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: 6182146
• 负责人: ELLEN L NEIDLE
• 金额: $ 18.09万
• 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/6182146
• 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：（改编自申请人的摘要）此项目提出了一个 了解芳香族化学和生物学的全面方法 来自土壤细菌的环羟基二加氧酶，acinetobacter sp。 ADP1。 这些酶催化芳香环的（顺式）二羟基化，因此 这样做，将两个O2原子掺入环中。该提案解决 这类酶的基本问题，包括催化 机理，金属中心的作用和分子决定因素 底物偏好，并将调查先前未表征的 芳族环羟基酶，炭疽菌1,2-二氧酶（ANT） 将催化和结构特性与相关苯甲酸酯的催化和结构特性进行比较 来自同一生物体的1,2-二氧酶（BEN）。这两个都是2组件 由含有rieske铁的A3B3六聚合加氧酶组成的系统 中心和一个亚基中的单核亚铁中心和一个还原酶 含有黄素和Fe/S中心的蛋白质。单核中心执行 Rieske Center作为氧化还原载体时的氧合酶反应。在 为了解决底物特异性问题，本金 研究人员建议使用一种包括随机突变和中的方法 体内选择以分离已修改的ben酶 利用炭疽菌作为有效的底物。氨基酸的比较 由此过程产生的几种突变的二加氧酶的序列 野生型酶的那些，以及结构和结构和 光谱数据将允许了解负责的功能 底物特异性。这些酶的催化机制和几种 还提出了该机制的测试。