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

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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.