Ero 1 encodes a flavoprotein oxidase that promotes oxidative protein folding through protein disulfide isomerase and generates reactive oxygen species (hydrogen peroxide) as byproducts. Ero 1 was cloned and sequenced from wheat (Triticum aestivum cv. Haruyutaka). Three homologous Ero 1 genes were obtained, which showed high sequence conservation. These Ero 1 genes were 455 amino acids in length with a conserved catalytic Cys-X-X-Cys-X-X-Cys motif (Cys370, Cys373, Cys376) and a transmembrane signal in the N-terminal domain. Wheat Ero 1 without the signal sequence was expressed in Escherichia coli as a His-tagged fusion protein. Recombinant Ero 1 was purified by affinity chromatography using a TALON resin column. The activity of purified recombinant Ero 1 was measured by both the catalytic reduction of FAD and an oxygen consumption assay. FAD was reduced in an Ero 1 concentration-dependent manner. Oxygen consumption at 0.1 μM wheat Ero 1 was slow; however, the rate increased with increased Ero 1 concentration. These results indicated that recombinant Ero 1 from wheat has FAD-reducing ability, and utilizes oxygen as a terminal electron accepter in the PDI-Ero 1 system.
