Bifunctionality of ActIV as a Cyclase-Thioesterase Revealed by in Vitro Reconstitution of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

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• 作者：Dr. Takaaki Taguchi ; Dr. Takayoshi Awakawa ; Yukitaka Nishihara ; Michiho Kawamura ; Prof. Dr. Yasuo Ohnishi and Prof. Dr. Koji Ichinose
• 刊名：ChemBioChem
• 出版年：2017
• 出版时间：February 1, 2017
• 年：2017
• 卷：18
• 期：3
• 页码：316-323
• 全文大小：1241K
• ISSN：1439-7633

文摘

Type II polyketide synthases iteratively generate a nascent polyketide thioester of the acyl carrier protein (ACP); this is structurally modified to produce an ACP-free intermediate towards the final metabolite. However, the timing of ACP off-loading is not well defined because of the lack of an apparent thioesterase (TE) among relevant biosynthetic enzymes. Here, ActIV, which had been assigned as a second ring cyclase (CYC) in actinorhodin (ACT) biosynthesis, was shown to possess TE activity in vitro with a model substrate, anthraquinone-2-carboxylic acid-N-acetylcysteamine. In order to investigate its function further, the ACT biosynthetic pathway in Streptomyces coelicolor A3(2) was reconstituted in vitro in a stepwise fashion up to (S)-DNPA, and the product of ActIV reaction was characterized as an ACP-free bicyclic intermediate. These findings indicate that ActIV is a bifunctional CYC-TE and provide clear evidence for the release timing of the intermediate from the ACP anchor.