Bifunctionality of ActIV as a Cyclase‐Thioesterase Revealed by in Vitro Reconstitution of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2) |
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| Authors: | Dr. Takaaki Taguchi Dr. Takayoshi Awakawa Yukitaka Nishihara Michiho Kawamura Prof. Dr. Yasuo Ohnishi Prof. Dr. Koji Ichinose |
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| Affiliation: | 1. Research Institute of Pharmaceutical Sciences, Musashino University, Nishitokyo-shi, Tokyo, Japan;2. Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan;3. Present address: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan |
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| Abstract: | 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. |
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| Keywords: | biosynthesis cyclization hydrolases polyketides Streptomyces |
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