The Molecular Basis of Regio-Specificity in Fungal Polyketide Synthase

描述（由申请人提供）：丝状真菌的聚酮化合物天然产物在化学结构和生物活性方面高度多样化，它们包括目前最畅销的药物，如洛伐他汀（用于降低胆固醇），以及强效毒素，如黄曲霉毒素和尾孢菌素。聚酮化合物是由一种多酶复合物聚酮化合物合成的.在将PKS结构与蛋白质-配体相互作用、酶催化和底物特异性相关联方面存在知识空白。这样的知识缺口严重阻碍了我们通过PKS工程生物合成新的基于聚酮的治疗剂的努力。为了解决这个问题，我们的目标是解决非还原PKS（NRPKS）的晶体结构，将产物与蛋白质结构相关联，并在结构-功能研究的基础上生物合成新的聚酮化合物。我们将确定NRPKS复合物和两个NRPKS结构域的序列-结构-功能关系，起始单元：ACP转酰酶（SAT）和产物模板（PT）。SAT和PT分别以高度特异性的方式催化聚酮化合物链的起始和环化。我们将努力实现以下具体目标：目标1。通过晶体结构和诱变不同的PT来确定NRPKS环化特异性的分子基础，这些PT将合成具有改变的环化模式的新聚酮化合物。目标2.通过晶体结构和诱变不同的SAT，然后通过组合生物合成产生具有不同起始单元和环化模式的新聚酮，确定NRPKS中起始单元特异性的分子基础。目标3。通过使用化学交联剂，通过稳定复合物并促进多结构域PKS复合物结晶的特异性交联探针，确定蛋白质-蛋白质相互作用对产品结果的重要性。我们已经获得了交联的多结构域PKS的衍射晶体（PKS领域的第一个）、PT的晶体结构、传递不同特异性的PT和SAT的衍射晶体、经验证的交联剂和优化的酶测定。从拟议的研究结果有两个方面的潜在的整体生物医学影响：（1）新的聚酮化合物具有不同的环化模式和起始单元可以筛选新的生物活性，（2）PT，SAT和NRPKS复合物的结构，生物产生毒素可以应用于基于结构的抑制剂设计，以确定新的化学预防剂对真菌毒素的生物合成。拟议研究的结果将具有很高的整体科学影响，因为它不仅将确定真菌PKS如何特异性环化（AIM 1）和启动（AIM 2）聚酮生物合成，而且还将导致PKS复合物的第一个晶体结构，并阐明mega合酶中的蛋白质-蛋白质相互作用如何影响产物结果（AIM 3）。

DESCRIPTION (provided by applicant): Polyketide natural products from filamentous fungi are highly diverse in both chemical structures and bioactivities, and they include the current top-selling drugs such as lovastatin (for cholesterol lowering), as well as potent toxins such as aflatoxin and cercosporin. Polyketides are biosynthesized by a multi- enzyme complex called polyketide synthase (PKS). There is a knowledge gap in correlating the PKS structures with protein-ligand interactions, enzyme catalysis, and substrate specificity. Such a knowledge gap has severely hampered our efforts to biosynthesize new polyketide-based therapeutics by PKS engineering. To address this issue, we aim to solve the crystal structures of non-reducing PKS (NRPKS), to correlate the product outcome with protein structures, and to biosynthesize new polyketides based on the structure-function studies. We will determine the sequence-structure-function relationship of the NRPKS complex and two NRPKS domains, the starter unit:ACP transacylase (SAT) and product template (PT). SAT and PT catalyze the polyketide chain initiation and cyclization, respectively, in a highly specific manner. We will pursue the following specific aims: Aim 1. Determine the molecular basis of cyclization specificity in NRPKS by crystal structures and mutagenesis different PTs that will synthesize new polyketides with altered cyclization patterns. Aim 2. Determine the molecular basis of starter unit specificity in NRPKS by crystal structures and mutagenesis different SATs followed by combinatorial biosynthesis to yield new polyketides with different starter units and cyclization patterns. Aim 3. Determine the importance of protein-protein interaction on product outcome using chemical crosslinkers by specific cross-linking probes that stabilize the complex and facilitate crystallization of multi-domain PKS complexes. We have already obtained diffracting crystals of crosslinked, multi-domain PKSs (a first in the PKS field), crystal structures of PTs, diffracting crystals of PTs and SATs conveying different specificities, validated crosslinkers, and optimized enzyme assays. Outcomes from the proposed research have two aspects of potential overall biomedical impact: (1) new polyketides with different cyclization patterns and starter units may be screened for new bioactivities, (2) the structures of PTs, SATs and NRPKS complex that biologically produces toxins can be applied to structure-based inhibitor design to identify new chemo-preventative agents against fungal toxin biosynthesis. Outcomes from the proposed research will have a high overall scientific impact, because it will not only determine how fungal PKSs specifically cyclize (AIM 1) and initiate (AIM 2) polyketide biosynthesis, but will also result in the first crystal structure of a PKS complex and elucidate how protein-protein interactions in the mega-synthase affects the product outcome (AIM 3).