STRUCTURE-FUNCTION OF PYRUVOYL & PLP-DEPENDENT ENZYMES

丙酮酰的结构-功能

• 批准号: 3277727
• 负责人: MARVIN L HACKERT
• 金额: $ 17.84万
• 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/3277727
• 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) 作为必需物质 辅因子而另一些则利用共价结合的α-酮酸。 拟议的 研究涉及每种辅因子类型的脱羧酶：丙酮酰- 依赖组氨酸脱羧酶 (Prv-HDC) 和 PLP 依赖鸟氨酸 脱羧酶 (PLP-ODC)，均来自革兰氏阳性乳杆菌 30α 存在于哺乳动物肠道内的细菌。 相应的哺乳动物 酶不太适合 X 射线衍射研究。 组氨酸脱羧是唯一已知的机制 组胺的生物合成。 组胺的生理作用包括 血管舒张、低血压、胃 HCl 分泌、过敏反应和 介导过敏反应。 L.30alpha 的 Prv-HDC 是 诱导酶，是一类不寻常的酶中研究最彻底的 从内部氨基酸产生自己的辅基的酶 残留物。 我们已经确定了低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。 HDC基因已被克隆 在 Jon Robertus 博士和几个位点特异性突变体的实验室中 准备好了。 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（S82A）来研究该酶的激活特性，并且 检查 HDC 的较高 pH（S 形动力学）形式。 高分辨率数据 这些系统的一部分将由我们的 Hamlin/Xuong 区域探测器收集 系统和在大学克雷超级计算机上改进的结构。 鸟氨酸脱羧酶 (ODC) 是一种 PLP 依赖性酶，可将 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，但在以下细胞中活性特别高 快速分裂的细胞。 它已被用作癌症靶点 化疗和抑制剂α-DL-二氟甲基鸟氨酸已被 成功用于治疗锥虫感染。 该酶是 通常因其与哺乳动物酶的快速周转率而闻名 稳定的半衰期仅为约1小时。 相比之下，来自 L. 30alpha 稳定，代表大亚基 (80kDa) PLP 脱羧酶。 我们的 ODC 晶体的衍射分辨率超过 2.4A，并且两个 潜在的重原子衍生物已被收集。 我们建议 确定ODC的晶体结构，对ODC基因进行克隆和测序， 然后将其表达用于对其活性进行定点诱变研究 地点。