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

MIOX是新生的混合价二价铁加氧酶(MVDO)亚家族的创始成员，也是迄今为止唯一确定的成员。它使用Fe2(II/III)辅助因子激活Fe(II)位上的O2和Fe(III)位上的肌醇，以进行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，其他几种蛋白质属于这一类。HD-结构域超家族中磷酸水解酶-MVDO边界的绘制和新MVDO的表征将有助于正确的功能分配，扩大这一新类别加氧酶的已知催化谱系，并揭示自然界在一个共同的蛋白质支架内从根本上不同功能的进化。该项目可能会从根本上发现新的反应，这些反应将扩大已知的一类新型加氧酶的催化反应，目前这种加氧酶只由一个已记录的成员组成。它将使人们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主要挑战：(1)对现有DNA序列的数据库中预测的大量蛋白质进行功能分配；(2)了解共享共同结构的蛋白质“超家族”中新功能的进化。它还将提供从生物信息学分析到分析和蛋白质生物化学、酶动力学和机理、蛋白质结晶学和生物光谱学等活动方面的杰出培训。该科学项目将为主要的外联活动--一个约有120人参加的两年一次的现代矿物生物化学培训“讲习班”注入新的案例研究，说明重要的概念，并努力提取这些概念和开发新的案例研究，这将突出受训者的科学重点。

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.