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.

The structural organization of substrate loading in iterative polyketide synthases

“…Although CTB1 is an iPKS, the locations of the N and C termini at opposite ends of the molecule would be compatible with extended connectivity in an assembly line. The only other known structure of a NR-PKS SAT, which was excised as an isolated domain from CazM 18 , reveals a C-terminal linker element that ends in a position similar to that of the SAT-KS in CTB1 despite a lack of sequence conservation in the linker (Supplementary Fig. 4a,b).…”

“…However, in NR-PKSs, soluble expression of KS-MAT is only achieved by including the SAT domain in a tridomain construct 16 , 17 , indicating that SAT, KS, and MAT form an integrated loading/condensing region architecture. SATs can be successfully isolated 18 and swapped in the context of the condensing region environment 19 . Together with an ACP, the SAT-KS-MAT core region of NR-PKSs is catalytically competent for synthesis of the complete polyketide backbone in vitro 7 , 10 , 17 .…”

Diversity-Oriented Combinatorial Biosynthesis of Hybrid Polyketide Scaffolds from Azaphilone and Benzenediol Lactone Biosynthons

“…2 For azaphilones such as 7 , ABH biosynthesis on the nrPKS is primed by an hrPKS-derived reduced polyketide chain in a sequential biosynthetic scheme analogous to that of BDLs (Figure 1). 6–8 Some other azaphilones use a convergent biosynthetic scheme, whereby the hrPKS product acylates a free pyranoquinone intermediate. 9–11 Chimeric BDL scaffolds were previously generated by us using iPKS subunit shuffling among noncognate BDL synthase (BDLS) hrPKS and nrPKS partners.…”

Comparison of 10,11‐Dehydrocurvularin Polyketide Synthases from Alternaria cinerariae and Aspergillus terreus Highlights Key Structural Motifs

“…[41] A key structural feature of SAT CazM is a “parallel” αP helix contained within the α/β-hydrolase-like subdomain (Supporting Information Figure S9) that is thought to be important in ACP-SAT interactions. We modeled SAT Dhc5 and SAT CurS2 based on this sequence and also without any structural constraints (Figure 5 and Supporting Information Figures S11 and S12).…”