Mechanisms of Catalysis and Cofactor Biosynthesis of Redox Enzymes with Unusual Cofactors

这项研究计划的重点是表征氧化还原酶与不寻常的 辅因子及其与其他电子转移（ET）蛋白的相互作用。这包括 不含外源辅因子的酶，而是具有“蛋白质衍生的”酶。 通过氨基酸的多重不可逆翻译后修饰形成的“辅因子”， 残基这项研究至少在生物学的三个领域做出了重大贡献。 和生物医学意义：阐明生物ET的机制; 蛋白质衍生辅因子的生物合成机制及其催化性质;以及 发现血红素功能和氧活化的新机制。未来的目标 包括三种新型氧化还原酶的表征，我们最近 鉴定Lod-A样蛋白具有半胱氨酸蒽醌（CTQ）辅因子，其来源于 特异性半胱氨酸和色氨酸残基。这些是不寻常的，因为它们是第一种酶， 作为氧化酶而不是脱氢酶起作用的蒽醌辅因子。他们是 还描述了第一种使用除黄素以外的辅因子进行催化的氨基酸氧化酶。 LodB样蛋白是催化翻译后修饰所需的黄素酶 用于CTQ生物合成的前体LodA样蛋白。这些反应必须通过 涉及远程ET的“远程催化”，因为被修饰的残基位于 蛋白质和不暴露于表面。因此，该过程的机制必须是新颖的 因为没有描述过由黄素酶催化的反应， 特别是通过远程催化。Rv 2633 c是来自结核分枝杆菌的蛋白质， 在感染期间响应宿主防御而上调。它有两个非血红素离子 根据序列预测它们形成氧桥连的hemerythrin样位点。我们发现 这是第一个具有过氧化氢酶功能的hemerythrin样蛋白，也是第一个 具有非血红素二铁辅因子的过氧化氢酶。这些研究的结果将进一步促进我们的 了解酶催化反应的范围和机理， 特别是涉及氧反应性和自由基中间体的那些。了解如何 酶在催化过程中控制这些活性物质，同时使氧化损伤最小化， 提供如何减轻氧化应激后果的见解。这也将扩大 或当前关于蛋白质进化和蛋白质结构-功能关系的观点，并提供 对蛋白质工程策略的见解，将新的官能团引入蛋白质。

This research program focuses on the characterization of redox enzymes with unusual cofactors, and their interactions with other electron transfer (ET) proteins. This includes enzymes that do not contain exogenous cofactors, but instead possess “protein-derived cofactors” that are formed by multiple irreversible post-translational modifications of amino acid residues. This research has made major contributions to at least three areas of broad biological and biomedical significance: elucidation of mechanisms of biological ET; characterization of mechanisms of biosynthesis of protein-derived cofactors and their catalytic properties; and discovery of novel mechanisms of heme function and oxygen activation. The future goals include characterization of three novel classes of redox enzymes that we have recently identified. Lod-A like proteins possess a cysteine tryptophylquinone (CTQ) cofactor derived from specific cysteine and tryptophan residues. These are unusual as they are the first enzymes with tryptophylquinone cofactors that function as oxidases rather than dehydrogenases. They are also the first amino acid oxidases described that uses a cofactor other than a flavin for catalysis. LodB-like proteins are flavoenzymes that catalyze the post-translational modifications required for CTQ biosynthesis on a precursor LodA-like protein. These reactions must be performed by “remote catalysis” that involves long-range ET, since the residues that are modified reside within the protein and are not surface exposed. As such, the mechanism of this process must be novel because no such reactions have been described that are catalyzed by a flavoenzyme, especially by remote catalysis. Rv2633c is a protein from Mycobacterium tuberculosis that is upregulated in response to the host defense during infection. It possesses two non-heme irons that are predicted from sequence to form an oxo-bridged hemerythrin-like site. We showed that this is the first hemerythrin-like protein to function as a catalase, and is the first example of a catalase with a non-heme di-iron cofactor. The results of these studies will further our understanding of the range and mechanisms of reactions that can be catalyzed by enzymes, particularly those involving oxygen reactivity and free radical intermediates. Understanding how enzymes control these reactive species during catalysis while minimizing oxidative damage will provide insights into how to mitigate the consequences of oxidative stress. This will also expand or current views about protein evolution and protein structure-function relationships, and provide insights for protein engineering strategies to introduce new functional groups into proteins.