The Role of RapP in the Biosynthesis of Rapamycin

描述(由申请人提供)：这项提案的重点是了解吡哌酸酯结合酶RAPP在雷帕霉素生物合成途径中的作用。雷帕霉素是由聚酮合成酶(PKS)和非核糖体多肽合成酶(NRPS)结合而成的具有免疫抑制作用的大环天然产物。这些酶已经引起了人们的极大关注，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，PKS/NRPS混合体系特别令人感兴趣，因为它们可以用来增加此类合成的多样性。RAPP是一个1542个氨基酸，四个结构域，160 kDa的NRPS，它将氨基酸哌甲酸添加到完整的聚酮链上，然后环化链形成大内酯。为了了解RAPP的四个结构域中的每一个的功能，本建议的具体目的是：(A)利用放射性掺入分析来表征腺化和多肽载体蛋白结构域的活性，(B)证明酰基底物和管道酰基酶中间体(C1域功能)之间形成多肽键，以及(C)使用各种合成的底物来阐明大内酯化的决定因素(C2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.