CHEMISTRY AND BIOLOGY OF NON-HEME IRON OXYGENASES

描述：（改编自申请人的摘要）本项目提出了一个 了解芳香族化合物的化学和生物学的综合方法 环羟化双加氧酶从土壤细菌，不动杆菌属ADP 1。 这些酶催化芳环的（顺式）二羟基化， 这样，将两个O2原子结合到环中。该提案涉及 这类酶的基本问题，包括催化 机制、金属中心的作用以及分子决定因素 基板的偏好，并将调查一个以前未表征 邻氨基苯甲酸1，2-双加氧酶 将催化和结构性质与相关苯甲酸酯的催化和结构性质进行比较 1，2-双加氧酶（Ben）来自同一种生物。这两种都是双组分的 由含有Rieske铁的A3 B3六聚体加氧酶组成的系统 A亚基中的单核亚铁中心和还原酶 含有黄素和Fe/S中心的蛋白质。单核细胞中心执行 加氧酶反应，而Rieske中心作为氧化还原载体。在 为了解决底物特异性的问题， 研究人员建议使用一种由随机突变和 体内选择以分离已经被修饰用于 利用邻氨基苯甲酸酯作为有效底物。比较氨基酸 由该方法产生的几种突变的双加氧酶的序列， 那些野生型酶，结合结构和 光谱数据将使我们能够理解 底物特异性这些酶的催化机制和几种 并提出了对该机理的检验方法。

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.