[P002] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P01-070] Functional analysis of unprecedented thioesterases in highly reducing type II polyketide synthase systems

○Kota Moriga¹, Seiji Kawai¹, Yohei Katsuyama^1,2, Yasuo Ohnishi^1,2 (1. Grad. Sch. of Agric. and Life Sci., The Univ. of Tokyo (Japan), 2. CRIIM, The Univ. of Tokyo (Japan))

Keywords:

Polyketide synthase,Thioesterase,iron-sulfur cluster,polyketide,Actinomycetes,biosynthesis,natural products,secondary metabolite

Chain-releasing reactions play a crucial role in polyketide biosynthesis because they control the chain length of the final product and provide versatile modifications. However, our understanding of chain-releasing reactions differs among polyketide synthase (PKS) systems, and the chain-releasing mechanisms of type II PKSs remain largely unknown. In this study, we report the identification of unprecedented iron–sulfur cluster-containing thioesterases (TEs) in highly reducing type II PKS systems involved in aryl-diene biosynthesis, namely, AvaPKS and AzoPKS, which are responsible for producing avenalumic acid^[1] and azoxymycin,^[2] respectively. By heterologous expression and in vitro analysis, a hypothetical protein, AzoE, was shown to be important for mature polyketide chain-releasing reaction. The azoE homologues are conserved in several highly reducing type II PKSs. And in AvaPKS, avaB is included as the homolog gene. Notably, purified recombinant AvaB and AzoE exhibited a brown color, indicating the presence of iron-sulfur clusters. Next, we attempted to solve the structure of the AvaB–AvaA2 (TE–acyl carrier protein [ACP]) complex. To obtain the AvaB–AvaA2 complex, we developed a maleimide-based cross-linking method by introducing a Cys residue in the active site. The obtained complex was applied to cryo-electron microscopy single-particle analysis. As a result, we succeeded in solving the structure of the AvaB–AvaA2 complex at a resolution of 3.78 Å. This is the first report of the TE–ACP complex structure in PKSs. Based on the complex structure, we performed Ala scanning into the interface of AvaB and AvaA2. By quantifying the cross-linking efficiency, several residues important for the AvaB–AvaA2 interaction were identified. Because these unprecedented TEs do not show amino acid sequence similarities to known hydrolase families, we expected that they have unreported reaction mechanisms. Therefore, we attempted to elucidate the reaction mechanism of these TEs by using AzoE as a model enzyme because it accepted N-acetylcysteamine (NAC) thioesters as a substrate. We conducted site-directed mutagenesis into the substrate-binding pocket of AzoE, suggesting that His32, Asp33, Asn53, and Tyr136 play important roles. Among them, Asp33 had the most critical function. We propose a unique hydrolysis mechanism involving hydroxide ion activation by a conserved Asp–His diad.

References
[1] S. Kawai et. al., Angew. Chem. 61:e202211728 (2022)
[2] Y.-Y. Guo et. al., Organic letters. 17, 6114–6117 (2015)