Mapping the hydrolase-oxygenase boundaries and range of catalytic capabilities in the HD-domain dinuclear metalloenzyme superfamily

MIOX是新生的混合价二铁加氧酶（MVDO）亚家族的创始成员，并且迄今为止是唯一建立的成员。它使用Fe 2（II/III）辅因子在Fe（II）位点激活O2，在Fe（III）位点激活肌醇，进行C-C键断裂，四电子氧化。最近的一篇论文和我们关于P-C键裂解酶PhnZ的初步数据强烈表明，它是MVDO的第二个例子，我们对现有序列和结构的分析表明，其他几种蛋白质也属于这一类。在HD结构域超家族中的磷酸水解酶-MVDO边界的映射和新的MVDO的表征将促进适当的功能分配，扩展这类新的加氧酶的已知催化库，并揭示自然界的进化在一个共同的蛋白质支架内的根本不同的功能。该项目可能会从根本上发现新的反应，这将扩大已知的一类新的加氧酶的催化库，目前只包括一个单一的记录成员。它将深入了解大自然如何以看似微妙的方式重塑蛋白质支架及其金属辅因子，以创造显着不同的催化活性。因此，该项目解决了分子生物科学中的两个核心挑战：（1）在可用DNA序列的terabases内预测的大量蛋白质的功能分配和（2）了解共享共同结构的蛋白质“超家族”内新功能的进化。它还将提供从生物信息学分析，分析和蛋白质生物化学，酶动力学和机制，蛋白质晶体学，生物光谱学等活动的优秀培训。科学项目将注入主要的推广活动，一个约120人参加的现代金属生物化学两年一次的培训“讲习班”，新的案例研究说明了重要的概念，提取这些概念和开发新的案例研究的努力将提高受训人员的科学重点。

MIOX is the founding and, to date, only established member of the nascent mixed-valent di-iron oxygenase (MVDO) sub-family. It uses a Fe2(II/III) cofactor to activate O2 at the Fe(II) site and myo-inositol at the Fe(III) site for a C-C-bond-cleaving, four-electron oxidation. A recent paper and our preliminary data on the P-C-bond cleaving enzyme, PhnZ, strongly suggest it is the second example of an MVDO, and our analysis of available sequences and structures suggests that several other proteins belong to this class. The mapping of the phosphohydrolase-MVDO boundaries in the HD-domain superfamily and characterization of new MVDOs will facilitate proper functional assignment, expand the known catalytic repertoire of this new class of oxygenase, and shed light on Nature's evolution of radically divergent functions within a common protein scaffold. The project will likely uncover fundamentally new reactions that will expand the known catalytic repertoire of a novel class of oxygenase that at present consists of just a single documented member. It will yield insight into how Nature remodels a protein scaffold and its metallocofactor in seemingly subtle ways to create markedly different catalytic activities. Thus this project addresses two central challenges in molecular biosciences: (1) functional assignment of the huge number of proteins predicted within the terabases of available DNA sequence and (2) understanding the evolution of new functions within a "superfamily" of proteins sharing a common architecture. It will also provide outstanding training in activities ranging from bioinformatic analysis, to analytical and protein biochemistry, to enzyme kinetics and mechanism, to protein crystallography, to biological spectroscopy. The scientific project will infuse the primary outreach activity, a ~120-participant, biennial training "workshop" in modern metallobiochemistry, with new case studies illustrating important concepts, and efforts to extract such concepts and develop new case studies will sharpen the scientific focus of the trainees.