Recently, the prestigious international journal Journal of the American Chemical Society (JACS) published online the collaborative research findings of Professor Xu-Dong Qu's team from the School of Life Sciences and Technology/Zhangjiang Advanced Research Institute at Shanghai Jiao Tong University and Professor Shu-Kun Luo's team from the School of Life Sciences and Technology. The paper, titled “A Stereoselective Decarboxylative Aromatase/Cyclase Directs the Biosynthesis of an Axially Chiral Biphenyl Framework in Fasamycin,” details the discovery of the biosynthetic pathway for an axially chiral biphenyl framework in fasamycin. Professor Xu-Dong Qu and Distinguished Research Fellow Shu-Kun Luo served as co-corresponding authors, with postdoctoral researcher Jiang Kai and doctoral candidate Cheng Zhu as co-first authors.

Aromatic polyketides represent a diverse class of natural products exhibiting multiple biological activities. Their biosynthesis is primarily catalyzed by type II polyketide synthases (PKSs), wherein minimal PKSs assemble polyketide chains, followed by aromatization/cyclization reactions catalyzed by aromatase/cyclase (ARO/CYC) enzymes to form fundamental aromatic skeletons. These skeletons serve as templates for further PKS post-modifications, conferring the complex structural diversity of aromatic polyketides. However, despite numerous post-modifications, only eight distinct planar conjugated structures have been identified for the basic aromatic skeleton (Figure 1). Therefore, discovering ARO/CYC enzymes capable of catalyzing novel cyclization patterns is crucial for expanding the structural and functional diversity of aromatic polyketides.

Fasamycin (FAS) and its analogues represent a class of aromatic polyketides garnering significant attention for their potent activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE). Unlike typical planar aromatic skeletons, FAS possesses a unique axial-chiral biphenyl skeleton (Figure 1), suggesting its biosynthesis involves a novel stereoselective ARO/CYC reaction and a distinctive biosynthetic mechanism. The research group recently elucidated the formation of the phenyldimethylanthrone (PDA) core in FAS, which is generated via a tetrahydrocyclization reaction catalyzed by a TcmI-type ARO/CYC (FasL) and influenced by a non-essential auxiliary ARO/CYC (FasD) (Proc. Natl. Acad. Sci. USA 2024, 121, e2321722121, Fig. 2). However, the mechanism governing the formation of the axial chiral biphenyl structure (i.e., the E ring) remains unclear.

Figure 1. Basic aromatic skeleton of type II aromatic polyketones. FAS exhibits a significantly distinct axial-chiral skeleton compared to other types.

In this study, the collaborative research team first reconstructed the biosynthetic pathway for the PDA skeleton shared by ABX and FAS pathways in vitro. By further optimizing the pH and temperature of the in vitro enzyme reaction system and incorporating ¹³C isotope labeling, they discovered that the previously unidentified biosynthetic intermediate 1 is the true substrate for FasU. In vitro experiments and in vivo complementation studies confirmed that FasU, a monooxygenase family protein, functions as a novel aromatase/cyclase (ARO/CYC) enzyme. It catalyzes the sequential decarboxylation and cyclization/aromatization of intermediate 1 with strict S-stereospecificity, thereby forming the axial-chiral biphenyl skeleton in FAS. To gain deeper insights into the unique decarboxylation/aromatization/cyclization/stereoselective mechanism of ARO/CYC, the collaborative research team further determined the crystal structure of FasU. Through site-directed mutagenesis of the FasU active site combined with molecular dynamics (MD) analysis, key catalytic residues were identified, confirming that FasU's stereospecificity arises from hydrophobic repulsion between the binding pocket and the C21 and C23 hydroxyl groups. This work not only achieved a major breakthrough in the diversity of ARO/CYC reactions on type II aromatic polyketide skeletons but also provides important insights for generating novel ARO/CYC and axial-chiral aromatic polyketide skeletons in the future.

Figure 2. FAS biosynthetic gene cluster and biosynthetic pathway. (A) FAS biosynthetic gene cluster. (B) FAS biosynthetic pathway and its derivatives.

This research was supported by the National Natural Science Foundation of China (32425033, 22377074, 22107069), the Shanghai Outstanding Academic Leader Program (22XD1421300), and the Shanghai Natural Science Foundation (23ZR1432800).

Paper link: https://pubs.acs.org/doi/10.1021/jacs.4c18376

Author: Xu Dong Qu's Team

Contributing Unit: Center for Innovation in Synthetic Science