Engineering Manganese Metalloenzymes

描述（由申请人提供）：为了提高我们对更大的锰需要酶家族的化学和生物学的理解，我们提出探索人类精氨酸酶I以及精氨酸酶相关的金属酶组蛋白去乙酰化酶8和多胺去乙酰化酶的结构-功能关系。人精氨酸酶I含有双核锰簇，这是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，催化是通过两种金属离子激活金属桥接氢氧化物离子作为催化亲核试剂的机制进行的。我们已经确定了这种酶的结构为1.29¿分辨率，我们将使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、哮喘和癌症等多种疾病中表达上调，因此我们的研究将扩大对人类疾病的治疗和诊断可能有用的化合物的宝库。鉴于精氨酸酶和金属依赖性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性。有趣的是，去乙酰化酶的Zn2+位点与精氨酸酶的Mn2+B位点相对应，但去乙酰化酶不含有与精氨酸酶的Mn2+ a相对应的金属结合位点。因此，金属结合的化学计量学在精氨酸酶和脱乙酰酶从一个共同的金属酶前体进化的过程中出现了分歧。有趣的是，人类组蛋白去乙酰酶-8的生物学首选金属离子被认为是Fe2+。因此，我们将确定Fe2+取代酶的结构，它的位点特异性变体，以及它的底物和抑制剂复合物。由于这种酶是一种经过验证的癌症化疗药物靶点，因此彻底了解体内金属酶的结构-功能关系非常重要。总的来说，本研究将对金属离子特异性（Mn2+, Zn2+, Fe2+）和精氨酸酶及精氨酸酶相关去乙酰化酶进化过程中的化学计量学有更深入的结构和功能理解。

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.