Structural rearrangements of a polyketide synthase module during its catalytic cycle

Abstract

The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents. The architecture of a full-length PKS module from the pikromycin pathway of Streptomyces venezuelae creates a reaction chamber for the intramodule acyl carrier protein (ACP) domain that carries building blocks and intermediates between acyltransferase, ketosynthase and ketoreductase active sites (see accompanying paper). Here we determine electron cryo-microscopy structures of a full-length pikromycin PKS module in three key biochemical states of its catalytic cycle. Each biochemical state was confirmed by bottom-up liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry. The ACP domain is differentially and precisely positioned after polyketide chain substrate loading on the active site of the ketosynthase, after extension to the β-keto intermediate, and after β-hydroxy product generation. The structures reveal the ACP dynamics for sequential interactions with catalytic domains within the reaction chamber, and for transferring the elongated and processed polyketide substrate to the next module in the PKS pathway. During the enzymatic cycle the ketoreductase domain undergoes dramatic conformational rearrangements that enable optimal positioning for reductive processing of the ACP-bound polyketide chain elongation intermediate. These findings have crucial implications for the design of functional PKS modules, and for the engineering of pathways to generate pharmacologically relevant molecules.

Figure 1

Cryo-EM structure of pentaketide-KS₅-PikAIII. a, Solid rendering (left) and transparent representation with modeled structures (right) of the pentaketide-KS₅-PikAIII cryo-EM map. b, Interface of AT and ACP docked within the cryo-EM density. ACP helix 2 (residues 1439–1452) contacts the two AT lid helices (residues 701–709 and 732–744). The phosphopantetheinylated Ser1438 of ACP is 35 Å from the AT active site. c, Comparison of the KR structure docked in the cryo-EM map for holo-PikAIII and pentaketide-KS₅-PikAIII. The dramatic rearrangement upon pentaketide-KS formation brings two helices (residues 1126–1138, 1156–1166) of the KR catalytic sub-domain (KR_cat, magenta) in contact with an AT helix (residues 760–775) and moves the structural sub-domain of KR (KR_struc, purple) away from AT.

Figure 2

Cryo-EM structure of β-ketohexaketide-PikAIII. a, Solid rendering and transparent representation with modeled structures of the β-ketohexaketide-PikAIII cryo-EM map. b, Comparison of the KS-AT interface between holo-PikAIII (left) and β-ketohexaketide-PikAIII (right). Polyketide chain extension results in an 8-Å shift and rotation of AT towards the KS. c, ACP interaction with KR. ACP interacts with the KR lid helix (residues 1302–1309) and lid loop (1293–1301) to position ACP Ser1438 near the NADPH binding cleft.

Figure 3

Cryo-EM structures of β-hydroxyhexaketide-PikAIII. a, Solid rendering of cryo-EM maps for the three conformers of β-hydroxyhexaketide-PikAIII. The ACP density is differentially colored with magenta, yellow, and cyan. The panel on the right shows a transparent representation with modeled structures of the adjacent β-hydroxyhexaketide-PikAIII conformer. b, Docking of the ACP structure in the corresponding density map of each β-hydroxyhexaketide-PikAIII conformer shows that Ser1438 invariably points away from the module. The ACP structures are colored according to the conformers in panel (a). c, Bottom view of overlay of the three cryo-EM maps of β-hydroxyhexaketide-PikAIII. The three different ACP positions cover a surface with a radius of 15 Å.

Figure 4

Schematic of substrate processing in PikAIII. Transfer of the upstream intermediate (pentaketide, yellow line) to the KS domain (blue with yellow active site) of holo-PikAIII (state 1) induces an end-to-end flip of the KR domain (purple, yellow active site) and positions the ACP domain (orange, yellow active serine) at the AT domain (green, yellow active site) (state 2). Methylmalonyl (MM, red line) loading positions the ACP at the bottom entrance of the KS (state 3). KS-catalyzed extension of the pentaketide to a β-ketohexaketide (red-yellow line) positions the ACP at the KR domain (state 4). KR-catalyzed reduction of the β-ketohexaketide to β-hydroxyhexaketide (green line) ejects the ACP out of the central chamber (state 5) and into position for intermediate transfer to PikAIV (state 6).