Mechanistic analysis of 6-deoxyerythronolide B synthase

描述（由申请人提供）：聚酮化合物是一个结构多样的天然产物大家族，具有广泛的生物活性。这些聚酮化合物，虽然结构复杂，立体化学丰富，优雅的生物系统中通过可预测的模块化聚酮化合物酶（PKS）的范例合成。PKS的模块化性质为新型化合物的工程化提供了重要的机会。然而，PKS的功能模块的合理开发目前受到限制的聚酮组装中所涉及的生化反应的不完全理解。拟议的研究的目标是确定动力学瓶颈，限制通过混合PKS的日益增长的聚酮化合物的处理。我们提出通过以下具体目标来实现这一目标：i）构建代表6-脱氧腺苷酸B合酶（DEBS）模块1和3的解离的双模块系统; ii）确定整个解离的双模块系统的反应动力学和每个不同的生化反应，以确定聚酮化合物加工中的限速步骤; iii）评估其他解离的双模杂合PKS中的速率限制生化反应，以确定聚酮化合物加工中的相同步骤是否在其他嵌合系统中产生瓶颈。这些目标将通过从DEBS PKS构建酰基载体蛋白结构域（ACP）和酮合酶-酰基转移酶双结构域（[KS][AT]）以构建解离的杂合PKS来实现。在这个系统中，域酰化和酮单元延伸的动力学将作为离散的催化事件，通过放射性SDS PAGE和放射性TLC进行研究。拟议的研究将确定目前利用嵌合PKS生产聚酮化合物的局限性。我们预计，拟议的研究结果将广泛适用于化学工程，提高目前的理解的基本原则，管理聚酮抗生素的生物合成。最终，我们希望我们的研究结果能够为合理设计具有潜在抗生素，抗肿瘤和其他生物活性的新型聚酮化合物提供重要工具。 公共卫生相关性：从拟议的机制研究的见解将被应用到识别和有效地产生新的聚酮化合物与生物活性相关的生物活性。

DESCRIPTION (provided by applicant): Polyketides are a large family of structurally diverse natural products with a broad range of biological activities. These polyketides, although structurally complex and rich in stereochemistry, are elegantly synthesized in biological systems via a predictable modular polyketide synthases (PKSs) paradigm. The modular nature of PKSs presents significant opportunities for the engineering of novel compounds. However, the rational exploitation of the functional modularity of PKSs is currently limited by an incomplete understanding of the biochemical reactions involved in the polyketide assembly. The goal of the proposed research is to identify the kinetic bottlenecks that limit the processing of a growing polyketide through hybrid PKSs. We propose to achieve this goal through the following Specific Aims: i) Construct a dissociated bimodular system that represents modules 1 and 3 of 6-deoxyerythronolide B synthase (DEBS); ii) Determine the reaction kinetics of the entire dissociated bimodular system and each distinct biochemical reaction to identify the rate-limiting steps in polyketide processing; ill) Evaluate the rate-liming biochemical reactions in other dissociated bimodular hybrid PKSs to ascertain if the same steps in polyketide processing create a bottleneck in other chimeric systems. These goals will be achieved by constructing acyl carrier protein domains (ACP) and keto synthase - acyl transferase didomains ([KS][AT]) from the DEBS PKS to build a dissociated hybrid PKS. Within this system, the kinetics of domain acylation and ketide-unit elongation will be studied as discrete catalytic events by radio-SDS PAGE and radio-TLC. The proposed research will identify the current limitations in harnessing chimeric PKSs for the production of polyketides. We anticipate that the results of the proposed study will be broadly applicable to chemical engineering by improving the current understanding of the fundamental principles that govern polyketide antibiotic biosynthesis. Ultimately, we expect our findings to contribute important tools for the rational engineering of novel polyketides with potential antibiotic, anti-tumor, and other pharmacologically relevant biological activities. PUBLIC HEALTH RELEVANCE: Insights from the proposed mechanistic study will be applied to identify and efficiently produce novel polyketides with pharmacologically relevant biological activities.