20 糸状菌メロテルペノイドの複雑骨格構築に関わるα-ケトグルタル酸依存性ジオキシゲナーゼの構造機能解析 (口頭発表の部)


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‘ĥÙā

(1) Matsuda, Y., Abe, I. Nat. Prod. Rep. 2016, 33, 26. (2) Geris, R., Simpson, T. J. Nat. Prod. Rep. 2009, 26, 1063.

(3) Matsuda, Y., Awakawa, T., Wakimoto, T., Abe, I. J. Am. Chem. Soc. 2013, 135, 10962. (4) Matsuda, Y., Iwabuchi, T., Fujimoto, T., Awakawa, T., Nakashima, Y., Mori, T., Zhang, H., Hayashi, F., Abe, I. J. Am. Chem. Soc. 2016, 138, 12671.



Structure-Function Analysis of
-Ketoglutarate-dependent Dioxygenases Involved in Fungal Meroterpenoids Biosynthesis

Yu Nakashimaa, Takahiro Moria, Takayoshi Awakawaa, Shotaro Hoshinoa, Masahiro Okadaa, Miki Sendab, Toshiya Sendab, and Ikuro Abea

(a Grad. Sch. of Pham. Sci., Univ. of Tokyo, Japan, b

SBRC, IMSS, KEK, Japan)

Non-heme iron/
-ketoglutarate (
KG) dependent dioxygenases catalyse key oxidation reactions to afford complex molecular structures in the fungal meroterpenoid biosynthesis. AusE and PrhA are two dioxygenases that catalyse intriguing oxidative rearrangement reactions in austinol and paraherquonin biosynthetic pathways, respectively. AusE and PrhA have high sequence similarity (~78%), and both enzymes accept the same preaustinoid A1 substrate but generate different products, preaustinoid A3 and berkeleydione. Here we describe the X-ray crystal structures of apo AusE and PrhA, as well as PrhA complexed to Fe(II),
KG, and preaustinoid A1 at under 2.1 Å resolution. Comparison of the crystal structures revealed several key active site residues that are proximal to the substrate and are different in AusE and PhrA. Mutation of the identified PhrA active site residues to mimic those of AusE (A232S/V150L) resulted in a PrhA-A232S/V150L mutant that catalyses an AusE-type reaction to produce preaustinoid A3. Subsequently, we solved the X-ray crystal structure of PrhA-A232S/V150L complexed to Fe(II),
KG, and preaustinoid A1 or preaustinoid A2. In the co-crystal structure with preaustinoid A1, the distance between C2 of preaustinoid A1 and Fe(II) was reduced by 0.9 Å compared to the wild type PrhA, providing evidence for the position of the initial hydrogen abstraction. Furthermore, we observed that the C5 position of preaustinoid A2 is proximal to the catalytic Fe center in the co-crystal structure of the PrhA mutant with preaustinoid A2, indicating that the hydrogen abstraction at C5 triggers the sequential reaction to form the spirolactone ring of preaustinoid A3. We also generated PrhA-A232S/V150L/M241V triple mutant and found that in this mutant generates several novel compounds through four steps oxidation from preaustinoid A1. This study presents the structural basis of a novel reaction mechanism in multifunctional dioxygenases, in combination with mutagenesis studies that successfully led to the engineering of a new function for these enzymes.