Engineering Proteins for Cell-Free Mimics of the Oxidative Burst Immune Strategy

A combination of combinatorial and rational biosynthetic strategies have been used to create a class of functional materials, based on enzymes, that mimics the oxidative burst immune strategy used by macrophages and neutrophils to kill bacteria and fungi. The NADPH oxidase (NOX) complex is part of the molecular-level machinery that creates oxidative bursts. This redox enzyme system consists of two membrane bound elements, three cytosolic proteins, and a G protein, and it is not routinely cloned or functionally assembled in its entirety outside of a cell. I have used directed evolution to alter cytochromes P450, enzymes with more favorable engineering properties, such that they functionally mimic NOX. I will present the identification and characterization of a panel of mutant enzymes that use NADPH to produce variable types and amounts of reactive oxygen species.