Mechanistic analysis of 6-deoxyerythronolide B synthase

描述（由申请人提供）：聚酮类化合物是一大类结构多样的天然产物，具有广泛的生物活性。这些聚酮虽然结构复杂且具有丰富的立体化学性质，但通过可预测的模块化聚酮合成酶（pks）模式，可以在生物系统中优雅地合成。pks的模块化特性为新化合物的工程设计提供了重要的机会。然而，由于对聚酮组装过程中所涉及的生化反应的不完全了解，目前对pks功能模块化的合理利用受到限制。提出的研究目标是确定动力学瓶颈，限制通过混合PKSs生长的聚酮的处理。我们建议通过以下具体目标来实现这一目标：i)构建一个解离双模系统，代表6-脱氧红素内酯B合成酶（DEBS）的模块1和模块3；ii)确定整个解离双模体系的反应动力学和每个不同的生化反应，以确定聚酮加工中的限速步骤；ii)评估其他解离双模杂交PKSs中的速率限制生化反应，以确定聚酮加工的相同步骤是否会在其他嵌合体系中造成瓶颈。这些目标将通过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]）来实现，构建解离杂交PKS。在该系统中，区域酰化和酮基单元延伸的动力学将通过放射性sds - PAGE和放射性tlc作为离散催化事件进行研究。拟议的研究将确定目前利用嵌合pks生产聚酮的局限性。我们预计，通过提高目前对控制聚酮类抗生素生物合成的基本原理的理解，所提出的研究结果将广泛适用于化学工程。最终，我们希望我们的发现能为合理设计具有潜在抗生素、抗肿瘤和其他药理学相关生物活性的新型聚酮提供重要工具。

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.