Molecular Basis for Polyketide Ketoreductase–Substrate Interactions

Abstract

Polyketides are a large class of structurally and functionally diverse natural products with important bioactivities. Many polyketides are synthesized by reducing type II polyketide synthases (PKSs), containing transiently interacting standalone enzymes. During synthesis, ketoreductase (KR) catalyzes regiospecific carbonyl to hydroxyl reduction, determining the product outcome, yet little is known about what drives specific KR–substrate interactions. In this study, computational approaches were used to explore KR–substrate interactions based on previously solved apo and mimic cocrystal structures. We found five key factors guiding KR–substrate binding. First, two major substrate binding motifs were identified. Second, substrate length is the key determinant of substrate binding position. Third, two key residues in chain length specificity were confirmed. Fourth, phosphorylation of substrates is critical for binding. Finally, packing/hydrophobic effects primarily determine the binding stability. The molecular bases revealed here will help further engineering of type II PKSs and directed biosynthesis of new polyketides.

The Path to Actinorhodin: Regio- and Stereoselective Ketone Reduction by a Type II Polyketide Ketoreductase Revealed in Atomistic Detail

“…6 Compared to the MD simulations without PPant-octaketide, the α6-α7 loops of actKR and adjacent residues (188-229) were positioned in a more 'closed' form, with the Tyr202 side chain projecting inside the active site (and a water molecule bridging Tyr202 and the octaketide). 44 For both stage I and II MD, all residues were in their standard protonation states (consistent with pKa predictions from PROPKA 3.1) 45 with actKR His162 protonated on Nδ1 and His153 and His201 on Nε2 (according to the surrounding Hbond network). All systems were solvated in a rectangular box extending at least 11 Å from any protein atom and neutralized by addition of Na + ions.…”

“…For the model selected after NMR assessment (M14), further MD simulations of the tetrameric complex were performed after introducing the PPant-octaketide moiety (2), with all combinations of the possible cyclization conformers (stage II, Figure 2; Scheme 2; Table S3). 2 was modelled from different starting positions in the active sites of each system by finding a balance between: 1) conformational agreement with the PPant of octaketide mimics crystallized with KRs; [42][43][44] and 2) maintaining catalytic interactions with residues Ser144 and Tyr157. The latter was not possible with C9 positioned for pro-R hydride attack; starting structures therefore had S prochirality in all cases.…”

“…In turn, each of these four isomers can access two low-energy chair conformers (Scheme 2), with the C7 -OH substituent oriented either axially (2OHax) or equatorially (2OHeq). actACP binding mode M1416IB was used and the actKR α6-α7 loop was remodeled prior to MD simulation, based on an actKR-octaketide mimic complex structure, 44 in line with the suggested role of this loop in substrate recognition. 5 By using previous structural information [42][43][44] and satisfying contacts between 2 and catalytic residues, initial placements for the PPant and the octaketide moieties were generated (two alternative starting positions were used in order to explore a greater portion of conformational space; modelling details and coordinates are included as Supporting Information).…”

“…A different hydrogen bond interaction that may be relevant for cyclization, between 2:O7/H7 and actKR:Tyr202's -OH group, is observed occasionally in simulations for most isomer-conformer pairs (Table S7, Table S8). This (highly conserved) 5 Tyr202 side chain, in its orientation towards the active site 44 (Figure 6a, Figure S2), could thus be involved in catalyzing regioselective C7-C12 cyclization, e.g. as proton donor to O7, or aiding proton transfer from the nearby His153 and His201.…”

Recent Developments in Free Energy Calculations for Drug Discovery

“…Manufacture of commercially valuable polyketides, secondary metabolites synthesized by multi-domain polyketide synthase complexes, has been investigated via comparison of thioesterase binding affinity to cyclized products to understand product release mechanisms of the antifungals amphotericin and nystatin ( Wang et al, 2021 ). Binding free energy analysis is also used to elucidate the molecular basis of ketoreductase chain length and regiospecificity ( Serapian and van der Kamp, 2019 ; Zhao et al, 2020 ). Potential insecticides targeting the ecdysone receptor to disrupt growth are identified ( Horoiwa et al, 2019 ).…”