Specificity and Activity of Fungal Polyketide-Nonribosomal Peptide Synthases

描述(申请人提供)：真菌天然产物是含有多种化学基元的复杂分子。这些天然产物中有许多与发病有关，许多具有药理活性。这项建议的重点是一种天然产物木犀草素的生物合成途径。这项研究的目标是收集特定的信息，这些信息将有助于努力对生物合成进行精确的改变。 这一途径将导致马钱子素结构的改变。像许多其他相关分子一样，马钱素是有毒的，并与一个重要的药物靶点(HIV-1整合酶，就马钱素而言)特异地相互作用。改变苦参素和相关分子结构的能力将有助于制造一般毒性较低或对特定靶点更有效的候选药物。包括木犀草素在内的真菌天然产品类别 是由聚酮合成酶/非核糖体肽合成酶(PKS-NRPS)混合酶产生的。这些大的酶包含许多结构域。在酶的合成过程中，其中两个结构域被用来将不断增长的木犀草素分子连接到酶上。在合成木犀草素分子的过程中，每个结构域都催化特定的反应。PKS-NRPS产品由聚酮(由PKS产生)和NRPS添加氨基酸组成。NRPS由四个域组成。一个结构域(T)是木犀草素分子的载体，第二结构域(A)需要激活氨基酸底物，第三结构域(C)催化氨基酸与聚酮的结合，最后一个结构域(R)催化产物从载体结构域释放。我的具体目标是：1)测试每个木犀草素合成酶NRPS结构域在确定哪种氨基酸作为底物时的作用；2)确定结构域中可以突变以改变底物专一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的机制催化产物的释放。实验方法将包括表达和纯化四个木犀草素合成酶NRPS结构域和来自PKS的载体域，以及来自另外两个PKS-NRPS酶的R结构域。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结合到木犀草素中的特定氨基酸，以及不同的氨基酸是否也可以结合。在这项研究的第二部分，我将利用木瓜素合成酶和一个密切相关的NRPS结构域来创造杂交NRPS酶，并使用随机和定向突变来提高这些杂交酶的活性。在第三个具体目标中，我将测试来自其他 PKS-NRPS酶的释放机制不同于木犀草素合成酶R结构域的释放机制，它将不同合成酶R结构域的催化活性与EQIS缩合和腺化结构域结合起来。

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.