Iron-Sulfur Clusters in Biological Radical Generation

描述(由申请人提供)：这项提案的重点是探索称为自由基SAM酶的酶的一个大的超家族的基本化学机制。这些酶在人类体内以不同的功能存在，如辅因子合成和抗病毒反应，并在自然界中存在于所有生命王国中。自由基SAM超家族很大，有数以千计的已知成员催化一系列反应；它们的共同之处是利用一种特定类型的金属簇([4Fe-4S]簇)和S-腺苷-L-蛋氨酸(SAM)在其底物上启动自由基化学。目前的建议将主要集中在具有不同功能的自由基SAM超家族的三个成员(丙酮酸甲酸裂解酶激活酶、赖氨酸2，3-氨基变位酶和蛇毒蛋白)，实验范围从旨在描绘化学机制细节的基础机制、光谱和结构研究到旨在了解哺乳动物自由基SAM酶的作用的基础功能研究。该建议的具体目的是：1)通过使用旨在捕捉反应中间体的技术来确定这些酶的催化机制中的具体步骤，2)探索这些酶在[4Fe-4S]簇中的价位、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，以及4)对参与抗病毒反应的人自由基SAM酶进行功能表征。

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.