From Fundamental Studies of Metalloproteins to Practical Applications

金属酶能够有效地调节金属离子的性质，以催化非常困难的化学物质。 变形。然而，指导蛋白质功能进化的决定因素仍然不完全。 明白了。对决定酶功能极限的相互作用的细微细节缺乏了解 我们合理设计催化剂的能力。我们将测试为什么相同的酶家族使用不同的 金属辅因子。金属酶NRDF属于一类核糖核苷酸还原酶(RNR)，是必需的 在所有生物体中发现的催化核苷酸转化为脱氧核苷酸的酶。RNR依赖于 金属将活性部位保守的半胱氨酸氧化成硫基自由基，硫基自由基随后引发核苷酸 还原。在各种生物体中已经发现了几种不同类别的RNRs，有趣的是，尽管 作为整个催化途径的显著保守，该酶可以利用不同的金属来实现这一点。 我们最近发现，病原生物体的Ib类RNRs中的双锰中心具有相同的功能 任务是人类体内的双铁中心。此外，其他I类RNR酶利用二铁和混合铁- 锰是功能的中心。这种金属偏好的分子和生物决定因素仍然是 仍然不为人所知，这是该领域一个重大的悬而未决的问题。为了解决这个问题，我们 制定了三个具体目标。目的1.阐明RNR中特异性金属化的起源。使用Ib类 RnR从血链球菌(SS)，我们将解决的基本问题是如何正确的金属化 酶是被控制的。目的2.功能性RNR模型的设计。我们将使用一个稳定而简单的蛋白质模型 RNR(DFsc)与各种金属离子结合，并为实际有用的反应生成催化剂。这一目标将 提供简单、廉价和生物相容的蛋白质催化剂，用于氧化还原转化和农药/化学品 环境中的武器补救，这在适用的环境中可以很容易地处理。目标3.结构 金属蛋白的表征。在这里，我们将测试金属离子如何影响蛋白质的结构。 这一目标中提出的工作将验证和关联关于天然酶的研究。

Metalloenzymes are capable of efficiently tuning the properties of a metal ion to catalyze very difficult chemical transformations. Yet the determinants of what guides the evolution of protein function still remain not fully understood. This lack of understanding of the subtle detail of interactions that determine enzymatic function limits our ability to rationally design catalysts. We will test why the same family of enzymes uses different metallocofactors. Metalloenzyme NrdF belongs to a class of ribonucleotide reductases (RNR), essential enzymes found in all organisms to catalyze the conversion of nucleotides to deoxynucleotides. RNRs rely on metals to oxidize a conserved cysteine in the active site into a thiyl radical, which then initiates nucleotide reduction. Several different classes of RNRs have been identified in various organisms and, interestingly, despite a remarkable conservation of the overall catalytic pathway, the enzyme can utilize different metals to achieve it. We have recently shown that dimanganese center in class Ib RNRs of pathogenic organisms performs the same task as the diiron center in humans. Moreover, other class I RNR enzymes utilize diiron and mixed iron- manganese centers for function. The molecular and biological determinants of this metal preference still remain unknown and present a major unanswered question in the field. To address this question, we devised three specific aims. Aim 1. Elucidation of the origins of specific metalation in RNR. Using class Ib RNR from Streptococcus sanguinis (Ss) we will address the fundamental question of how correct metalation of enzymes is controlled. Aim 2. Design of functional RNR models. We will use a stable and simple protein model of RNR (DFsc) to bind various metal ions and generate catalysts for practically useful reactions. This aim will give simple, inexpensive and biocompatible protein catalysts for redox transformations and pesticide/chemical weapons remediation in the environment, that could be easily handled in the applied setting. Aim 3. Structural characterization of metalloproteins. Here we will test how metal ions influence the structure of the protein. The work proposed in this aim will validate and correlate the studies on the natural enzymes.