Metalloenzyme structure, function and assembly

摘要 金属离子与蛋白质的结合提供了独特的化学反应，这是许多 自然界最惊人的化学变化之一。我的实验室询问了金属酶是如何利用 超级亲核者和自由基辅因子的反应性，同时保护自己免受潜在的损害。它 是研究金属酶的一个令人难以置信的令人兴奋的时刻。生物信息学和基因组研究 以令人眼花缭乱的速度鉴定新的可能的金属酶，现在有超过10万个独特的序列 与自由基S-腺苷蛋氨酸(SAM)酶家族有关。对这些的描述 酶揭示了前所未有的化学和新的辅因子结合结构基序。令人印象深刻的是，许多人 在这些自由基中，SAM(RS)酶是生物合成途径的一部分，产生天然产物 新型分子支架和良好的药物性能(包括抗生素、抗病毒和抗病毒) 肿瘤属性)。我的实验室正在使用我们最喜欢的X射线结晶学技术来探测 这个家族中的序列空间，目的是了解RS酶如何利用自由基物种 进行具有化学挑战性的反应。在接下来的五年里，我们将利用最近的成功，继续 研究钴胺依赖的RS酶的结构和功能。这个有7000名成员的RS子群 代表了一系列新的挑战和机遇，以了解自然如何调整和控制这两个激进的 以及超亲核反应性。最近备受关注的不仅仅是RS酶家族； 甘氨酰自由基酶(GRE)家族也受到越来越多的关注。在后一种情况下，人类 微生物组计划正在提供有关GREs在人类中的重要性和丰富程度的新信息 肠道和口腔。例如，在肠道中发现的最丰富的未鉴定的酶是GRE！在……里面 在接下来的五年里，我们计划调查几个新发现的GRE家族成员，它们似乎 成为人类微生物群落中的关键角色。我们的目标是使用我们的结构工具来审问 有助于微生物新陈代谢的基于自由基的化学的分子基础，并有可能 发病机制，在人类的肠道中。许多这样的GRE在常见的病原体中发现，比如艰难梭菌， 是潜在的毒品目标。最后，这是一个研究“生命的大集群”的伟大时期，它们是 负责固定碳(C-簇/A-簇)、氮(MoFe簇)和氢(H-簇)。 我的实验室专注于碳固定和一氧化碳脱氢酶/乙酰基的C-和A-簇- 辅酶A合成酶。最近的进展提供了重组系统，使我们能够探测星系团 以一种以前不可能实现的方式进行组装、反应机理和氧气敏感性。氧气- 敏感性是复杂金属蛋白的致命弱点，我们计划使用我们的结构工具箱 探讨C-团簇氧敏感性的分子基础。

Abstract The combination of metal ions with proteins offers unique chemical reactivities, which are at the heart of many of Nature's most amazing chemical transformations. My laboratory interrogates how metalloenzymes harness the reactivity of supernucleophiles and radical cofactors, while protecting themselves from potential damage. It is an incredibly exciting time to be studying metalloenzymes. Bioinformatics and genomic studies are identifying new putative metalloenzymes at a dizzying pace, with more than 100,000 unique sequences now associated with the Radical S-adenosylmethionine (SAM) enzyme family alone. Characterization of these enzymes is revealing unprecedented chemistry and new cofactor-binding structural motifs. Impressively, many of these Radical SAM (RS) enzymes are part of biosynthetic pathways that produce natural products with novel molecular scaffolds and promising pharmaceutical properties (including antibiotic, antiviral, and anti- tumor properties). My laboratory is employing our favorite technique of X-ray crystallography to probe sequence space within this family with the goal of understanding how RS enzymes harness radical-species to perform chemically challenging reactions. In the next five years, we will leverage recent success and continue to investigate the structure/function of cobalamin-dependent RS enzymes. This 7000-membered RS subgroup represents a new set of challenges and opportunities to understand how Nature tunes and controls both radical and supernucleophile reactivities. It is not only the RS enzyme family that has been in the spotlight recently; the glycyl radical enzyme (GRE) family is also receiving increased attention. In this latter case, the human microbiome project is providing new information as to the importance and abundance of GREs in the human gut and oral cavities. For example, the most abundant uncharacterized enzyme found in the gut is a GRE! In the next five years, we plan to investigate several newly discovered members of the GRE family that appear to be key players in human microbial communities. Our goal is to use our structural tools to interrogate the molecular basis for the radical-based chemistry that contributes to microbial metabolism, and potentially pathogenesis, in the human gut. A number of these GREs are found in common pathogens, like C. difficile, and are potential drug targets. Finally, it is a great period to be working on the “great clusters of life,” which are responsible for the fixation of carbon (C-cluster/A-cluster), nitrogen (MoFe cluster) and hydrogen (H-cluster). My laboratory focuses on carbon fixation and the C- and A-clusters of carbon monoxide dehydrogenase/acetyl- CoA synthase. Recent advances have afforded recombinant systems that are allowing us to probe cluster assembly, reaction mechanism, and oxygen-sensitivity in a manner that was not possible previously. Oxygen- sensitivity is the Achilles heel of a complex metalloprotein and we plan to use our structural toolbox to investigate the molecular basis of C-cluster oxygen-sensitivity.