Mechanistic analysis of 6-deoxyerythronolide B synthase

6-脱氧赤酮内酯B合酶的机理分析

• 批准号: 8131690
• 负责人: Louise Karine Charkoudian
• 金额: $ 5.3万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8131690
• 关键词: 6 Deoxyerythronolide B Synthase; Acyl Carrier Protein; Acylation; Anabolism; Antibiotics; Biochemical Reaction; Biological; Biological Factors; Chemical Engineering; Complex; Engineering; Event; Family; Goals; Hybrids; Kinetics; Limes; Nature; Process; Production; Radio; Reaction; Research; Sodium Dodecyl Sulfate-PAGE; System; Tertiary Protein Structure; Transferase; biological systems; improved; insight; novel; polyketide synthase; public health relevance; stereochemistry; tool; tumor

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.

描述（由申请人提供）：聚酮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。这些聚酮化合物虽然结构复杂且立体化学丰富，但可以通过可预测的模块化聚酮化合物合酶（PKS）范式在生物系统中优雅地合成。 PKS 的模块化性质为新型化合物的工程设计提供了重要的机会。然而，目前对 PKS 功能模块性的合理开发由于对聚酮化合物组装中涉及的生化反应的不完全理解而受到限制。拟议研究的目标是确定限制通过混合 PKS 加工生长聚酮化合物的动力学瓶颈。我们建议通过以下具体目标来实现这一目标： i) 构建一个代表 6-脱氧赤酮内酯 B 合酶 (DEBS) 模块 1 和 3 的解离双模系统； ii) 确定整个解离双模系统和每个不同生化反应的反应动力学，以确定聚酮化合物加工中的限速步骤； ill) 评估其他解离双模混合 PKS 中的限速生化反应，以确定聚酮化合物加工中的相同步骤是否会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。在该系统中，域酰化和酮化物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 生产聚酮化合物的局限性。我们预计，通过提高目前对控制聚酮化合物抗生素生物合成基本原理的理解，拟议研究的结果将广泛适用于化学工程。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、抗肿瘤和其他药理学相关生物活性的新型聚酮化合物提供重要工具。 公共健康相关性：拟议的机理研究的见解将用于识别和有效生产具有药理学相关生物活性的新型聚酮化合物。