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

Abstract

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 (TE_S148C) with improved reaction kinetics and gain-of-function processing of an unnatural, epimerized hexaketide. Quantum mechanical comparison of model TE_S148C and TE_WT reaction coordinate diagrams revealed a change in mechanism from a stepwise addition-elimination (TE_WT) to a lower energy concerted acyl substitution (TE_S148C), 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.

Figure 1

Macrolactonization or hydrolysis of an ACP-tethered polyketide intermediate.

Figure 2

Pik TE displays a high level of substrate stereoselectivity. (a) Pik hexaketides used in this study to probe Pik TE substrate flexibility. 3 is generated in situ by photolysis of the 2-nitrobenzyloxymethyl ether (NBOM) protected native hexaketide. (b) Incubation of C-11-epimerized Pik hexaketide 5 results exclusively in hydrolysis.

Figure 3

Acyl-enzyme starting structures for the MD simulations of 4 (Pik TE-4) and C-11-epimerized 5 (Pik TE-5).

Figure 4

Comparison of the reactive conformations for each acyl-enzyme intermediate obtained from clustering analysis of MD simulations with Pik TE_WT: (a) Pik TE-4 and (b) Pik TE-5. Pik TE-4 conformations I and II contain a hexaketide orientation most conducive to macrolactonization with the C-11 OH in close proximity to both His268 and the C-1 carbonyl. The corresponding conformation (cluster I′) in the Pik TE-5 simulation likely represents a larger barrier to macrolactonization as the distance between the C-11 OH and His268 has increased and the resulting geometry hinders deprotonation. The Pik TE-5 hexaketide continues to evolve toward a linear conformation until the final cluster III′ is reached which places the C-11 OH distal to both His268 and the C-1 carbonyl and in an orientation susceptible to hydrolysis. The catalytic triad His268 and Asp176 residues are colored yellow. For each conformation the distance in angstroms from the nucleophilic hydroxyl oxygen to the Nε nitrogen of His268 and the ester C-1 carbonyl is displayed above the dashed lines. Clusters containing catalytically productive conformations contain red dashed lines. (c) The distance of the nucleophilic hydroxyl oxygen and the ester C-1 carbonyl plotted for each frame of the MD simulation with each data point colored according to the corresponding clustered conformation. The vertical dashed line at 50 ns indicates when the distance constraints were released.

Figure 5

Procatalytic sampling of Pik TE during MD simulations. (a,b) Low-energy QM optimized transition states for macrolactonization of Pik hexaketides (a) Pik TE-4 and (b) Pik TE-5. Nonpolar hydrogens have been removed for clarity. (c,d) Deviations of the key catalytic distances (x axis) and angles (y axis) in the MD simulations of (c) Pik TE-4 and (d) Pik TE-5 from their respective optimized transition structure (green square at the origin of coordinates). Each point represents a single frame from the 550 ns simulation, while the shaded rectangles represent frames from the MD that are likely in a catalytically productive state.

Scheme 1. Yamaguchi Macrolactonization of Methyl-Protected Hexaketides

Conversion of 8 to 6 and 7 to 9 was monitored by HPLC, with data represented as the mean ± standard deviation, where n = 3.

Scheme 2. Evaluation of Pik TE_S148C with Methyl-Protected Hexaketides 4 and 5

Enzymatic reaction conditions: 1 mM hexaketide, 8 mM 2-vinylpyridine, purified Pik TE_S148C (10 μM), 4 h, stationary, RT. Conversion of 4 to 6 and 5 to 9 was monitored (HPLC), with data represented as the mean ± standard deviation, where n = 3.

Scheme 3. Reaction of PikAIII-TE_S148C with C-9-Epimerized Pentaketide 10

Enzymatic reaction conditions: 1 mM Pik pentaketide, 20 mM (20 equiv) MM-NAC, 8 mM (8 equiv) 2-vinylpyridine, 0.5 mM (50 mol %) NADP⁺, 2.5 mM (2.5 equiv) glucose-6-phosphate, glucose-6-phosphate dehydrogenase (2 units/mL), 3 μM (0.3 mol %) PikAIII-TE_S148C, 8 h, stationary, RT.

Figure 6

Reaction coordinate diagram representing the relative free energies for Pik TE-catalyzed macrolactonization of hexaketides 13 and 14. Calculations were performed at the PCM/M06-2X/6-31+G(d,p) level using reduced models that define the enzymatic active site (theozymes). Relative free energies are in kcal mol^–1. Only the lowest energy conformers are represented; see Supporting Information for details and all the calculated structures. *This intermediate is higher in energy than its preceding TS due to conformational differences between both stationary points.

Figure 7

Lowest energy rate-limiting transition structures calculated with PCM/M06-2X/6-31+G(d,p) for the abbreviated active site models (theozymes) of the Pik TE_WT (left) and Pik TE_S148C (right) catalyzed macrolactonization of native hexaketide 13 (top, in blue) and C-11-epi hexaketide 14 (bottom, in red). Activation free energies (ΔG^⧧) calculated from the corresponding Pik TE hexaketides are given in kcal mol^–1 and distances in angstroms. Relevant breaking/forming C–O and C–S bonds are shown in boldface. Nonpolar hydrogens have been removed for clarity. See Supporting Information for all calculated structures along the reaction pathway.