BIOCHEMICAL GENETICS OF CYCLIC PEPTIDE BIOSYNTHESIS

环肽生物合成的生化遗传学

• 批准号: 2183471
• 负责人: JONATHAN WALTON
• 金额: $ 11.09万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-15 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2183471
• 关键词: Fungi; biological products; cyclic peptides; enzyme mechanism; epoxides; genetic mapping; host organism interaction; mass spectrometry; molecular cloning; molecular genetics; mutant; mycotoxins; nucleic acid sequence; open reading frames; polymerase chain reaction; protein sequence; protein structure function; southern blotting; toxin metabolism

Cyclic peptides comprise a large and diverse group of secondary metabolites many of which are important as toxins and as antibiotics. Molecular genetic studies of cyclic peptide biosynthesis could lead to the genetic engineering of novel compounds with potential medical usefulness. HC-toxin is a cyclic tetrapeptide involved in the disease interaction between the producing fungus, Cochliobolus carbonum, and its host, maize, and is also cytostatic at nanomolar concentrations against cultured mammalian cancer cells. It contains an unusual epoxide-containing amino acid called Aeo which is required for activity. Four other, unrelated fungi are known to produce cyclic tetrapeptides containing Aeo. The production of HC-toxin is controlled by a single Mendelian locus called TOX2. We have recently cloned and sequenced a 22-kb region of DNA that is found only in isolates of the fungus that make HC-toxin. This region contains three genes, one of which is a 15.7-kb open reading frame (called HTS1) encoding a 570 kDa tetrapartite cyclic peptide synthetase called HC- toxin synthetase. This enzyme is the central enzyme in the assembly of HC- toxin, but does not synthesize Aeo. We propose to (1) study the structure of HC-toxin synthetase using biochemical and molecular genetic tools, in order to understand better the function of this kind of ecologically and pharmacologically-important enzyme, (2) study the biosynthesis of Aeo, relying on molecular genetics and the purification and identification of a novel Aeo precursor which accumulates when the genes for HC-toxin synthetase are mutated, (3) use the knowledge gained from our study of HTS1 and Aeo biosynthesis to isolate the corresponding genes from the four unrelated fungi that make closely related cyclic tetrapeptides. This will provide essential comparative information that will help us analyze the structure and function of all cyclic peptide synthetases, with the ultimate goal of biosynthesizing novel cyclic peptides. It will also allow us to test the hypothesis that HTS1 and TOX2 moved into C. carbonum from one of the other fungi (or vice versa) by horizontal gene transfer.

环肽包括一个大的和不同的组的二级结构。 代谢物，其中许多是重要的毒素和抗生素。 环肽生物合成的分子遗传学研究可能导致 具有潜在医学用途的新型化合物的基因工程。 HC-毒素是一种参与疾病相互作用的环状四肽 在产生真菌Cochliobolus carbonum和它的宿主玉米之间， 并且在纳摩尔浓度下对培养的 哺乳动物癌细胞它含有一种不寻常的含环氧化物的氨基 一种叫做Aeo的酸，它是活性所必需的。 4、无关 已知真菌产生含有Aeo的环四肽。的 HC-毒素的产生由一个称为 TOX2.我们最近克隆并测序了一个22 kb的DNA区域， 只在制造HC毒素的真菌中发现这一地区 包含三个基因，其中一个是15.7 kb的开放阅读框架（称为 HTS 1）编码570 kDa的四部分环肽合成酶，称为HC- 毒素合成酶这种酶是HC-1组装的中心酶。 毒素，但不合成Aeo。我们建议（1）研究结构 的HC-毒素合成酶使用生物化学和分子遗传学工具， 为了更好地了解这种生态功能， 药理学上重要的酶，（2）研究Aeo的生物合成， 依靠分子遗传学和纯化和鉴定 一种新的Aeo前体，当HC毒素基因 合成酶突变，（3）使用我们研究中获得的知识， HTS 1和Aeo的生物合成，以从这四个基因中分离出相应的基因 产生密切相关的环四肽的不相关的真菌。这将 提供必要的比较信息，帮助我们分析 所有环肽合成酶的结构和功能， 生物合成新型环肽的最终目标。它还将允许 我们来检验HTS 1和TOX 2进入C.碳从 通过水平基因转移，将一种真菌转化为另一种真菌（反之亦然）。