The Role of RapP in the Biosynthesis of Rapamycin

描述（由申请人提供）：该提案的重点是了解管道酸结合酶RapP在雷帕霉素生物合成途径中的作用。雷帕霉素是由多酮合成酶（pks）和非核糖体肽合成酶RapP （NRPS）合成的一种具有强免疫抑制特性的天然大环产物。这些酶引起了广泛的关注，因为它们的模块化性质可能为定向组合合成提供了机会。特别是，混合PKS/NRPS系统特别令人感兴趣，因为它们可以用来增加这种合成的多样性。RapP是一种含有1542个氨基酸，4个结构域，160 kDa的NRPS，它将氨基酸胡椒酸酯添加到完整的聚酮链上，然后将该链环化形成大内酯。为了理解RapP的四个领域的功能，本提案的具体目标是：(A)利用放射性结合试验表征腺苷化和肽基载体蛋白结构域的活性，(B)证明酰基底物和管聚酶中间体之间肽键的形成（C1结构域功能），(C)利用各种合成制备的底物阐明大内酯化的决定因素（C2结构域功能）。

DESCRIPTION (provided by applicant): This proposal is focused on understanding the role of the pipecolate-incorporating enzyme RapP in the rapamycin biosynthetic pathway. Rapamycin, a macrocyclic natural product with potent immunosuppressive properties, is synthesized by a combination of polyketide synthases (PKSs) and RapP, a nonribosomal peptide synthetase (NRPS). These enzymes have generated much attention because their modular nature may provide the opportunity for directed combinatorial syntheses. In particular, hybrid PKS/NRPS systems are of special interest as they could be utilized to increase the diversity of such syntheses. RapP is a 1542 amino acid, four domain, 160 kDa NRPS that adds the amino acid pipecolate to the completed polyketide chain, then cyclizes the chain to form the macrolactone. With the goal of understanding the function of each of the four domains of RapP, the specific aims of this proposal are to: (A) characterize the activity of the adenylation and peptidyl carrier protein domains using radioactivity incorporation assays, (B) demonstrate the formation of a peptide bond between acyl substrates and the pipecolyl-enzyme intermediate (C1 domain function), and (C) elucidate the determinants of macrolactonization (C2 domain function) using a variety of synthetically prepared substrates.