Department of Chemical and Biomolecular Engineering, University of California Los Angeles, 420 Westwood Plaza, Los Angeles, California 90095, United States.
J Am Chem Soc. 2011 Oct 5;133(39):15773-85. doi: 10.1021/ja206906d. Epub 2011 Sep 14.
The linear tetracyclic TAN-1612 (1) and BMS-192548 (2) were isolated from different filamentous fungal strains and have been examined as potential neuropeptide Y and neurokinin-1 receptor antagonists, respectively. Although the biosynthesis of fungal aromatic polyketides has attracted much interest in recent years, the biosynthetic mechanism for such naphthacenedione-containing products has not been established. Using a targeted genome mining approach, we first located the ada gene cluster responsible for the biosynthesis of 1 in Aspergillus niger ATCC 1015. The connection between 1 and the ada pathway was verified through overexpression of the Zn(2)Cys(6)-type pathway-specific transcriptional regulator AdaR and subsequent gene expression analysis. The enzymes encoded in the ada gene cluster share high sequence similarities to the known apt pathway linked to the biosynthesis of anthraquinone asperthecin 3. Subsequent comparative investigation of these two highly homologous gene clusters by heterologous pathway reconstitution in Saccharomyces cerevisiae revealed a novel α-hydroxylation-dependent Claisen cyclization cascade, which involves a flavin-dependent monooxygenase that hydroxylates the α-carbon of an acyl carrier protein-bound polyketide and a bifunctional metallo-β-lactamase-type thioesterase (MβL-TE). The bifunctional MβL-TE catalyzes the fourth ring cyclization to afford the naphthacenedione scaffold upon α-hydroxylation, whereas it performs hydrolytic release of an anthracenone product in the absence of α-hydroxylation. Through in vitro biochemical characterizations and metal analyses, we verified that the apt MβL-TE is a dimanganese enzyme and requires both Mn(2+) cations for the observed activities. The MβL-TE is the first example of a thioesterase in polyketide biosynthesis that catalyzes the Claisen-like condensation without an α/β hydrolase fold and forms no covalent bond with the substrate. These mechanistic features should be general to the biosynthesis of tetracyclic naphthacenedione compounds in fungi.
线性四环 TAN-1612（1）和 BMS-192548（2）分别从不同丝状真菌菌株中分离出来，被分别鉴定为潜在的神经肽 Y 和神经激肽-1 受体拮抗剂。尽管近年来真菌芳香聚酮的生物合成引起了广泛关注，但这种含萘醌二酮的产物的生物合成机制尚未建立。通过靶向基因组挖掘方法，我们首先在黑曲霉 ATCC 1015 中定位了负责合成 1 的 ada 基因簇。通过过表达 Zn(2)Cys(6)-型途径特异性转录调节剂 AdaR 并随后进行基因表达分析，验证了 1 与 ada 途径的联系。ada 基因簇中编码的酶与已知的 apt 途径具有很高的序列相似性，该途径与蒽醌类化合物 asperthecin 3 的生物合成有关。随后，在酿酒酵母中通过异源途径重建对这两个高度同源的基因簇进行比较研究，揭示了一种新的α-羟化依赖性 Claisen 环化级联反应，其中涉及一种黄素依赖性单加氧酶，该酶羟化酰基辅酶 A 结合的聚酮的α-碳，以及一种双功能金属β-内酰胺酶型硫酯酶（MβL-TE）。双功能 MβL-TE 在α-羟化后催化第四环环化，生成萘醌二酮骨架，而在没有α-羟化的情况下，它会水解释放出蒽醌产物。通过体外生化特性和金属分析，我们验证了 apt MβL-TE 是一种二锰酶，并且观察到的活性需要两个 Mn(2+)阳离子。MβL-TE 是聚酮生物合成中第一个催化 Claisen 样缩合而没有 α/β 水解酶折叠并与底物不形成共价键的硫酯酶。这些机制特征应该适用于真菌中环四萘醌二酮化合物的生物合成。
