STRUCTURE-FUNCTION OF PYRUVOYL & PLP-DEPENDENT ENZYMES

丙酮酰的结构-功能

• 批准号: 3277722
• 负责人: MARVIN L HACKERT
• 金额: $ 17.76万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-02-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3277722
• 关键词: Lactobacillus; X ray crystallography; acidity /alkalinity; chemical binding; cofactor; conformation; covalent bond; crystallization; enzyme mechanism; enzyme structure; histidine decarboxylase; molecular cloning; mutant; ornithine decarboxylase; protein engineering; protein sequence; protein structure function; pyridoxal phosphate; pyruvates; site directed mutagenesis

The structure-function relationships of basic amino acid decarboxylases will be studied by X-ray diffraction and protein engineering techniques. Most decarboxylases require pyridoxal phosphate (PLP) as an essential cofactor while others utilize a covalently bound a-keto acid. The proposed studies involve a decarboxylase of each cofactor type: a pyruvoyl- dependent histidine decarboxylase (Prv-HDC) and a PLP-dependent ornithine decarboxylase (PLP-ODC), both from Lactobacillus 30alpha, a Gram-positive bacterium resident in the mammalian gut. The corresponding mammalian enzymes are less amenable for X-ray diffraction studies. The decarboxylation of histidine is the only known mechanism for the biosynthesis of histamine. Physiological effects of histamine include vasodilation, hypotension, gastric HCl secretion, anaphylaxis, and mediation of the allergic response. The Prv-HDC from L. 30alpha is an inducible enzyme, and the most thoroughly studied of an unusual class of enzymes that produce their own prosthetic group from an internal amino acid residue. We have determined the X-ray structure of a low pH (hyperbolic kinetics) form of Prv-HDC and have proposed a mechanism of action on the basis of product and inhibitor complexes. The gene for HDC has been cloned in the laboratory of Dr. Jon Robertus and several site-specific mutants prepared. We have isolated and crystallized several such mutants for crystallographic study. In addition, we propose to produce a stable proHDC mutant (S82A) to study the activation properties of this enzyme and also examine a higher pH (sigmoidal kinetics) form of HDC. High resolution data of these systems will be collected with our Hamlin/Xuong area detector system and the structures refined on the University's Cray supercomputer. Ornithine decarboxylase (ODC) is a PLP-dependent enzyme that converts ornithine to putrescine which is a precursor of the polyamines spermine and spermidine. ODC is found in all cells but in particularly high activity in rapidly dividing cells. It has been used as a target for cancer chemotherapy and the inhibitor alpha-DL-difluoromethylornithine has been used successfully to treat Trypanosomal infections. The enzyme is generally noted for its rapid turnover rate with the mammalian enzyme having a stable half-life of only about 1 hour. In contrast, ODC from L. 30alpha is stable and representative of large subunit (80kDa) PLP decarboxylase. Our ODC crystals diffract to beyond 2.4A resolution and two potential heavy atom derivatives have been collected. We propose to determine the crystal structure of ODC, to clone and sequence the ODC gene, and later express it for site-directed mutagenesis studies on its active site.

碱性氨基酸脱羧酶的结构与功能关系 将通过X射线衍射和蛋白质工程技术进行研究。 大多数脱羧酶需要磷酸吡哆醛（PLP）作为必需的 辅因子，而其它的利用共价结合的α-酮酸。 拟议 研究涉及每种辅因子类型的脱羧酶：脱羧酰- 依赖性组氨酸脱羧酶（Prv-HDC）和PLP依赖性鸟氨酸 脱羧酶（PLP-ODC），两者均来自乳杆菌30 α，革兰氏阳性 哺乳动物肠道中的细菌。 相应的哺乳动物 酶不太适合于X射线衍射研究。 组氨酸的脱羧作用是唯一已知的 组胺的生物合成。 组胺的生理作用包括 血管舒张、低血压、胃酸分泌、过敏反应和 调节过敏反应。 从L. 30阿尔法是一个 诱导酶，和最彻底的研究一类不寻常的 从内部氨基酸产生自身辅基的酶 残余物 我们测定了低pH（双曲线）的X射线结构 动力学）形式的Prv-HDC，并提出了一种作用机制， 产品和抑制剂复合物的基础。 人树突状细胞的基因已经克隆 在乔恩·罗伯特斯博士的实验室里， 制备 我们已经分离并结晶了几个这样的突变体， 晶体学研究 此外，我们建议生产稳定的proHDC 突变体（S82 A），以研究该酶的活化特性， 检查HDC的较高pH（S形动力学）形式。 高分辨率数据 这些系统将收集与我们的哈姆林/Xuong地区探测器 系统和结构在该大学的克雷超级计算机上进行了优化。 鸟氨酸脱羧酶（ODC）是一种PLP依赖性酶， - 将鸟氨酸转化为腐胺，腐胺是多胺精胺的前体， 亚精胺 ODC存在于所有细胞中，但在 快速分裂的细胞 它已被用作癌症的靶点 化疗和抑制剂α-DL-二氟甲基鸟氨酸已经被 成功用于治疗锥虫感染。 所述酶是 通常以其与哺乳动物酶的快速周转率而闻名 具有仅约1小时的稳定半衰期。 相反，L. 30 α是稳定的，代表大亚基（80 kDa）PLP 脱羧酶 我们的ODC晶体可提供超过2.4A的分辨率， 已经收集了潜在的重原子衍生物。 我们建议 确定ODC的晶体结构，克隆ODC基因并测序， 然后表达它，用于定点突变研究其活性， 绝佳的价钱