Studies on the Mechanism of Constructing Molecular Skeletons of Bioactive Polyether Metabolites

天然聚醚代谢物具有广泛的生物活性，已从土壤细菌(离子载体抗生素)、海洋甲藻(梯形聚醚毒素)、海洋藻类(三萜类)和番荔枝科植物(抗肿瘤乙酰基)等多种生物中分离出来。在聚醚代谢物的生物合成中，多烯前体被立体选择性地环氧化，然后随后的连续开环将形成聚醚骨架。在离子载体抗生素的生物合成中，用同位素标记的前体进行喂养实验支持了这一有见地的假设。最近关于莫能菌素生物合成的遗传学研究进一步支持了这一假说。尽管已经做出了巨大的努力来证明这一假说，但还没有获得关于这些酶转化的直接证据。本项目对拉沙利链霉菌的抗球虫药拉沙洛西德A进行了详细的生物合成研究，以阐明聚醚骨架的酶促结构。首先，我们成功地鉴定了拉沙洛西德生物合成基因簇，并开发了拉沙洛西德及其环氧化物的立体控制合成，这是拉索西德的生物合成前体。拉沙洛西德聚醚形成中最有趣的特征是能量上不受欢迎的6-内切选择性环化反应。我们在大肠杆菌中过表达的酶Lsd18和Lsd19对看似合理的生物合成前体的酶转化的研究明确地表明，拉沙洛西的复杂分子骨架是由相应的环氧化物与单一的环氧化物水解酶Lsd19顺序开环构建的。这是第一个在酶作用下形成聚醚骨架的例子。

Naturally occurring polyether metabolites show a broad range of biological activities and have been isolated from a number of organisms including soil bacteria (ionophore antibiotics), marine dinoflagelate (ladder polyether toxins), marine algae (triterpenes) and annonaceae plants (antitumor acetogenins). In the biosynthesis of polyether metabolites, it is proposed that a polyene precursor is stereoselectively epoxidized and then the subsequent sequential ring openings would afford polyether skeleton. This insightful hypothesis was supported by feeding experiments with isotopically labeled precursors in the biosynthesis of ionophore antibiotics. Recent genetic studies on monensin biosynththesis provided further supports for this hypothesis. Although enormous efforts to prove this hypothesis have been made, direct evidence on these enzymatic transformations has not been obtained. In this project, detailed biosynthetic studies on lasalocid A, an anticoccidium agent from Stretomyces lasaliensis, has carried out to elucidate the enzymatic construction of polyether skeleton.At first, we have succeeded identification of lasalocid biosynthetic gene cluster spanning 82 kb, and have also developed the stereocontroled synthesis of prelasalocid and its epoxide, which are plausible biosynthetic precursors of lasalocid. The most interesting feature in the polyether formation of lasalocid is an energetically disfavored 6-endo selective cyclization. Our efforts on enzymatic conversions of plausible biosynthetic precursors with the enzymes Lsd18 and Lsd19 overexpressed in Escherichia coli unambiguously showed that complex molecular skeleton of lasalocid is constructed by a sequencial ring opening of the corresponding epoxides with a single epoxide hydrolase Lsd19. This is the first example of the enzymatic formation of polyether skeleton.