: H2O, solvent B: MeCN, 10% B 0-1 min, 10-40% B linear gradient 1-10 min, 100% B 10-11 min, 10% B 11-12 min. A time course for the cyclization reaction with each TE was accomplished by incubating assay mixtures containing 400 mM phosphate buffer (pH 7.2), 10% (v/v) DMSO, 0.125 mM 3, and 0.3 μM TE S148C or 1.0 μM TE WT in a total reaction volume of 350 μL at room temperature for intervals of 5, 10, 20, and 30 min, at which point they were quenched and analyzed via HPLC. The NBOM protected substrate was deprotected and utilized in enzymatic reactions as previously reported 1 , briefly: a solution of ascorbic acid (25 mM final concentration), sodium metabisulfite (1 mM final concentration), NBOM protected hexaketide (1 mM final concentration), and H 2 O (requisite dead volume) was irradiated under a consumer facial tanning lamp at a height of 14 cm (Verseo #AH129c) for 20 min to furnish the deprotected Pik hexaketide 3. After photolysis, the solution was diluted with enzymatic reaction buffer, and catalysis was initiated via the addition of enzyme. Reactions were run in duplicate and the velocities were determined to be linear by linear regression analysis of the data over a period of 20 min ( Figure S10).
Samples were quantified by linear regression analysis comparing the product peak areas to those generated from the corresponding standard curves.

Pik TE structure preparation
The wild type apo Pik TE structure was obtained from the RCSB Protein Data Bank (PDB: 1MNA) 9 . Structures for the wild type and mutant enzymes were prepared from WT Pik TE using PyMOL 1.3 10 . Chain B of PDB file 1MNA and the crystallographic water were removed, and 5 missing protein residues (T109-T113) were built into chain A using PyMOL 1.3. 10 Parameters for the apo TE were generated using the antechamber module of AMBER 14 11 with the widely tested Stony Brook modification of the Amber 99 force field (ff99SB) 12 force field. The initial substrate conformations were generated by modeling the linear hexaketide chain as neutral acyl serine or cysteine amino acids with N-terminal (-CO-CH 3 ) and C-terminal (-NH-CH 3 ) caps in an approximately cyclic orientation using the solved crystal structures of the derivatized macrolactone products as templates. The structure reported in reference 1 served as the template for Pik TE-4 and S2 was used for modeling the Pik TE-5. The initial hexaketide conformations were then optimized by performing a geometry optimization with the M06-2X functional 13 level using the Gaussian 09 package 14 . The charges and parameters for the hexaketide substrates were then determined using the general AMBER force field (GAFF) 15 with partial charges set to fit the electrostatic potential generated at the HF/6-31G(d) level by the RESP model 16 . The charges were calculated according to the Merz-Singh-Kollman scheme 17,18 , using Gaussian 09. The N and C-terminal caps were then removed and the parameterized hexaketides were used to replace Ser148 of the TE to generate Pik TE modeled as acyl-enzyme intermediates. Parameter, topology, and coordinate files for the complex were compiled for molecular dynamics (MD) simulation using the tleap 11 module. Following parameterization, the Pik TE models with bound hexaketides were immersed in a preequilibrated truncated cuboid box with a 10 Å buffer of TIP3P 19 water molecules using tleap and the systems were neutralized by addition of explicit counter ions (Na + and Clˉ).

Molecular Dynamics simulations
MD simulations were performed using the AMBER 14 package. 11 A cutoff of 8 Å was used for all Lennard-Jones and electrostatic interactions. Water molecules were treated with the SHAKE algorithm such that the angle between the hydrogen atoms was kept fixed. Periodic boundary conditions simulated the effects of a larger system size. Long-range electrostatic effects were modelled using the particle-mesh-Ewald method. 20 Prior to performing the production MD simulations, the solvated systems were subjected to two steps each of minimization and equilibration to relax and correct any possibly unrealistic arrangements: two minimizations were performed first over a total of 5000 cycles each, with the method being switched from the steepest descent algorithm to conjugate gradient after 2500 cycles. The first minimization relaxed the positions of the solvent molecules and ions by imposing positional restraints on the solute by a harmonic potential with a force constant of 500 kcal mol -1 Å -2 . Following the first minimization, a distance restraint of 2.3 Å between the hexaketide nucleophilic hydroxyl hydrogen and the Nε nitrogen of His268 was imposed and maintained throughout the second minimization, both equilibrations, and first production simulation, the restraint was then released S20 for the second production simulation. This restraint maintains the hexaketide and the catalytic histidine residue -His268 acts as the general base-in an arrangement close to that calculated for the macrolactonization transition state, while allowing the hexaketide backbone to explore both reactive and non-reactive conformations. The restraint also served to minimize any potential biases resulting from the initial substrate positioning. After the distance restraint was imposed, a second stage minimization was performed on all the atoms in the simulation cell without cartesian atomic restraints. Post minimization, two equilibrations were performed. In the first equilibration step, a 0.1 ns MD simulation was performed with a constant pressure of 1 atm and a constraint of 10 kcal mol -1 Å -2 on the protein and substrate. The Andersen thermostat equilibration scheme was used to control and equalize the temperature by gradually increasing the system temperature from 0 to 300 K over the course of the first equilibration run. The system volume was then fixed and a second equilibration step was achieved with 2 ns MD simulation conducted without cartesian atomic restraints with a temperature maintained at 300 K using the Andersen thermostat.
Post equilibration, two additional MD simulations were carried out utilizing the above conditions: Initially, a 50 ns simulation was performed with the aforementioned distance restraint intact to ensure proper shape complementarity of the TE active site for the acyl intermediate forced in a pre-catalytic state. Next, the substrate-protein distance restraint was removed and a final production simulation was run for an additional 500 ns. This portion of the simulation represents the conformation of both the substrate and the protein environment in the covalently bound Michaelis-Menten complex prior to reaction. An integration time step of 2 fs was utilized with structural snapshots being extracted every 50 steps during both production simulations. The resulting production trajectories were analyzed using the cpptraj program in Ambertools14 employing the default options to describe hydrogen-bonding, hydrophobic interactions, and clustering analysis. Clustering analysis of the resulting simulations based on the conformation of the acyl-enzyme intermediate was used to bin each frame into representative clusters of structures. The clusters were subsequently examined for hydrogen-bond and hydrophobic interactions involving the substrate and TE using LigPlot+ 21 ( Figure S13).
Examination of the overall protein root-mean-square deviation (RMSD) and per-residue rootmean-square fluctuation (RMSF) values for the trajectories of each TE complex demonstrated stable dynamic behavior throughout the course of the simulations, with Pik TE-5 maintaining a slightly increased level of flexibility ( Figure S14). Investigation of the Pik TE-5 residues that displayed higher RMSF values revealed these residues were located predominantly in loops and linker regions with higher intrinsic flexibility and had limited impact on the conformation of the hexaketide. Pik TE-4 clusters I and II stabilize the hexaketide in a catalytically productive conformation mainly by hydrophobic packing leading to a high level of shape-complementarity and hydrogen bonding between the nucleophilic hydroxyl and the catalytic histidine. Conversely, cluster III shows decreased hydrophobic contacts and the hexaketide adopts a more linear conformation making it unreactive towards macrolactonization. Pik TE-5 Cluster I' stabilizes the hexaketide in a cyclic conformation via hydrophobic packing; however the hexaketide adopts a conformation that impedes macrocyclization due to formation of an intramolecular hydrogen bond. Clusters II' and III' show a gradual loss of hydrophobic packing as the hexaketide adopts a linear conformation that makes it vulnerable to the experimentally observed hydrolysis. The catalytic triad His268 and Asp176 residues are colored yellow while the active site residues found to interact with the acyl-intermediate are colored green. Residues involved in hydrophobic interactions are displayed in surface representation; dashed lines indicated hydrogen bonds to the labeled residues.

QM Calculations
TE catalyzed macrolactonization QM Computational Details. Full geometry optimizations were carried out with Gaussian 09 14 using the M06-2X hybrid functional 13 and 6-31+G(d,p) basis set. Solvation was considered implicitly through the PCM polarizable continuum model 22 in order to better reproduce the electrostatic environment inside the enzyme active site (ε=4). The possibility of different conformations was taken into account for all structures. All stationary points were characterized by a frequency analysis performed at the same level used in the geometry optimizations from which thermal corrections were obtained at 298.15 K. Local minima and first order saddle points were identified by the number of imaginary vibrational frequencies. The quasiharmonic approximation reported by Truhlar et al. was used to replace the harmonic oscillator approximation for the calculation of the vibrational contribution to enthalpy and entropy. 23 Scaled frequencies were not considered. Mass-weighted intrinsic reaction coordinate (IRC) calculations were carried out by using the Gonzalez and Schlegel scheme 24,25 in order to ensure that the TSs indeed connected the appropriate reactants and products. Gibbs free energies (ΔG) were used for the discussion on the relative stabilities of the considered structures. Cartesian coordinates, electronic energies, entropies, enthalpies, Gibbs free energies, and lowest frequencies of the calculated structures are available below.
Attempts to locate transition states for the initial covalent binding step (i.e. Ser/Cys148 acylation) from thioester models of Pik TE WT and Pik TE S148C were unsuccessful, due to the very flat topology found for the corresponding potential energy surfaces. Hence, very low activation barriers are expected for the initial substrate binding, and macrocyclization was assumed to be the rate-limiting reaction.