Structural and mechanistic insights into Streptococcus pneumoniae NADPH oxidase

Abstract

NADPH oxidases (NOXs) play a major role in the physiology of eukaryotic cells by mediating the production of reactive oxygen species (ROS). Evolutionarily distant proteins sharing the NOX catalytic core have been recently described in Bacteria. Among them, the Streptococcus pneumoniae NOX (SpNOX) has been proposed as a model for the study of NOXs due to its high activity and stability in detergent micelles. Here, we report high-resolution cryo-EM structures of substrate-free and stably reduced NADH-bound SpNOX, and of the NADPH-bound SpNOX and a Phe397Ala mutant under turnover conditions. In combination with structure-guided mutagenesis and biochemical analyses, we provide the structural basis for constitutive activity, the lack of substrate specificity towards NADPH and the electron transfer pathway. Additionally, we shed light on the catalytic regulation by the C-terminal tail residue Phe397 and the potential in vivo function of this protein.