CHEMISTRY AND BIOLOGY OF NON-HEME IRON OXYGENASES

非血红素铁加氧酶的化学和生物学

• 批准号: 6386590
• 负责人: ELLEN L NEIDLE
• 金额: $ 18.53万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386590
• 关键词: Mossbauer spectrometry; Neisseriaceae; X ray crystallography; bacterial proteins; bioengineering /biomedical engineering; catalyst; circular magnetic dichroism; electron spin resonance spectroscopy; enzyme activity; enzyme structure; enzyme substrate; iron sulfur protein; nucleic acid sequence; o aminobenzoate; oxygenases; phenotype; protein binding; protein sequence; stoichiometry

DESCRIPTION: (adapted from applicant's abstract) This project proposes a comprehensive approach to understanding the chemistry and biology of aromatic ring-hydroxylating dioxygenases from the soil bacteria, Acinetobacter sp. ADP1. These enzymes catalyze the (cis) dihydroxylation of aromatic rings and, in so doing, incorporate both atoms of O2 into the ring. The proposal addresses fundamental questions for this class of enzymes including catalytic mechanism(s), the roles of the metal centers, and the molecular determinants of substrate preferences, and will investigate a previously uncharacterized aromatic ring-hydroxylating enzyme, anthranilate 1,2-dioxygenase (Ant) comparing catalytic and structural properties to those of the related benzoate 1,2-dioxygenase (Ben) from the same organism. Both of these are 2-component systems consisting of a A3B3 hexameric oxygenase containing a Rieske iron center and a mononuclear ferrous center in the A subunit, and a reductase protein containing flavin and Fe/S centers. The mononuclear center carries out the oxygenase reaction while the Rieske center acts as a redox carrier. In order to address the question of substrate specificity, the principal investigator proposes to use an approach consisting of random mutation and in vivo selection to isolate Ben enzymes that have been modified for the utilization of anthranilate as an efficient substrate. Comparison of amino acid sequences of several mutated dioxygenases resulting from this procedure with those of the wild-type enzymes, in conjunction with structural and spectroscopic data will allow an understanding of the features responsible for substrate specificity. A catalytic mechanism for these enzymes and several tests of the mechanism are also proposed.

描述：(改编自申请者摘要)本项目提出了一种 了解芳香族化合物化学和生物学的综合方法 土壤细菌不动杆菌的环羟化双加氧酶。ADP1。 这些酶催化芳香环的(顺式)二羟基化，在SO 这样做，将O2的两个原子结合到环中。该提案涉及 关于这类酶的基本问题，包括催化 机理(S)，金属中心的作用，以及分子决定因素 底物偏好，并将调查一种以前未被表征的 芳香族环羟化酶，邻氨基苯甲酸酯1，2-双加氧酶(Ant) 与相关苯甲酸酯的催化性能和结构性能的比较 来自同一生物体的1，2-双加氧酶(BEN)。这两个都是双组分的 由含有Rieske铁的A3B3六聚体加氧酶组成的体系 A亚基上有一个中心和一个单核铁中心，以及一个还原酶 含有黄素和铁/S中心的蛋白质。单核中心进行 加氧酶反应，而Rieske中心作为氧化还原载体。在……里面 为了解决底物专一性的问题，主要的 研究人员建议使用一种由随机突变和 活体选择以分离已被修饰的BEN酶 邻氨基苯甲酸酯作为高效底物的利用。氨基酸的比较 几种突变的双加氧酶的序列 野生型酶的那些，结合结构和 光谱数据将允许理解负责的特征 底物专一性。这些酶和几种酶的催化机理 文中还提出了对该机制的测试。