Structural basis of specificity in affinity-labeled polyketide synthase didomains

描述（由申请人提供）：由于多重耐药细菌的传播，抗菌药物获得性耐药是一个日益严重的公共卫生问题。该项目的长期目标是建立知识工程的新组合的聚酮合酶（PKS）催化结构域的化学和立体化学的结果的可靠预测。模块化PKS是“组合生物合成”最理想的靶途径之一，因为其产物具有丰富的化学多样性。由于其模块化组织，它们也是最适合这种方法的途径之一。PKS结构域的简化，以建立新的化合物的合成途径，需要了解的基础上，底物特异性和立体化学的结果，为每个催化结构域，这是目前缺乏的。一些结构域的晶体结构是可用的，但是，除了针对该应用的初步研究之外，这些结构缺乏结合的底物或类似物。亲和标记已被证明是建立吡克霉素硫酯酶（Pik TE）机制的有效手段。该研究的中心假设是基于聚酮的亲和标记是探测分离的PKS双结构域的结构和机制的有力工具。本申请的具体目的是：（1）确定几个NADP依赖性酮还原酶（KR）结构域的底物特异性和立体化学结果的结构基础，（2）探索KR结构域对于非天然底物的底物范围，以及（3）理解通过末端硫酯酶（TE）结构域形成大环内酯的机制。研究设计和方法将利用双结构域构建体的酰基载体蛋白（ACP），KR-ACP和ACP-TE，将底物和产物模拟物递送到KR和TE活性位点。天然KR-ACP和ACP-TE双结构域将被乙烯基酮亲和标记模拟物共价修饰，并测定其晶体结构。将检查来自吡克罗霉素、红霉素和泰乐菌素的PKS系统的双羟香豆素，因为这些分子是抗生素药物发现的既定先导化合物。拟议的研究是重要的，因为PKS系统的合理工程有望提供新的大环内酯类抗生素，以打击多药耐药细菌的上升趋势。公共卫生相关性：被称为聚酮酶（PKS）的复杂巨型酶机器产生许多抗生素和其他生物活性分子。通过适当的工程改造，这些蛋白质是新药物的潜在丰富来源。本项目将研究底物与几个PKS结构域相互作用的细节。将合成共价连接到酶的底物模拟物，并且将解析捕获在酶中的底物模拟物的晶体结构。其目标是了解每个反应的底物特异性和范围，从而允许设计新的酶来制造新的生物活性分子。

DESCRIPTION (provided by applicant): Acquired resistance to antibacterial drugs is a growing public health problem due to the spread of multidrug-resistant bacteria. The long-term objective of this project is to build the knowledge to engineer new combinations of polyketide synthase (PKS) catalytic domains with reliable predictions of the chemical and stereochemical outcome. Modular PKSs are among the most desirable target pathways for "combinatorial biosynthesis" because of the rich chemical diversity of their products. They are also among the most amenable pathways to this approach because of their modular organization. Recombination of PKS domains to build synthetic pathways for novel compounds requires an understanding of the basis for substrate specificity and stereochemical outcome for each catalytic domain, which at present is lacking. Crystal structures of some domains are available, but, apart from the preliminary studies for this application, the structures lack bound substrates or analogs. Affinity labels have been demonstrated to be an effective means to establish the mechanism of pikromycin thioesterase (Pik TE). The central hypothesis of the proposed research is that polyketide-based affinity labels are powerful tools to probe the structure and mechanism of isolated PKS didomains. The specific aims of this application are: (1) to determine the structural basis for substrate specificity and stereochemical outcome of several NADP-dependent ketoreductase (KR) domains, (2) to explore the substrate range of KR domains for non-natural substrates, and (3) to understand the mechanisms of macrolactone formation by terminal thioesterase (TE) domains. The research design and methods will utilize the acyl carrier protein (ACP) of didomain constructs, KR-ACP and ACP-TE, to deliver substrate and product mimics to the KR and TE active sites. Natural KR-ACP and ACP-TE didomains will be covalently modified by vinyl ketone affinity label mimics, and their crystal structures determined. Didomains from the PKS systems for pikromycin, erythromycin, and tylosin will be examined since these molecules are established lead compounds for antibiotic drug discovery. The proposed research is significant because the rational engineering of PKS systems is expected to provide novel macrolide antibiotics to combat the rising tide of multidrug-resistant bacteria. PUBLIC HEALTH RELEVANCE: Complex mega-enzyme machines known as polyketide synthases (PKS) produce many antibiotics and other bio-active molecules. With appropriate engineering, these proteins are potentially rich sources of new pharmaceuticals. This project will examine the details of substrate interactions with several PKS domains. Substrate mimics will be synthesized that covalently attach to the enzyme, and crystal structures of substrate mimic trapped in the enzyme will be solved. The goal is to understand how the substrate specificity and range for each reaction, and thereby allow design of new enzymes to make new bio-active molecules.