GENETICS OF TRYPTOPHAN TRYPTOPHYLQUINONE BIOSYNTHESIS

色氨酸色氨酸醌生物合成的遗传学

• 批准号: 2191288
• 负责人: ANDREI Y CHISTOSERDOV
• 金额: $ 13.98万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2191288
• 关键词: Bacillus; SDS polyacrylamide gel electrophoresis; amine oxidoreductase; bacterial genetics; cofactor; enzyme activity; enzyme biosynthesis; formaldehyde; gene expression; high performance liquid chromatography; methylamines; molecular cloning; mutant; proteolysis; quinones; site directed mutagenesis; tryptophan

Amines play an important role in global cycling of carbon and nitrogen in nature, they are apparently involved in the production of greenhouse gases and are widespread contaminants of water and soil. Despite the fact that many amines are normal constituents of human blood, they have been connected with cases of poisoning and have been shown to cause vascular tissue pathology. Enzymes participating in amine oxidation are widespread in both eukaryotes and prokaryotes. One group of enzymes oxidizing amines are quinoproteins with quinone groups synthesized posttranslationally from a tyrosine or tryptophan residue of the polypeptide chain. The periplasmic methylamine dehydrogenase (MADH, encoded by the mau-genes) is the only known member of this unique class of quinoproteins containing tryptophan tryptophylquinone (TTQ) as a cofactor, and the enzyme has so far been found only in prokaryotes. The long term objective of this proposal is the study of TTQ cofactor biosynthesis in methylotrophic bacteria. The specific aims of the initial portion of this long-term work is to generate and characterize mutants in the mau genes to identify those that may be involved in TTQ synthesis and to develop an in vivo expression system in order to reconstruct events of TTQ biosynthesis in a heterologous host. A series of chemically induced Mau- mutants of Methylobacillus flagellatum KT will be isolated and the impaired genes will be identified in all of the mutants. Those genes that do not map within the known mau gene cluster will be cloned. Site-specific mutagenesis will also be carried out on MADH structural genes to determine targets and temporal order for modifications. Physiological and biochemical studies of all mutants will be carried out to elucidate their participation in specific steps of MADH assembly and cofactor biosynthesis. The following characteristics of Mau- mutants will be studied: l. activities of MADH, its electron acceptor and NAD(P) and dye-linked formaldehyde and formate dehydrogenases. This will allow the elimination of some of the mutants as not pertaining to MADH assembly and TTQ synthesis. 2. the presence of the MADH small and large subunit polypeptides in the mutants, the form of each (mature or premature), and the cell compartment in which they are present. 3. the presence of a quinone in MADH after SDS-PAGE separation of the mutant enzymes (by specific staining). MADHs from the mutants that appear to be defective in cofactor biosynthesis or MADH assembly will be isolated and characterized by visible spectroscopy and the presence of the TTQ cross- link will be assessed by proteolytic analysis of the small subunit. These studies should delineate the steps in cofactor biosynthesis and their temporal and spatial characteristics. The second approach will be to develop in vivo expression systems for mau genes. Two systems will be considered: P(L)-dependent expression of the mau genes from M.flagellatum KT in E. coli and expression of the mau genes from Methylobacterium extorquens AM1 in other Methylobacterium strains which do not grow on methylamine naturally. The information obtained will allow us to refine the existing working model of TTQ biosynthesis, to determine the pathway for TTQ biosynthesis, and to begin to define its place in the process of MADH assembly as a whole. This knowledge will facilitate studying the synthesis and assembly of other post-translationally modified quinoproteins including those of medical importance.

胺在碳和氮的全球循环中起重要作用 大自然，他们显然参与了温室气体的生产 并且是水和土壤的广泛污染物。尽管事实 许多胺是人血的正常成分，它们一直是 与中毒病例有关，已被证明引起血管 组织病理学。参与胺氧化的酶很普遍 在真核生物和原核生物中。一组氧化胺的酶 是喹他酮与喹酮基团合成后翻译后的奎诺蛋白是从 多肽链的酪氨酸或色氨酸残基。周质 甲胺脱氢酶（MADH，由Mau-Genes编码）是唯一的 这类含有色氨酸的独特类昆诺蛋白的已知成员 色氨酸酮酮（TTQ）作为辅因子，酶已经是迄今为止的 仅在原核生物中发现。该提议的长期目标是 甲基营养细菌中TTQ辅因子生物合成的研究。这 这项长期工作的初始部分的具体目的是生成 并表征毛毛基因中的突变体，以识别那些可能是的突变体 参与TTQ合成并在 为了重建异源宿主中TTQ生物合成事件。一个 一系列化学诱导的甲基曲杆菌鞭毛突变体 KT将被隔离，并且将在所有的基因中识别出受损的基因 突变体。那些未在已知的MAU基因簇中绘制的基因 将被克隆。网站特异性诱变也将在MADH上进行 结构性基因确定目标和时间顺序 修改。所有突变体的生理和生化研究将 被执行以阐明他们参与MADH的特定步骤 组装和辅因子生物合成。毛毛的以下特征 将研究突变体：l。 Madh的活动，其电子受体和 NAD（P）和染料连接的甲醛和甲酸甲酸脱氢酶。这会 允许消除一些与MADH有关的突变体 组装和TTQ合成。 2。大小的Madh的存在 突变体中的亚基多肽，每种形式（成熟或成熟或 过早的），以及它们存在的细胞室。 3 SDS-PAGE分离突变体后MADH中存在醌 酶（通过特定染色）。来自突变体的madhs 辅因子生物合成或MADH组件的缺陷将被隔离，并且 以可见光谱和TTQ交叉的存在为特征 链接将通过对小亚基的蛋白水解分析进行评估。这些 研究应描述辅因子生物合成及其的步骤 时间和空间特征。第二种方法是 开发用于毛生基因的体内表达系统。两个系统将是 考虑的：p（l）依赖性表达毛毛虫基因来自m.flagellatum 大肠杆菌中的Kt和甲基杆菌的毛基因的表达 其他不生长在其他甲基杆菌菌株中的AM1 甲胺自然。获得的信息将使我们能够完善 TTQ生物合成的现有工作模型，以确定途径 为了进行TTQ生物合成，并开始在此过程中定义其位置 整个集会。这些知识将促进研究 其他翻译后修改的合成和组装 奎诺蛋白包括具有医学重要性的蛋白质。