3 糸状菌ポリケタイド合成酵素の機能解析 : Aspergillus fumigatsuのテトラヒドロキシナフタレン合成酵素系(口頭発表の部)

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Aspergillus fumigatus is an important opportunistic pathogen causing aspergillosis. One of its virulence factors, DHN-melanin, is biosynthesized from pentaketide T4HN. We recently reported that naphthopyrone YWA1, a product of heptaketide synthase Alblp, is converted to pentaketide T4HN by a chain-length shortening enzyme Ayglp. To characterize this novel T4HN biosynthesis system in A. fumigatus, functional studies on Alblp and Ayglp were carried out. Alblp is a typical iterative type I PKS with KS, AT, tandem ACPs, and CYC domains. We first established the functional expression system of this fungal PKS in yeast using pYES-DEST52 expression vector with co-expression of Bacillus subtilis sfp. The yeast transformant produced heptaketide naphthopyrone YWA1 by GAL1 promoter induction. The Alblp C-terminus deletion experiments revealed that the deletion mutant Cd-3 which lacked ACP2 and CYC still produced heptaketide DHCI. Co-expression of Cd-3 with C-terminus region reconstituted the naphthopyrone YWA1 production, indicating that CYC domain can work as a separate enzyme on the heptaketide intermediate anchored on PKS. The Ayglp was purified to homogeneity from the Aspergillus oryzae overexpression transformant. The purified Ayglp converted the heptaketide naphthopyrone YWA1 to the pentaketide T4HN with a release of the diketide acetoacetic acid. Ayglp showed strict substrate specificity to the heptaketide YWA1 and was strongly inhibited by the serine protease inhibitors. Site-directed mutagenesis confirmed that the Ser^<257> residue is the active site of the enzyme. Thus, Ayglp appears to catalyze the hydrolytic cleavage of the C-C bond between the naphthalene ring and the side-chain carbonyl of YWA1 which is attacked by a hydroxyl anion of the Ser^<257> in the catalytic center.
天然有機化合物討論会の論文
2003-09-01

3 糸状菌ポリケタイド合成酵素の機能解析 : Aspergillus fumigatsuのテトラヒドロキシナフタレン合成酵素系(口頭発表の部)

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