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: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=667670
• 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界面的目标是：a）了解真菌聚酮化合物的酶结构和组装机制，B）获得抗菌肽及其与细菌膜和靶分子（如脂质II）相互作用的结构见解。在第一个领域，真菌聚酮组装的详细模式将被检查。靶点包括5种还原多环代谢物，即降胆固醇剂洛伐他汀、cladosporin（抗疟疾）、dehydrocurvularin（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）。与以装配线方式使用多种蛋白质的细菌聚酮合酶（PKS）相反，真菌迭代PKS酶重复地使用单一蛋白质中的相同活性位点。最终，它们的功能是将乙酸酯和其他前体转化为具有特定长度、官能度和立体化学的链。我们已经鉴定并表达了所有5种代谢产物的真菌PKS蛋白。真菌迭代PKS酶的功能受蛋白质序列/结构以及作为硫酯共价连接到蛋白质上的生长底物链的长度和功能控制。我们将使用完全纯化的构成酶的迭代I型PKS，检查的速率和机制的详细组装在每个阶段的链延长。这将通过加载标记的推定中间体来实现。我们建议确定的域，催化的Diels桤木反应过程中洛伐他汀形成LovB。我们建议将CoA-SH转化为CoA-NH 2，并使用它来产生酰基载体蛋白（ACP）结构域衍生物，该结构域衍生物将与其他结构域（例如酮合酶（KS））交联，以在单个离散阶段“冷冻”移动的酶，用于X射线晶体学分析。在第二个方面，我们建议通过NMR研究在十二烷基磷酸胆碱（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，我们将首先研究4种靶肽，tridecaptin，lacticin 3147 A1和A2，以及paenicidin。由此产生的3D结构应该有助于阐明作用机制，并有助于设计更简单有效的抗菌剂。抗菌肽已被广泛用于保存食品，并在人类和兽医学中用于治疗对当前疗法有抵抗力的感染方面具有巨大潜力。该方法涉及合成特异性标记的13 C十二烷基磷酸胆碱（DPC）和同位素编辑的NOESY。靶点包括tridecaptin，lacticin 3147 A1 & A2，carnocyclin和潜在的paenicidin和enterocin 7A /7 B。