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BIOCHEMICAL GENETICS OF CYCLIC PEPTIDE BIOSYNTHESIS

环肽生物合成的生化遗传学

基本信息

批准号：
2711238
负责人：
JONATHAN WALTON
金额：
$ 3.14万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
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

项目摘要

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.

环肽由一大群不同的次生素组成代谢物，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疗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生产真菌Cochliobolus Carbon um和它的宿主玉米之间，在纳摩尔浓度下对培养的细胞也有抑制作用哺乳动物癌细胞。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。另外四个，无关的众所周知，真菌可以产生含有AEO的环状四肽。这个 HC-毒素的产生受一个孟德尔基因座控制，称为 TOX2。我们最近克隆了一个22kb的DNA区域并对其进行了测序仅在产生HC-毒素的真菌分离株中发现。这一地区包含三个基因，其中一个是15.7kb的开放阅读框架(称为 HTS1)编码一个称为HC-的570 kDa四组分环肽合成酶毒素合成酶。这种酶是组装HC-的中心酶。毒素，但不合成AEO。我们建议(1)研究其结构使用生化和分子遗传学工具研究HC-毒素合成酶为了更好地从生态和生态角度认识这种生物多样性的作用药理上重要的酶，(2)研究AEO的生物合成，依靠分子遗传学和分离纯化鉴定当HC-毒素基因积聚时一种新的AEO前体合成酶是突变的，(3)使用我们从研究 HTS1和AEO生物合成来分离四个菌株的相应基因不相关的真菌，能产生密切相关的环状四肽。这将是提供基本的比较信息，帮助我们分析所有环肽合成酶的结构和功能，以及生物合成新型环肽的最终目标。它还将允许美国检验HTS1和TOX2从碳链霉菌转移到碳链霉菌的假设另一种真菌(或反之亦然)通过水平基因转移。