Specificity and Activity of Fungal Polyketide-Nonribosomal Peptide Synthases

真菌聚酮化合物-非核糖体肽合成酶的特异性和活性

• 批准号: 8527480
• 负责人: ELIZABETH PIERCE
• 金额: $ 4.92万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8527480
• 关键词: Acids; Aldehydes; Amino Acids; Bacteria; Binding; Biological Factors; Categories; Chemicals; Cholesterol; Complex; Cyclization; Data; Drug Design; Drug Targeting; Drug usage; Engineering; Enzymes; Genes; Goals; HIV-1; Hybrids; Integrase; Keto Acids; Kinetics; Learning; Lovastatin; Mutagenesis; Mutate; Organism; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Physical condensation; Property; Reaction; Research; Role; Serum; Site; Specificity; Structure; Substrate Specificity; System; Testing; Work; cross reactivity; design; drug candidate; equisetin; fungus; hybrid enzyme; improved; interest; non-ribosomal peptide synthase; polyketide synthase; public health relevance; research study

DESCRIPTION (provided by applicant): Fungal natural products are complex molecules containing a wide range of chemical motifs. Many of these natural products are involved in pathogenesis, and many have pharmacological activity. This proposal focuses on the biosynthetic pathway of one natural product, equisetin. The goal of the research is to gather specific information that will be useful in efforts to make precise alterations to the biosynthetic pathway that will result in alterations to the structure of equisetin. Like many other related molecules, equisetin is toxic and interacts specifically with an important drug target (HIV-1 integrase, in the case of equisetin). The ability to alter the structures of equisetin and related molecules would assist in making drug candidates that are less generally toxic or are more effective against specific targets. The category of fungal natural products that includes equisetin is produced by hybrid polyketide synthase/non-ribosomal peptide synthase (PKS-NRPS) enzymes. These large enzymes contain many domains. Two of the domains are used to attach the growing equisetin molecule to the enzyme during its synthesis. Each other domain catalyzes a specific reaction in the synthesis of the equisetin molecule. PKS-NRPS products are made up of a polyketide (produced by the PKS) to which the NRPS adds an amino acid. The NRPS consists of four domains. One domain (T) is a carrier for the equisetin molecule, a second domain (A) is required for activating the amino acid substrate, a third (C) catalyzes the attachment of the amino acid to the polyketide, and the last domain (R) catalyzes the release of the product from the carrier domain. My specific aims are 1) to test the role of each equisetin synthase NRPS domain in determining which amino acid is used as a substrate, 2) to identify regions and specific residues in the domains that can be mutated to alter substrate specificity and 3) to test whether other fungal PKS-NRPS enzymes' R domains catalyze product release by different mechanisms. The experimental approach will involve expression and purification of the four equisetin synthase NRPS domains along with the carrier domain from the PKS, and R domains from two other PKS-NRPS enzymes. In the first specific aim, I will learn about the amino acid substrate specificities of the different domains, with the goal of understanding which parts of the whole PKS-NRPS are responsible for favoring the particular amino acid that is incorporated into equisetin, and whether different amino acids could be incorporated as well. In the second part of the research, I will create hybrid NRPS enzymes with domains from equisetin synthase and a closely related NRPS, and use random and directed mutagenesis to improve the activities of these hybrid enzymes. In the third specific aim, I will test R domains from other PKS-NRPS enzymes for a release mechanism different from the mechanism used by the equisetin synthase R domain, and will combine the catalytic activities of R domains from different synthases with the EqiS condensation and adenylation domains.

描述（由申请人提供）：真菌天然产物是含有多种化学基序的复杂分子。许多这些天然产物参与发病机制，并且许多具有药理活性。该提案重点关注一种天然产物木贼黄素的生物合成途径。该研究的目的是收集有助于对生物合成进行精确改变的具体信息 途径将导致木贼黄素结构的改变。与许多其他相关分子一样，木贼黄素具有毒性，并且与重要的药物靶点（就木贼黄素而言，为 HIV-1 整合酶）发生特异性相互作用。改变木贼黄素和相关分子结构的能力将有助于制造毒性较小或针对特定靶点更有效的候选药物。包含木贼黄素的真菌天然产物类别 由杂合聚酮合酶/非核糖体肽合酶 (PKS-NRPS) 酶产生。这些大酶包含许多结构域。其中两个结构域用于在合成过程中将不断生长的木贼素分子附着到酶上。每个其他结构域都催化木贼黄素分子合成中的特定反应。 PKS-NRPS 产品由聚酮化合物（由 PKS 生产）制成，其中 NRPS 添加了氨基酸。 NRPS 由四个领域组成。第一个结构域 (T) 是木贼黄素分子的载体，第二个结构域 (A) 是激活氨基酸底物所必需的，第三个结构域 (C) 催化氨基酸与聚酮化合物的连接，最后一个结构域 (R) 催化产物从载体结构域中释放。我的具体目标是 1) 测试每个木贼素合酶 NRPS 结构域在确定使用哪种氨基酸作为底物时的作用，2) 识别结构域中可以突变以改变底物特异性的区域和特定残基，3) 测试其他真菌 PKS-NRPS 酶的 R 结构域是否通过不同机制催化产物释放。实验方法将涉及四个木贼素合酶 NRPS 结构域以及来自 PKS 的载体结构域和来自其他两种 PKS-NRPS 酶的 R 结构域的表达和纯化。在第一个具体目标中，我将了解不同结构域的氨基酸底物特异性，目的是了解整个 PKS-NRPS 的哪些部分负责有利于掺入木贼素中的特定氨基酸，以及是否也可以掺入不同的氨基酸。在研究的第二部分中，我将创建具有木贼素合酶结构域和密切相关的 NRPS 的杂合 NRPS 酶，并使用随机和定向诱变来提高这些杂合酶的活性。在第三个具体目标中，我将测试其他的 R 域 PKS-NRPS 酶的释放机制不同于木贼素合酶 R 结构域所使用的机制，并且将不同合酶的 R 结构域的催化活性与 EqiS 缩合和腺苷酸化结构域结合起来。