The crystal structure of six-transmembrane epithelial antigen of the prostate 4 (Steap4), a ferri/cuprireductase, suggests a novel interdomain flavin-binding site.

Steap4 is a cell surface metalloreductase linked to obesity-associated insulin resistance. Initial characterization of its cell surface metalloreductase activity has been reported, but thorough biochemical characterization of this activity is lacking. Here, we report detailed kinetic analysis of the Steap4 cell surface metalloreductase activities. Steap4 shows physiologically relevant Km values for both Fe(3+) and Cu(2+) and retains activity at acidic pH, suggesting it may also function within intracellular organelles to reduce these metals. Flavin-dependent NADPH oxidase activity that was much greater than the equivalent Steap3 construct was observed for the isolated N-terminal oxidoreductase domain. The crystal structure of the Steap4 oxidoreductase domain was determined, providing a structural explanation for these differing activities. Structure-function work also suggested Steap4 utilizes an interdomain flavin-binding site to shuttle electrons between the oxidoreductase and transmembrane domains, and it showed that the disordered N-terminal residues do not contribute to enzymatic activity.