The Ketoreduction and Cyclization of Aromatic Polyketide Biosynthesis

描述(由申请人提供)：此R01修订申请回答RFA NOT-OD-09-058(通知标题：NIH宣布恢复法案资金可用于竞争性修订申请)。这是R01授权GM076330的修订申请，题为“芳香族聚酮生物合成的酮还原和环化”，其具体目的是1.确定酮还原酶(KR)的序列-结构-功能关系，从而导致其独特的还原专一性。2.确定了芳香酶/环化酶(ARO/CyC)的序列-结构-功能关系，这导致了其独特的环化专一性。3.确定蛋白质相互作用在KR和ARO/CyC序列-结构-功能关系中的重要性。我们高兴地报告，我们在实现这三个目标方面的进展已经超过了拟议的时间表。为了进一步加快理解聚酮还原和环化的研究步伐，我们提出了以下三个不重叠的目标，而是与父R01目标相辅相成： 目的1.用化学探针法确定酮还原反应的分子基础。 目的：用化学探针法确定芳香族聚酮环化反应的分子基础。 目的3.解决化学交联的多域PKS络合物的晶体结构。这项拟议的研究意义重大，因为其结果将回答有关如何精确控制聚酮还原和环化的重要问题。它还在分子水平上提供了关于KR和ARO/CyC的新信息，这也是创新之处。长期的生物医学意义在于，聚酮研究界可以应用这一建议确定的序列-结构-功能关系，通过蛋白质工程使“非天然”天然产物的种群多样化，从而可以产生具有不同酮还原和环化模式的新型聚酮文库。这次修订将加快聚酮合成酶(PKS)的科学研究步伐，允许为三名研究生创造和保留就业机会，并聘请一名新的博士后研究员，并采购实验室设备，以加快拟议研究的节奏。 与公共健康相关：这预计将对公共健康产生积极影响，因为它将允许开发新的“非天然”天然产品，可以为新的药物活动进行筛选。同时，这项建议中关于PKS序列结构与聚酮酮还原和环化过程中的催化、底物专一性和蛋白质-蛋白质相互作用的基本新知识有望对天然产物研究界产生重大影响。

DESCRIPTION (provided by applicant):This R01 revision application answers RFA NOT-OD-09-058 (Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications). This is a revision application for the R01 grant GM076330, titled "The Ketoreduction and Cyclization of Aromatic Polyketide Biosynthesis", whose specific aims were 1. Determine the sequence-structure-function relationship of ketoreductase (KR) that leads to its unique reduction specificity. 2. Determine the sequence-structure-function relationship of aromatase/cyclase (ARO/CYC) that leads to its unique cyclization specificity. 3. Determine the importance of protein-protein interactions on the sequence-structure-function relationship between KR and ARO/CYC. We were pleased to report that our progress towards these three aims has exceeded the proposed timelines. In order to further accelerate the research pace towards understanding polyketide ketoreduction and cyclization, we proposed the following three aims that do not overlap, but rather complementary to the parent R01 aims: AIM 1. Determine the molecular basis of ketoreduction using chemical probes. AIM 2. Determine the molecular basis of aromatic polyketide cyclization using chemical probes. AIM 3. Solve crystal structures of chemically cross-linked multi-domain PKS complexes. The proposed research is significant, because the outcome will answer important questions about how polyketide reduction and cyclization are precisely controlled. It is also innovative by providing new information about KR and ARO/CYC at a molecular level not achieved previously. The long-term biomedical relevance is that the polyketide research community can apply the sequence-structure-function relationships determined from this proposal to diversify the population of "unnatural" natural products via protein engineering, such that a library of novel polyketides with different ketoreduction and cyclization patterns can be produced. The revision will accelerate the tempo of scientific research on polyketide synthase (PKS) and allow for job creation and retention for three graduate students and the hiring of one new postdoctoral researcher, and procuring laboratory equipments to accelerate the tempo of the proposed research. PUBLIC HEALTH RELEVANCE: This is expected to positively affect public health, because it will allow the development of new "unnatural" natural products that can be screened for new pharmaceutical activities. Meanwhile, the fundamental new knowledge obtained in this proposal on correlating PKS sequence-structure with the catalysis, substrate specificity and protein-protein interactions during polyketide ketoreduction and cyclization is expected to have a high impact on the natural product research communities.