title: Bifunctionality of ActIV as a Cyclase-Thioesterase Revealed by in Vitro Reconstitution of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2).
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.
authors.affiliation: Research Institute of Pharmaceutical Sciences, Musashino University, Shinmachi, Nishitokyo-shi, Tokyo, 202-8585, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Taguchi
authors.affiliation: Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.|Present address: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-8657, Japan.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Awakawa
authors.affiliation: Research Institute of Pharmaceutical Sciences, Musashino University, Shinmachi, Nishitokyo-shi, Tokyo, 202-8585, Japan.
authors.forename: Yukitaka
authors.initials: Y
authors.lastname: Nishihara
authors.affiliation: Research Institute of Pharmaceutical Sciences, Musashino University, Shinmachi, Nishitokyo-shi, Tokyo, 202-8585, Japan.
authors.forename: Michiho
authors.initials: M
authors.lastname: Kawamura
authors.affiliation: Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ohnishi
authors.affiliation: Research Institute of Pharmaceutical Sciences, Musashino University, Shinmachi, Nishitokyo-shi, Tokyo, 202-8585, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Ichinose
mesh_terms: D000880:Anthraquinones / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D008316:Malonyl Coenzyme A / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D048630:Polyketide Synthases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D048372:Streptomyces coelicolor / Q000235:genetics / Q000378:metabolism*; D013869:Thiolester Hydrolases / Q000235:genetics / Q000378:metabolism; D000095622:Benzoisochromanequinones
chemical_list: D000880:Anthraquinones; D001426:Bacterial Proteins; D008316:Malonyl Coenzyme A; D010088:Oxidoreductases; D048630:Polyketide Synthases; D011994:Recombinant Proteins; D013869:Thiolester Hydrolases; C013390:actinorhodin; D000095622:Benzoisochromanequinones
keywords: Streptomyces; biosynthesis; cyclization; hydrolases; polyketides