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

被引:27
|
作者
Taguchi, Takaaki [1 ]
Awakawa, Takayoshi [2 ,3 ]
Nishihara, Yukitaka [1 ]
Kawamura, Michiho [1 ]
Ohnishi, Yasuo [2 ]
Ichinose, Koji [1 ]
机构
[1] Musashino Univ, Pharmaceut Sci Res Inst, Shinmachi, Nishitokyo, Tokyo 2028585, Japan
[2] Univ Tokyo, Grad Sch Agr & Life Sci, Bunkyo Ku, Tokyo 1130032, Japan
[3] Univ Tokyo, Grad Sch Pharmaceut Sci, Bunkyo Ku, Tokyo 1138657, Japan
关键词
biosynthesis; cyclization; hydrolases; polyketides; Streptomyces; POLYKETIDE SYNTHASE; GENE-CLUSTER; ANTIBIOTIC ACTINORHODIN; ENGINEERED BIOSYNTHESIS; BETA-LACTAMASE; HETEROLOGOUS EXPRESSION; NUCLEOTIDE-SEQUENCE; VIOLACEORUBER TU22; DEDUCED FUNCTIONS; RING FORMATION;
D O I
10.1002/cbic.201600589
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 offloading 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.
引用
收藏
页码:316 / 323
页数:8
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