CATALYTIC FUNCTIONS AND METABOLISM OF VITAMIN B6

维生素 B6 的催化功能和代谢

• 批准号: 3226576
• 负责人: ESMOND E SNELL
• 金额: $ 9.94万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-12-01 至 1989-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3226576
• 关键词: Escherichia coli; Klebsiella pneumoniae; Lactobacillaceae; X ray crystallography; chemical structure function; chromatography; cofactor; enzyme mechanism; enzyme structure; gel electrophoresis; gene expression; genetic manipulation; genetic regulation; gram negative bacteria; high performance liquid chromatography; histidine decarboxylase; molecular cloning; nucleic acid sequence; nutrition related tag; point mutation; protein engineering; protein sequence; pyridoxal phosphate; pyridoxine; tissue /cell culture; vitamin metabolism

Histidine decarboxylase (Hdc) is responsible for histamine formation in living organisms. Although histamine produces many physiological effects in animals (e.g. vasodilation, hypotension, HC1 secretion and gastric ulcer formation, anaphylaxis, allergic effects, etc.), Hdc has been studied in pure form only recently. Studies have shown that two classes of Hdc enzymes exist. One class, present in Gram-positive bacteria, (e.g. Lactobacillus sp.) has a catalytically-essential pyruvoyl residue at the active site and no pyridoxal 5'-phosphate (PLP). In the other class, found in Gram-negative bacteria (e.g. Morganella AM-15) and animals, PLP is the essential cofactor. Amino acid sequences of a single Hdc from each class are known; they are not homologous and the quaternary structures are very different. Our specific goals, all of which are aimed at determining the detailed reaction mechanisms of these enzymes, are: (1) Obtain expression of the hdc gene from Morganella in E. coli and develop procedures for purification of the expressed protein in convenient amounts for covalent modification procedures and for crystallization. If the latter is achieved, a collaborating crystallography group will study the 3-dimensional structure of the enzyme. (2) If (1) is successful, then, by use of synthetic mismatched probes and cloning techniques to obtain site-specific mutations in this hdc gene, isolate the resulting expressed Hdc's, and determine their properties to assist in judging the essentiality of specific amino acid residues that have been implicated in enzyme action by other data. These include lysine 232, which forms the azomethine link to PLP in the native enzyme; histidine 231, which precedes lysine in the PLP-binding site of Hdc, arginine decarboxylase, lysine decarboxylase and glutamic acid decarboxylase; serine 229, which also is invariant in the PLP-binding site of these enzymes; and serine 322, which is destroyed when the suicide inhibitor, yield-fluoromethylhistidine, acts on Hdc. Crystals of such modified enzymes, if obtained, will also be subjected to x-ray examination. (3) We will clone and sequence hdc genes from Clostridium perfringens and from Klebsiella pneumoniae. The encoded protein sequences will provide an independent approach to determining amino acid residues that must be conserved for activity of Class I and Class II Hdc's.

组氨酸脱羧酶 (Hdc) 负责组胺的形成 活的有机体。 尽管组胺会产生许多生理效应 动物（例如血管舒张、低血压、HC1 分泌和胃溃疡） 的形成、过敏反应、过敏反应等），Hdc 已在 纯粹的形式只是最近才出现的。 研究表明，两类 Hdc 酶的存在。 一类存在于革兰氏阳性细菌中（例如 乳杆菌属 (Lactobacillus sp.) 在 活性位点且不含吡哆醛 5'-磷酸 (PLP)。 在其他班级，发现 在革兰氏阴性菌（例如摩根氏菌 AM-15）和动物中，PLP 是 必需的辅助因子。 每个类别的单个 Hdc 的氨基酸序列 是已知的；它们不是同源的，四级结构非常不同 不同的。 我们的具体目标，所有这些目标都是为了确定 这些酶的详细反应机制是： (1)获得摩根氏菌的hdc基因在大肠杆菌中的表达，并 开发方便的纯化表达蛋白的程序 共价修饰程序和结晶的量。 如果 后者实现后，一个合作晶体学小组将研究 酶的三维结构。 (2) 如果(1)成功，那么，通过使用合成的错配探针和 克隆技术以获得该 hdc 基因的位点特异性突变， 分离所得表达的 Hdc，并确定其特性 协助判断特定氨基酸残基的重要性 其他数据表明与酶的作用有关。 这些包括赖氨酸 232，与天然酶中的 PLP 形成偶氮甲碱连接；组氨酸 231，位于 Hdc、精氨酸的 PLP 结合位点赖氨酸之前 脱羧酶、赖氨酸脱羧酶和谷氨酸脱羧酶；丝氨酸 229，这些酶的 PLP 结合位点也是不变的；和 丝氨酸 322，当自杀抑制剂被破坏时， 产量-氟甲基组氨酸，作用于 Hdc。 这种改性的晶体 如果获得酶，也将接受 X 射线检查。 (3) 我们将克隆产气荚膜梭菌的hdc基因并测序 来自肺炎克雷伯菌。 编码的蛋白质序列将提供 确定必须的氨基酸残基的独立方法 保留 I 类和 II 类 Hdc 的活性。