Engineering Manganese Metalloenzymes

描述(申请人提供)：为了促进我们对需要锰的酶大家族的化学和生物学的了解，我们建议探索人类精氨酸酶I以及与精氨酸酶相关的金属酶组蛋白脱乙酰基酶8和多胺脱乙酰基酶的结构-功能关系。人精氨酸酶I含有一个双核锰簇，是L-精氨酸水解生成L-鸟氨酸和尿素所必需的，我们的研究表明，催化作用是通过一种机制进行的，在该机制中，两种金属离子都作为催化亲核试剂激活了金属桥联的氢氧离子。我们已经确定了这种酶的结构，分辨率为1.29？，我们将利用这种结构来指导抑制剂和生物传感器的设计。最近的发现表明，精氨酸酶在动脉粥样硬化、哮喘和癌症等各种疾病中表达上调，因此我们的研究将扩大化合物的谱系，这些化合物可能对人类疾病的治疗和诊断有用。鉴于精氨酸酶和金属依赖脱乙酰酶之间新发现的和意想不到的结构关系，我们的结构和功能研究将阐明这些酶家族之间重要的机制相似之处。有趣的是，脱乙酰酶的锌离子部位对应于精氨酸酶的锰2+B部位，但脱乙酰酶并不含有精氨酸酶的锰2+A相对应的金属结合部位。因此，在精氨酸酶和脱乙酰酶从一个共同的金属酶前体进化而来的过程中，金属结合的化学计量学有所不同。有趣的是，人类组蛋白脱乙酰基酶-8的生物偏好金属离子被认为是Fe2+。因此，我们将确定Fe2+取代酶的结构，它的特定位点变体，以及它的底物和抑制剂复合体。由于这种酶是癌症化疗的有效药物靶点，因此彻底了解体内发现的金属酶的结构与功能关系是很重要的。总体而言，拟议的研究将对精氨酸酶和精氨酸酶相关脱乙酰酶进化中的金属离子特异性(Mn2+、Zn2+、Fe2+)和化学计量学有更好的结构和功能理解。 公共卫生相关性：对人类精氨酸酶I、人类组蛋白脱乙酰酶-8和细菌多胺脱乙酰酶的结构和功能研究将促进潜在新药的设计，这些新药可用于治疗动脉粥样硬化、哮喘和癌症。此外，我们的研究将使生物传感器的设计和开发成为可能，这些传感器可能对人类疾病的早期诊断有用。

DESCRIPTION (provided by applicant): In order to advance our understanding of the chemistry and biology of the greater family of manganese- requiring enzymes, we propose to explore structure-function relationships in human arginase I as well as the arginase-related metalloenzymes histone deacetylase 8 and polyamine deacetylase. Human arginase I contains a binuclear manganese cluster required for the hydrolysis of L-arginine to form L-ornithine and urea, and our studies indicate that catalysis proceeds through a mechanism in which both metal ions function to activate a metal-bridging hydroxide ion as the catalytic nucleophile. We have determined the structure of this enzyme to 1.29 ¿ resolution, and we will use this structure to guide the design of inhibitors and biosensors. Recent discoveries show that arginase is upregulated in various diseases such as atherosclerosis, asthma, and cancer, so our studies will expand the repertoire of chemical compounds that will potentially be useful for the treatment and diagnosis of human disease. Given the newly-discovered and unexpected structural relationship between the arginases and metal- dependent deacetylases, our structural and functional studies will illuminate important mechanistic parallels between these enzyme families. Intriguingly, the Zn2+ site of the deacetylase corresponds to the Mn2+B site of arginase, but the deacetylase does not contain a metal binding site corresponding to Mn2+A of arginase. Thus, the stoichiometry of metal binding has diverged in the evolution of the arginases and the deacetylases from a common metalloenzyme precursor. Intriguingly, the biologically preferred metal ion of human histone deacetylase-8 is believed to be Fe2+. Therefore, we will determine the structures of the Fe2+-substituted enzyme, its site-specific variants, and its substrate and inhibitor complexes. Since this enzyme is a validated drug target for cancer chemotherapy, it is important to thoroughly understand structure-function relationships in the form of the metalloenzyme that is found in vivo. Overall, the proposed research will provide a greater structural and functional understanding of metal ion specificity (Mn2+, Zn2+, Fe2+) and stoichiometry in the evolution of the arginases and the arginase-related deacetylases. PUBLIC HEALTH RELEVANCE: Structural and functional studies of human arginase I, human histone deacetylase-8, and bacterial polyamine deacetylase will facilitate the design of potential new drugs that can be used to treat atherosclerosis, asthma, and cancer. Additionally, our studies will enable the design and development of biosensors that may be useful in the early diagnosis of human disease.