Iron-Sulfur Clusters in Biological Radical Generation

描述（由申请人提供）：该提案的重点是探索称为自由基SAM酶的酶的大超家族的基本化学机制。这些酶存在于人类中，具有多种功能，如辅因子合成和抗病毒反应，并且在自然界中的所有生命王国中发现。自由基SAM超家族很大，有数千个已鉴定的成员催化一系列反应;它们的共同点是利用特定类型的金属簇（[4Fe-4S]簇）和S-腺苷-L-甲硫氨酸（SAM）在其底物上引发自由基化学。目前的建议将主要集中在三个成员的自由基SAM超家族（丙酮酸甲酸裂解酶激活酶，赖氨酸2，3-aminomutase，和蝰蛇蛋白），具有不同的功能，实验范围将从基本的机械，光谱和结构的研究，旨在描绘的化学机制的细节，以基本的功能研究，旨在了解一个重要的哺乳动物自由基SAM酶的作用。该提案的具体目标是：1）通过使用设计用于捕获反应中间体的技术来识别这些酶的催化机制中的特定步骤，2）探索这些酶的[4Fe-4S]簇中的价定位、其原因及其对功能的影响，3）从结构上表征一种自由基SAM酶与其蛋白质底物之间的相互作用，和4）功能性表征参与抗病毒应答的人自由基SAM酶。

DESCRIPTION (provided by applicant): This proposal is focused on exploring the fundamental chemical mechanism of a large superfamily of enzymes called radical SAM enzymes. These enzymes occur in humans in such diverse functions as cofactor synthesis and the antiviral response, and are found in nature in all kingdoms of life. The radical SAM superfamily is large, with thousands of identified members catalyzing a range of reactions; they have in common the utilization of a specific type of metal cluster (a [4Fe-4S] cluster) and S-adenosyl-L-methionine (SAM) to initiate radical chemistry on their substrates. The current proposal will focus primarily on three members of the radical SAM superfamily (pyruvate formate-lyase activating enzyme, lysine 2,3-aminomutase, and viperin) that have diverse functions, and experiments will range from fundamental mechanistic, spectroscopic, and structural studies aimed at delineating details of the chemical mechanisms to basic functional studies aimed at understanding the role of an important mammalian radical SAM enzyme. The specific aims of the proposal are: 1) To identify specific steps in the catalytic mechanisms of these enzymes by using techniques designed to trap reaction intermediates, 2) To explore valence localization in the [4Fe-4S] cluster of these enzymes, its causes, and its implications for function, 3) To structurally characterize the interaction between one radical SAM enzyme and its protein substrate, and 4) to functionally characterize a human radical SAM enzyme involved in the antiviral response.