Taguchi Takaaki, Awakawa Takayoshi, Nishihara Yukitaka, Kawamura Michiho, Ohnishi Yasuo, Ichinose Koji
Research Institute of Pharmaceutical Sciences, Musashino University, Shinmachi, Nishitokyo-shi, Tokyo, 202-8585, Japan.
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.
Chembiochem. 2017 Feb 1;18(3):316-323. doi: 10.1002/cbic.201600589. Epub 2017 Jan 12.
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.
II型聚酮合酶以迭代方式生成酰基载体蛋白（ACP）的新生聚酮硫酯；该硫酯会进行结构修饰，以产生一种不含ACP的中间体，进而生成最终代谢产物。然而，由于相关生物合成酶中缺乏明显的硫酯酶（TE），ACP卸载的时机尚不清楚。在这里，在放线紫红素（ACT）生物合成中被指定为第二环化酶（CYC）的ActIV，在体外对模型底物蒽醌-2-羧酸-N-乙酰半胱氨酸表现出TE活性。为了进一步研究其功能，在体外逐步重建了天蓝色链霉菌A3(2)中的ACT生物合成途径，直至生成（S）-DNPA，并将ActIV反应的产物表征为一种不含ACP的双环中间体。这些发现表明ActIV是一种双功能CYC-TE，并为中间体从ACP锚定物上释放的时机提供了明确证据。
