The Role of RapP in the Biosynthesis of Rapamycin

RapP 在雷帕霉素生物合成中的作用

• 批准号: 6693148
• 负责人: GREGORY J GATTO
• 金额: $ 3.97万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2006-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6693148
• 关键词: acid aminoacid ligase; biosynthesis; chemical bond; combinatorial chemistry; enzyme activity; enzyme substrate; pharmacology; postdoctoral investigator; sirolimus

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.

描述（由申请人提供）：该提案的重点是了解帕皮霉素生物合成途径中的蛋白酶结合酶Rapp的作用。雷帕霉素是一种具有有效免疫抑制特性的大环霉素，是通过聚酮化合物合酶（PKSS）和RAPP（非透叶胶肽合成酶（NRPS））的组合合成的。这些酶引起了很多关注，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PK/NRPS系统具有特别的兴趣，因为它们可以用于增加此类合成的多样性。 RAPP是一种1542氨基酸，四个结构域，160 kDa NRP，将氨基酸圆柱状添加到完整的聚酮化合物链中，然后循环链条形成大分子。为了理解Rapp的四个领域的功能，该提案的具体目的是：（a）使用放射性掺入分析鉴定腺苷酸化和肽基载体蛋白结构域的活性，（b）证明了EL糖基底物和果胶基因组合的肽之间的肽键的形成（b）使用多种合成制备的底物使用大分裂（C2结构域功能）。