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

这项研究计划集中在氧化还原酶的特性与不寻常的 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，而是具有“蛋白质来源”的酶 由多次不可逆的氨基酸翻译后修饰形成的“辅因子” 残留物。这项研究至少在三个广泛的生物学领域做出了重大贡献 和生物医学意义：阐明生物ET的机制；表征 蛋白质辅因子的生物合成机制及其催化性能；以及 发现血红素功能和氧活化的新机制。未来的目标 包括我们最近获得的三类新的氧化还原酶的特性 已确认身份。类LOD-A蛋白含有一种半胱氨酸色氨酸苯二酚(CTQ)辅因子，来源于 特定的半胱氨酸和色氨酸残基。这些是不同寻常的，因为它们是第一种具有 作为氧化酶而不是脱氢酶起作用的色氨酸辅因子。他们是 还描述了第一种使用黄素以外的辅因子进行催化的氨基酸氧化酶。 LodB样蛋白是一种黄素酶，可催化所需的翻译后修饰 用于前体类Loda蛋白的CTQ生物合成。这些反应必须由 “远程催化”涉及远程ET，因为被修饰的残基驻留在 这种蛋白质不会暴露在表面。因此，这一过程的机制必须是新颖的 因为没有描述由黄素酶催化的这种反应， 尤其是通过远程催化。Rv2633c是一种来自结核分枝杆菌的蛋白质 在感染过程中，由于宿主的防御而上调。它有两个非血红素铁 根据序列预测形成氧桥联的氢氰菊酯样位点。我们向大家展示了 这是第一个发挥过氧化氢酶功能的类似氢氰菊酯的蛋白质，也是第一个 含非血红素二铁辅因子的过氧化氢酶。这些研究的结果将推动我们的 了解可由酶催化的反应的范围和机理， 尤其是那些涉及氧气反应性和自由基中间体的化合物。了解如何 酶在催化过程中控制这些活性物种，同时将氧化损伤降至最低 提供有关如何减轻氧化应激后果的见解。这也将扩大 或关于蛋白质进化和蛋白质结构-功能关系的当前观点，并提供 对蛋白质工程策略的见解，为蛋白质引入新的官能团。

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.