Biochemical and Structural Basis for Controlling Chemical Modularity in Fungal Polyketide Biosynthesis

Abstract

Modular collaboration between iterative fungal polyketide synthases (IPKSs) is an important mechanism for generating structural diversity of polyketide natural products. Inter-PKS communication and substrate channeling are controlled in large by the starter unit acyl carrier protein transacylase (SAT) domain found in the accepting IPKS module. Here, we reconstituted the modular biosynthesis of the benzaldehyde core of the chaetoviridin and chaetomugilin azaphilone natural products using the IPKSs CazF and CazM. Our studies revealed a critical role of CazM's SAT domain in selectively transferring a highly reduced triketide product from CazF. In contrast, a more oxidized triketide that is also produced by CazF and required in later stages of biosynthesis of the final product is not recognized by the SAT domain. The structural basis for the acyl unit selectivity was uncovered by the first X-ray structure of a fungal SAT domain, highlighted by a covalent hexanoyl thioester intermediate in the SAT active site. The crystal structure of SAT domain will enable protein engineering efforts aimed at mixing and matching different IPKS modules for the biosynthesis of new compounds.

Figure 1

Chemical modularity in building fungal polyketides. A) Representative molecules. The red portion indicates reduced products of upstream synthases that are transferred to downstream NR-PKSs. B) Proposed biosynthesis of chaetoviridin A (6) and chaetomugilin A (7). Domain abbreviations:ketosynthase (KS), malonyl-CoA:acyl carrier protein acyltransferase (MAT), dehydratase (DH), methyltransferase (MT), enoylreductase (ER), ketoreductase (KR), acyl carrier protein (ACP), starter-unit:ACP-transacylase (SAT), product template (PT), and reductive domain (R). 11-SNAC was synthesized and used to directly load the KS of CazM.

Figure 2

Modular CazF-CazM interaction and cazaldehyde production. HPLC analysis (λ=290 nM) of polyketide products when i) CazM was incubated with acetyl-CoA, malonyl-CoA, SAM and NADPH; ii) CazM was incubated with CazF, malonyl-CoA, SAM and NADPH; iii) CazM was incubated with 11-SNAC, malonyl-CoA, SAM and NADPH; iv) CazM was incubated with 11-SNAC, malonyl-CoA and SAM; and v) CazM was co-expressed with CazF in S. cerevisiae BJ5464-NpgA. Two benzaldehydes 10 and 12 were observed.

Figure 3

Trans SAT complementation assays. HPLC analysis (λ= 290 nm) of 10 synthesized when i) CazM H277A was incubated with CazF, malonyl-CoA, SAM and NADPH; ii) CazM H277A was incubated with 11-SNAC, malonyl-CoA, SAM and NADPH; iii-vi)CazM H277A was incubated with CazF, malonyl-CoA, SAM, NADPH and standalone SAT domains.

Figure 4

Crystal structure of the CazM SAT domain. A) The monomeric SAT domain consists of a large subdomain containing an α/β-hydrolase-like fold and a small subdomain containing a ferredoxin-like fold. B)Structure of the avermectin AT loading domain Ave-AT° (PDB 4RL1). The two teal helices indicate a change in orientation compared to αJ and αP in the SAT structure and the active site S120 is shown as a stick. C) Electrostatic surface representation of the SAT domain showing a cross section of the cavity leading to the active site and bound hexanoyl. D) Residues located within 5 Å of bound hexanoyl and distances of residues lining the bottom of the active site cavity to C6 of hexanoyl (yellow dashed lines). Polar contacts are shown as black dashes. E) Active site residue comparison between the apo and hexanoyl-bound SAT. The apo SAT residues are shown in gray, whereas the hexanoyl-bound residues are shown in purple. Bound hexanoyl is shown in ball and stick form. F) Electrostatic surface maps of the apo-SAT (left) and CazF ACP (right) docking interfaces. Colors range from blue (positive) to white to red (negative). The homology model for CazF ACP was generated using I-TASSER and docking simulations were performed with PatchDock. TheACP is rotated 180° such that A–C on the SAT and ACP interfaces should match up. On the SAT, A is R374, B is R243 and C is R366. On the ACP, A is N61, B is E58 and C is D31.G) The homology model of CazF ACP (colored in yellow) was used as a ligand protein to dock with the apo SAT structure (colored in white). Helix αPof the SAT is highlighted in purple and surface residues that may be responsible for protein-protein interaction are shown as sticks. The active site Ser in the ACP and Cys in the SAT are in bold. All electrostatic surface maps were generated using PyMOL.

Scheme 1

Synthesis of 11-SNAC using the SNAC- HWE reagent 13 and 2-(S)-methyl-1-butanol 14.