The Ketoreduction and Cycilization of Aromatic Polyketide Biosynthesis

描述（由申请人提供）：本提案的目的是确定称为聚酮化合物合酶（PKS）的蛋白质复合物的序列-结构-功能关系，PKS是一种由5 - 10个不同结构域组成的酶复合物，可产生药学上重要的天然产物。聚酮化合物的多样性是通过控制扩链单元、链长、环化和还原模式的变化来实现的。本研究的重点是确定酮还原酶（KR）和双功能芳香化酶/环化酶（ARO/CYC）两个芳香族PKS结构域的晶体结构和序列结构功能关系。KR和ARO/CYC分别以高度特异性的方式催化聚酮化合物链的还原和环化。本研究的中心假设是，我们可以利用结构定向突变以可预测的方式改变底物特异性和酶活性。我们已经制定了这一假设的基础上，我们的初步结果的14个晶体结构。对放线菌紫素KR（actKR）和四环霉素ARO/CYC（tcmARO）的30个突变体、5种体外底物和动力学进行了研究。我们将努力实现以下具体目标：目标1。确定酮还原酶（KR）的序列-结构-功能关系，导致其独特的还原特异性，其中我们将（1）确定actKR和底物/抑制剂的共晶结构，以确定对催化和蛋白质-配体相互作用重要的结构特征，（2）用多羰基化合物作为“分子标尺”探测actKR的活性位点几何形状，以便将不同的配体结合基序与活性位点几何形状相关联，和（3）通过动力学测定和结构定向诱变鉴定对酶活性、立体特异性和区域特异性重要的残基。AIM 2.确定芳香化酶/环化酶（ARO/CYC）的序列-结构-功能关系，这导致其独特的环化特异性，其中我们将（1）确定tcmARO和抑制剂的共晶体结构，以剖析蛋白质-配体相互作用，（2）确定不同ARO/CYC的晶体结构，以确定不同生物功能的重要序列-结构特征，和（3）通过动力学分析和结构定向诱变鉴定对催化和环化特异性重要的tcmARO残基; AIM 3.确定蛋白质-蛋白质相互作用对KR和ARO/CYC之间的序列-结构-功能关系的重要性，其中我们将PKS结构域之间的蛋白质-蛋白质相互作用与酶活性和区域特异性相关联。这项研究意义重大，因为其结果将回答有关如何精确控制聚酮化合物还原和环化的重要问题。它也是创新的，提供了新的信息KR和ARO/CYC在分子水平上以前没有实现。长期的生物医学相关性是聚酮化合物研究界可以应用由该提议确定的序列-结构-功能关系，以通过蛋白质工程使“非天然”天然产物的群体多样化，使得可以产生具有不同酮还原和环化模式的新型聚酮化合物的文库。预计这将对公众健康产生积极影响，因为它将允许开发新的“非天然”天然产品，可以筛选新的药物活性。与此同时，在该提案中获得的关于将PKS序列结构与聚酮酮还原和环化过程中的催化、底物特异性和蛋白质-蛋白质相互作用相关联的基础新知识预计将对天然产物研究界产生重大影响。

DESCRIPTION (provided by applicant): The objective of this proposal is to determine sequence-structure-function relationships of a protein complex called polyketide synthase (PKS), an enzyme complex comprised of 5 - 10 distinct domains that produce pharmaceutically important natural products. Polyketide diversity is achieved via a controlled variation of extender units, chain length, cyclization and reduction patterns. The focus of this proposal is to determine the crystal structures and sequence-structure-function relationship of two aromatic PKS domains, the ketoreductase (KR) and bifunctional aromatase/cyclase (ARO/CYC). KR and ARO/CYC catalyze the polyketide chain reduction and cyclization, respectively, in a highly specific manner. The central hypothesis 'or the proposed research is that we can use structure-directed mutagenesis to change the substrate specificity and enzyme activity in a predictable manner. We have formulated this hypothesis based on our preliminary results of 14 crystal structures. 30 mutants, five in vitro substrates and kinetic studies of the actinorhodin KR (actKR) and tetracenomycin ARO/CYC (tcmARO). We will pursue the following specific aims: AIM 1. Determine the Sequence-Structure-Function Relationship of Ketoreductase (KR) That Leads to its Unique Reduction Specificity, in which we will (1) determine the cocrystal structures of actKR and substrates/inhibitors in order to identify structural features important for catalysis and protein-ligand nteractions, (2) probe the active site geometry of actKR with polycarbonyl compounds as the "molecular ruler" in order to correlate different ligand binding motifs with active site geometry, and (3) identify residues important for enzyme activity, stereo-specificity and regio-specificity by kinetic assays and structure-directed mutagenesis. AIM 2. Determine the Sequence-Structure-Function Relationship of Aromatase/Cyclase (ARO/CYC) That Leads to its Unique Cyclization Specificity, in which we will (1) determine the co-crystal structures of tcmARO and inhibitors in order to dissect the protein-ligand interactions, (2) determine the crystal structures of different ARO/CYCs in order to identify important sequence-structure features for different biological functions, and (3) identify tcmARO residues important for catalysis and cyclization specificity by kinetic assays and structure-directed mutagenesis; AIM 3. Determine the Importance of Protein-Protein Interactions on the Sequence-Structure-Function Relationship between KR and ARO/CYC, in which we will correlate protein-protein interactions between PKS domains with enzyme activity and regio-specificity. The proposed research is significant, because the outcome will answer important questions about how polyketide reduction and cyclization are precisely controlled. It is also innovative by providing new information about KR and ARO/CYC at a molecular level not achieved previously. The long- term biomedical relevance is that the polyketide research community can apply the sequence-structure- function relationships determined from this proposal to diversify the population of "unnatural" natural products via protein engineering, such that a library of novel polyketides with different ketoreduction and cyclization patterns can be produced. This is expected to positively affect public health, because it will allow the development of new "unnatural" natural products that can be screened for new pharmaceutical activities. Meanwhile, the fundamental new knowledge obtained in this proposal on correlating PKS sequence-structure with the catalysis, substrate specificity and protein-protein interactions during polyketide ketoreduction and cyclization is expected to have a high impact on the natural product research communities.