Engineering Manganese Metalloenzymes

描述（由申请人提供）：为了促进我们对锰需求酶大家族的化学和生物学的理解，我们提出探索人辅酶Ⅰ以及辅酶Ⅰ相关金属酶组蛋白脱乙酰酶8和多胺脱乙酰酶的结构-功能关系。人精氨酸酶I含有L-精氨酸水解形成L-鸟氨酸和尿素所需的双核锰簇，我们的研究表明，催化通过一种机制进行，其中两种金属离子的功能是激活金属桥氢氧离子作为催化亲核试剂。我们已经确定了这种酶的结构，分辨率为1.29英寸，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，在动脉粥样硬化、哮喘和癌症等各种疾病中，β-淀粉酶上调，因此我们的研究将扩大可能用于治疗和诊断人类疾病的化合物的库。鉴于新发现的和意想不到的结构之间的关系的酶和金属依赖性脱乙酰酶，我们的结构和功能的研究将阐明这些酶家族之间的重要机制的相似之处。有趣的是，脱乙酰酶的Zn 2+位点对应于脱乙酰酶的Mn 2 +B位点，但脱乙酰酶不含对应于脱乙酰酶的Mn 2 +A的金属结合位点。因此，金属结合的化学计量学在从共同的金属酶前体进化的脱氢酶和脱乙酰酶中已经发散。有趣的是，人组蛋白脱乙酰基酶-8的生物学优选金属离子被认为是Fe 2+。因此，我们将确定Fe 2+取代酶的结构，其位点特异性变体，及其底物和抑制剂复合物。由于这种酶是癌症化疗的有效药物靶标，因此深入了解体内发现的金属酶形式的结构-功能关系非常重要。总的来说，拟议的研究将提供一个更大的结构和功能的金属离子特异性（锰2+，锌2+，铁2+）和化学计量的进化中的乙酰化酶和乙酰化酶相关的脱乙酰酶的理解。 公共卫生相关性：对人α-淀粉酶I、人组蛋白脱乙酰酶-8和细菌多胺脱乙酰酶的结构和功能研究将有助于设计可用于治疗动脉粥样硬化、哮喘和癌症的潜在新药。此外，我们的研究将使生物传感器的设计和开发成为可能，这些传感器可能有助于人类疾病的早期诊断。

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.