Diaryl Ether Formation by a Versatile Thioesterase Domain

Abstract

Oxidative coupling and oxidative rearrangement are two of the most common biosynthetic strategies to form diaryl ethers. In contrast, enzymatic diaryl ether generation that proceeds in a nonoxidative manner has not been characterized thus far. Here, we discovered a versatile thioesterase (TE) domain from the nonreducing polyketide synthase (nrPKS) AN7909, which catalyzes diaryl ether formation through a series of successive steps involving esterification, a Smiles rearrangement, and hydrolysis. Further mutations and biochemical analyses with synthetic mimic substrates provide insight into the proposed catalytic process of the TE domain.

Cleavage Off-Loading and Post-assembly-Line Conversions Yield Products with Unusual Termini during Biosynthesis

“…Alternatively, PxbG Cy 3 catalyzes the β-sulfhydryl group of cysteinyl–S–T 5 forming a thioester with intermediate B, followed by a rapid intramolecular S- to N-acyl transfer, resulting in the amide bond formation. Atypical TE domains have been increasingly identified, exemplified by TE domains catalyzing transesterification, , Claisen condensation, and multiple reactions, and therefore, we tentatively assigned PxbG TE the role of off-loading the T domain-bound residue of cleavage products, which was indicated by the remarkable decrease in production titers of 3 – 5 in the PxbG TE S3793A mutant.…”

Depside Bond Formation by the Starter-Unit Acyltransferase Domain of a Fungal Polyketide Synthase

“…8 A recent report described the in-depth characterization of AN7909, a depside-forming PKS whose major product is diaryl ether diorcinolic acid and whose minor products are lecanoric acid and orsellinic acid. 9 Intriguingly, it was found that the formation of the depside bond and diaryl ether linkage are both catalyzed by the thioesterase (TE) domain of AN7909 (Figure 1B). However, it is unclear whether other PKSs that synthesize a depside as a major product are capable of TE-mediated formation of depside bonds.…”

“…We next sought to elucidate the biosynthesis of 1, particularly the mechanism of depside bond formation. Following a recent study on AN7909, 9 we initially hypothesized that the TE domain of DrcA is responsible for ester bond formation via 3-methylorsellinyl-and 3,5-dimethylorsellinyl-ACPs and for the subsequent chain hydrolysis required to release 1. To investigate this hypothesis, we synthesized the Nacetylcysteamine (NAC) thioesters of 3-MOA and DMOA, respectively, as surrogates of the ACP-bound reaction intermediates (Figure 3C), 14 and purified the TE domain of DrcA from an Escherichia coli expression system.…”

“…Importantly, we elucidated that the SAT domain of DrcA is involved in depside bond formation, which is unprecedented SAT domain chemistry. Although another fungal PKS, AN7909, was recently found to catalyze depside bond formation, 9 DrcA uses a unique strategy to create a depside bond. Our finding suggests that mining and characterizing unexploited fungal NR-PKSs may reveal additional SAT domains that are responsible for unusual chemical transformations.…”

Hot off the Press

“…A thioesterase domain from the fungal non-reducing polyketide synthase (PKS) AN7909 responsible for the biosynthesis of diorcinolic acid 24 has been shown to catalyse diaryl ether formation in a nonoxidative manner. 19 Biochemical analyses with a group of SNAC substrates revealed diaryl ether formation via a series of steps involving esterification, a Smiles rearrangement and hydrolysis. Biosynthetic studies of the mushroom Cortinarius odorifer have revealed a new class of non-reducing PKS involved in the biosynthesis of a key anthraquinone precursor, atrochrysone carboxylic acid 25 .…”