Biochemical characterization of the six-transmembrane epithelial antigen of the prostate family of metalloreductases
Kleven, Mark Daniel
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Iron and copper are the two most abundant transition metals in humans and are mediators of many essential cellular processes. The entry of these metals into cells require controlled processes, including their reduction prior to uptake. A group of integral membrane enzymes, the six-transmembrane epithelial antigen of the prostate (Steap) family, are able to perform this function. Steap3, in particular, functions as the primary ferric reductase in the transferrin cycle, the dominant mode of erythrocyte iron uptake. How these enzymes perform these functions has remained ill-defined. Here, the biochemical underpinnings of Steap metalloreductase activity have been investigated. To elucidate these mechanisms, expression systems for Steap3 and Steap4 have been developed in bacterial, insect, and human cell lines and purified to varying degrees. By analyzing the truncated cytoplasmic oxidoreductase domain of Steap4, it was found that NADPH is oxidized by transferring a pair of electrons to a flavin. With this truncation, however, flavin only binds weakly and the construct shows no ability to preferentially bind one type of flavin. In contrast, when the full length Steap3 was partially purified, it exhibits high-affinity FAD-binding, indicating that the transmembrane region of the protein contains the major structural features of the FAD binding site. Further, it was found that the cytoplasm-oriented loops between transmembrane helices formed the site. The next cofactor in the electron transport chain is a single b-type heme. Two strictly conserved histidines were identified that coordinate the heme and both are required for heme incorporation. The metal binding site at the extracellular face of the membrane was also characterized. Here, it was found that Steap3 and Steap4 share a conserved high-affinity iron binding site. Additionally, iron and copper both bind with similar affinities to Steap4. Two critical residues of the metal binding site were determined and their predicted proximity to the heme cofactor suggests that the electron is transfer is direct between cofactor and metal. Finally, it was found that Steap's are able to dimerize in the cells, forming homo- and heterodimers Together, the enzymatic mechanism has been characterized in-depth for the first time for these physiologically-significant enzymes.