Mechanisms of Polyketide, Amino Acid and Polypeptide Biosynthesis

聚酮化合物、氨基酸和多肽生物合成机制

• 批准号: RGPIN-2015-05163
• 负责人: Vederas, John
• 金额: $ 9.11万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=613389
• 关键词: Mechanisms; Polyketide; Amino; Acid; Polypeptide

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.

该计划在化学和生物学的界面上的目标是：a)了解真菌聚酮的酶结构和组装机制；b)深入了解抗菌肽及其与细菌膜和靶分子(如Lipid II)的相互作用。 在第一个区域中，将研究真菌聚酮组装的详细模式。靶点包括5个还原的多环代谢物，即降胆固醇药洛伐他汀、枝孢菌素(抗疟疾)、脱氢曲霉素素(免疫调节植物毒素)、细胞松弛素E(抗血管生成抗癌药物)和降霉素(激酶抑制剂和抗癌药物)。与以流水线方式使用多个蛋白质的细菌聚酮合成酶(PKS)酶不同，真菌迭代的PKS酶重复使用单个蛋白质中相同的活性部位。最终，它们的功能是将醋酸酯和其他前体聚合成具有特定长度、官能度和立体化学的链。我们已经鉴定并表达了所有5种代谢物的真菌PKS蛋白。真菌重复PKS酶的功能既受蛋白质序列/结构控制，也受生长底物链的长度和功能控制，底物链以硫酯的形式共价连接到蛋白质上。我们将使用完全纯化的I型PKS的组成酶，来检测在链延长的每个阶段详细组装的速度和机制。这将通过装载有标签的推定中间体来实现。我们建议确定在LovB形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，并用它来创建酰基载体蛋白(ACP)结构域的衍生物，这些衍生物将与其他结构域(如酮合成酶(KS))交联，以在单个离散阶段冻结可移动的酶，用于X射线结晶学分析。 在第二个领域，我们建议用核磁共振技术研究十二烷基磷胆碱(DPC)胶束中与类脂II(细菌肽多糖前体)结合的抗菌肽的结构。我们将首先研究四个靶标多肽，十三碳蛋白，乳糖素3147A1和A2，以及巴尼西丁。由此得到的3D结构应该有助于阐明作用机制，并有助于设计更简单、更有效的抗菌剂。抗菌肽已经被广泛用于保存食品，并在人类和兽医中具有巨大的潜力，用于治疗对当前治疗方法耐药的感染。此外，与抗菌剂结合的胶束脂类和与类脂II类似物的复合体的结构将通过核磁共振进行检测。该方法包括合成专门标记的13C十二烷基磷胆碱(DPC)和同位素编辑的NOESY。这些靶标包括十三碳联素、乳糖素3147A1和A2、卡诺环素以及可能的巴尼西丁和肠球菌素7A/7B。