Project Title

Characterizing SpNox Enzyme Chimeras

Presenters

Academic department under which the project should be listed

CSM - Molecular and Cellular Biology

Faculty Sponsor Name

Susan Smith

Abstract (300 words maximum)

NADPH oxidases (NOXes) are a family of eukaryotic enzymes that produce reactive oxygen species (ROS). These transmembrane proteins move electrons from NADPH through FAD and across the membrane to the final electron acceptor O2, forming the superoxide anion O2-. Superoxide and downstream ROS are crucial signaling and cytotoxic molecules whose misregulation is associated with disease states ranging from diabetes to cancer. SpNox is a recently discovered prokaryotic NOX homolog from Streptoccous pneumoniae that is robust to bacterial expression and isolation, making it a good model system for study of the NOX family. The focus of this research project is to characterize the biochemical properties of SpNox using mutants and chimeras. The mutants are SpNox F397S and F397W, which in homologous systems affect affinity for the substrate NADPH and/or the cofactor FAD. The chimeras are composed of the wild type prokaryotic SpNox transmembrane domain and a human dehydrogenase domain of NOX 2 or NOX 4. With each NOX type, there are 4 different chimeras; each contains a slight variation in the composition of their cytosolic loops that connect the transmembrane helices. We are using these chimeras because mutations in the human NOXes will create structural constraints during electron transport, allowing us to more easily study this fundamental NOX function. Our results indicate differences in activity and in cofactor and substrate affinity among the various constructs used.

Project Type

Oral Presentation (15-min time slots)

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Characterizing SpNox Enzyme Chimeras

NADPH oxidases (NOXes) are a family of eukaryotic enzymes that produce reactive oxygen species (ROS). These transmembrane proteins move electrons from NADPH through FAD and across the membrane to the final electron acceptor O2, forming the superoxide anion O2-. Superoxide and downstream ROS are crucial signaling and cytotoxic molecules whose misregulation is associated with disease states ranging from diabetes to cancer. SpNox is a recently discovered prokaryotic NOX homolog from Streptoccous pneumoniae that is robust to bacterial expression and isolation, making it a good model system for study of the NOX family. The focus of this research project is to characterize the biochemical properties of SpNox using mutants and chimeras. The mutants are SpNox F397S and F397W, which in homologous systems affect affinity for the substrate NADPH and/or the cofactor FAD. The chimeras are composed of the wild type prokaryotic SpNox transmembrane domain and a human dehydrogenase domain of NOX 2 or NOX 4. With each NOX type, there are 4 different chimeras; each contains a slight variation in the composition of their cytosolic loops that connect the transmembrane helices. We are using these chimeras because mutations in the human NOXes will create structural constraints during electron transport, allowing us to more easily study this fundamental NOX function. Our results indicate differences in activity and in cofactor and substrate affinity among the various constructs used.