Indispensible Roles of an "Inactive" Domain in Catalase-Peroxidase Structure and Catalysis: Applications for Enzyme Engineering

过氧化氢酶-过氧化物酶是酶工程的理想起点，部分原因是它们为探索酶结构/功能的一个知之甚少的方面提供了一个窗口。 也就是说，多远的蛋白质结构可以用来调节活性位点的功能。 所有的过氧化氢酶-过氧化物酶都具有一种新的基因复制的双结构域结构。 C-末端结构域不再结合血红素或催化任何反应，但很明显，尽管其外观退化，但它对于距离约30 A的活性位点的功能是必不可少的。 过氧化氢酶-过氧化物酶可被导向酶工程化的方式有三种：1）现有的活性位点可被精炼用于新的氧化转化，2）过氧化氢酶-过氧化物酶组分可被适当地用于构建新的血红素酶，以及3）过氧化氢酶-过氧化物酶功能中明显的策略可用于工程化其他血红素酶。 由于C-末端结构域可能在所有三个维度上都是有用的，因此本项目的目标是确定C-末端结构域在酶工程应用中的过氧化氢酶-过氧化物酶催化中的作用。实验计划是评估和表征E.大肠杆菌过氧化氢酶-过氧化物酶及其独立结构域的催化和结构特性的诊断变化。 阐明的每个角色代表了工程新血红素酶的潜在策略。 此外，C末端结构域本身在结构上准备保留自己的活性位点，为新催化剂的产生开辟了更多选择。通过设计具有独特功能的新酶，可以解决有毒污染物对环境造成的污染等全球性的紧迫问题。本项目包括通过为社区大学生的教师提供研究经验，加强对社区大学生的科学教育的具体计划。 这是通过邀请教师花周末从事研究项目来实现的。 这些经验已经并将继续扩大教师在课程中涵盖尖端技术/概念的能力，因此，吸引社区学院学生进入科学和工程职业轨道。 这种方法对该地区的少数民族学生产生了直接影响，因为在亚拉巴马社区/初级学院注册的学生中有24%是非洲裔美国人，这一比例远远高于该州的主要大学。

Catalase-peroxidases are ideal starting points for enzyme engineering, in part, because they offer a window to explore a poorly understood aspect of enzyme structure/function. Namely, how distant protein structures can be used to modulate active site function. All catalase-peroxidases have a novel gene-duplicated two-domain structure. The C-terminal domain no longer binds heme or catalyzes any reaction, but it is clear that in spite of its vestigial appearance, it is essential for the function of the active site some 30 A away. There are three ways that catalase-peroxidases may be directed toward enzyme engineering: 1) the existing active site may be refined for new oxidative transformations, 2) catalase-peroxidase components may be appropriated to build new heme enzymes, and 3) strategies evident in catalase-peroxidase function may be used for engineering other heme enzymes. Because the C-terminal domain may be useful in all three dimensions, the objective of this project is to determine the role(s) of the C-terminal domain in catalase-peroxidase catalysis in light of application to enzyme engineering. The experimental plan is to evaluate and characterize variants of E. coli catalase-peroxidase and its freestanding domains for diagnostic changes in catalytic and structural properties. Each role elucidated represents a potential strategy for engineering new heme enzymes. Moreover, the C-terminal domain itself is structurally poised to hold its own active site, opening even more options for generation of new catalysts. The ability to engineer new enzymes for unique functions holds great promise for addressing urgent concerns that are global in their scope and impact, such as contamination of the environment with toxic pollutants.This project includes specific plans to enhance science education for community college students by providing research experiences for their instructors. This is accomplished by inviting instructors to spend weekends engaged in research on the project. These experiences have already and will continue to expand instructors' ability to cover cutting-edge techniques/concepts in their courses, and as a result, draw community college students into science and engineering career tracks. This approach provides for immediate impact on minority students in this region because 24% of students enrolled in Alabama community/junior colleges are African-American, a proportion much greater than the major universities in the state.