BIOCHEMICAL GENETICS OF CYCLIC PEPTIDE BIOSYNTHESIS

环肽由大量不同的二级肽组成 其中许多代谢物作为毒素和抗生素很重要。 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 产菌炭炭菌 (Cochlioboluscarbonum) 与其宿主玉米之间 并且在纳摩尔浓度下对培养的细胞也具有细胞抑制作用 哺乳动物癌细胞。它含有一种不寻常的含环氧化物的氨基 称为 Aeo 的酸，是活性所必需的。 其他四个，不相关 已知真菌产生含有Aeo的环状四肽。的 HC 毒素的产生由一个孟德尔基因座控制，该基因座称为 毒性2。我们最近克隆并测序了一个 22 kb 的 DNA 区域， 仅在产生 HC 毒素的真菌分离株中发现。这个地区 包含三个基因，其中之一是一个 15.7 kb 的开放阅读框（称为 HTS1) 编码 570 kDa 四联环肽合成酶，称为 HC- 毒素合成酶。该酶是 HC- 组装过程中的核心酶。 毒素，但不合成Aeo。我们建议（1）研究结构 使用生化和分子遗传学工具对 HC 毒素合成酶进行研究 为了更好地了解这种生态和功能 药理上重要的酶，（2）研究Aeo的生物合成， 依靠分子遗传学以及纯化和鉴定 一种新的 Aeo 前体，当 HC 毒素基因出现时会积累 合成酶发生突变，（3）利用我们从研究中获得的知识 HTS1和Aeo生物合成从四个中分离出相应的基因 产生密切相关的环状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 我们检验 HTS1 和 TOX2 从 通过水平基因转移转移到其他真菌之一（或反之亦然）。

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.