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）：真菌天然产物是包含各种化学基序的复杂分子。这些天然产物中有许多参与发病机理，许多天然产物具有药理活性。该提议着重于一种天然产物的生物合成途径。研究的目的是收集特定信息，这些信息将在对生物合成的精确更改中有用 途径将导致对奎汀结构的改变。与许多其他相关分子一样，雌激素是有毒的，并且与重要的药物靶靶（HIV-1积分酶，在Equisetin的情况下）特别相互作用。改变奎汀和相关分子的结构的能力将有助于使候选药物通常毒性较小或对特定靶标更有效。包括Equisetin的真菌天然产品类别 由杂化聚酮化酶/非核糖体肽合酶（PKS-NRPS）酶产生。这些大型酶包含许多域。其中两个结构域用于在合成过程中将生长的均值分子连接到酶。彼此的域在合成均值分子的合成中催化了特定的反应。 PKS-NRPS产品由NRPS添加氨基酸的聚酮化合物（由PKS产生）组成。 NRP由四个域组成。一个结构域（t）是均衡分子的载体，激活氨基酸底物需要第二个结构域（a），三分之一（c）催化氨基酸的附着在聚酮化合物上，最后一个结构域（R）催化了从载体域中释放产品的释放。我的具体目的是1）测试每个均衡素合成酶NRPS结构域在确定哪种氨基酸用作底物中的作用，2）识别域中可以突变以更改底物特异性的区域和特定残基，以更改底物特异性，以测试其他真菌PKS-NRPS酶rps rps酶的rps ryains rps rys rps ryains ryains ryains ryains是否通过不同的机构释放。实验方法将涉及四个奎汀合酶NRPS结构域以及来自PK的载体域以及来自PKS的R域的表达和纯化，以及其他两个PKS-NRPS酶的R域。在第一个特定目的中，我将了解不同域的氨基酸底物特异性，目的是了解整个PKS-NRP的哪些部分负责偏爱将特定的氨基酸掺入Equisetin中，以及是否也可以掺入不同的氨基酸。在研究的第二部分中，我将创建具有含量素合酶和紧密相关的NRP结构域的混合NRPS酶，并使用随机和定向的诱变来改善这些混合酶的活性。在第三个特定目标中，我将测试其他 释放机制的PKS-NRPS酶不同于均质素合酶R结构域使用的机制，并将不同合酶的R结构域的催化活性与EQIS凝结和腺苷酸化结构域相结合。