Mechanisms of Polyketide, Amino Acid and Polypeptide Biosynthesis

该项目在化学和生物学领域的目标是：a)了解真菌聚酮的酶结构和组装机制；b)获得抗菌肽的结构洞察及其与细菌膜和靶分子（如脂质II）的相互作用。***在第一个领域，将研究真菌聚酮组装的详细模式。靶点包括5种减少的多环代谢物，即降胆固醇剂洛伐他汀、克拉多孢素（抗疟疾）、脱氢曲柳素（免疫调节植物毒素）、细胞松弛素E（抗血管生成、抗癌）和hypothemycin（激酶抑制剂和抗癌）。细菌聚酮合成酶（PKS）以流水线方式使用多种蛋白质，与之相反，真菌迭代PKS酶在单个蛋白质中重复使用相同的活性位点。它们的最终功能是将醋酸酯和其他前体聚合成具有特定长度、功能和立体化学的链。我们已经鉴定并表达了所有5种代谢物的真菌PKS蛋白。真菌迭代PKS酶的功能受蛋白质序列/结构以及生长的底物链的长度和功能控制，该底物链以硫酯的形式共价附着在蛋白质上。我们将使用完全纯化的迭代I型PKS组成酶，来检查链延伸每个阶段的详细组装速率和机制。这将通过装载标记的假定中间体来完成。我们建议鉴定LovB生成洛伐他汀过程中催化Diels Alder反应的结构域。我们建议将CoA-SH转化为CoA-NH2，并使用它来创建酰基载体蛋白（ACP）结构域衍生物，该衍生物将与其他结构域（例如酮合酶（KS））交联，以“冻结”移动酶在单个离散阶段进行x射线晶体学分析。***在第二个领域，我们建议通过核磁共振检查在十二烷基磷脂胆碱（DPC）胶束中与脂质II（细菌肽聚糖前体）类似物结合的抗菌肽的结构。我们将首先看一下4个目标肽，tridecaptin，乳酸菌蛋白3147 A1和A2，和paenicidin。由此产生的三维结构应该有助于阐明作用机制，并协助设计更简单的有效抗菌药物。抗菌肽已广泛用于保存食品，在人类和兽医学中具有巨大潜力，可用于治疗对当前疗法具有耐药性的感染。胶束脂结合抗菌剂和配合物与脂类II类似物的结构将被核磁共振检查。该方法包括合成特异性标记的13C十二烷基磷胆碱（DPC）和同位素编辑的noesi。靶点包括tridaptin、乳酸菌素3147 A1和A2、肉毒杆菌素和潜在的paenicidin和肠球菌素7A / 7b ***********

The objectives of this program at the interface of chemistry and biology are: a) to understand the enzyme structures and mechanisms of assembly of fungal polyketides and b) to obtain structural insight into antimicrobial peptides and their interactions with bacterial membranes and target molecules (e.g. Lipid II).***In the first area, the detailed mode of fungal polyketide assembly will be examined. The targets include 5 reduced polycyclic metabolites, namely the cholesterol-lowering agent lovastatin, cladosporin (anti-malarial), dehydrocurvularin (immunomodulatory phytotoxin), cytochalasin E ( anti-angiogenesis anticancer) and hypothemycin (kinase inhibitor & anticancer). In contrast to bacterial polyketide synthase (PKS) enzymes that employ multiple proteins in an assembly line fashion, the fungal iterative PKS enzymes use the same active sites in a single protein repeatedly. Ultimately their function is to polymerize acetate and other precursors into chains having specific length, functionality & stereochemistry. We have already identified and expressed the fungal PKS proteins for all 5 metabolites. The function of fungal iterative PKS enzymes is controlled by both the protein sequence/structure as well as by the length and functionality of the growing substrate chain that is attached covalently as a thioester to the protein. We will use fully purified constituent enzymes of the iterative type I PKS, to examine the rates and mechanism of detailed assembly at each stage of chain elongation. This will be accomplished by loading labelled putative intermediates. We propose to identify the domain that catalyzes the Diels Alder reaction during lovastatin formation by LovB. We propose to convert CoA-SH to CoA-NH2 and use this to create acyl carrier protein (ACP) domain derivatives that will cross link to other domains (e.g. ketosynthase (KS)) to "freeze" the mobile enzymes at a single discrete stage for x-ray crystallographic analysis.***In a second area, we propose to examine by NMR structures of antimicrobial peptides bound to analogs of Lipid II (bacterial peptidoglycan precursor) in dodecyl phosphocholine (DPC) micelles.We will first look at 4 target peptides, tridecaptin, lacticin 3147 A1 and A2, and paenicidin. The resulting 3D structures should help elucidate the mechanism of action and assist design of simpler potent antimicrobial agents. Antimicrobial peptides are already extensively used to preserve food and have great potential in human and veterinary medicine for treatment of infections resistant to current therapy. The structures of micelle lipids bound to antimicrobials and complexes with Lipid II analogs will be examined by NMR. The approach involves synthesis of specifically labelled 13C dodecyl phosphocholine (DPC) and isotope-edited NOESY. The targets include tridecaptin, lacticin 3147 A1 & A2, carnocyclin and potentially paenicidin and enterocin 7A / 7B.***********