Main observation and conclusion The aminoglycoside antibiotic apramycin contains a unique bicyclic octose moiety, and biosynthesis of this moiety involves an oxidoreductase AprQ. Unlike other known Q series proteins involved in aminoglycosides biosynthesis, AprQ does not work with an aminotransferase partner, and performs a four-electron oxidation that converts a CH2OH moiety to a carboxylate group. In this study, we report mechanistic investigation of AprQ. We showed AprQ contains a flavin mononucleotide (FMN) cofactor, which is different from other known Q series enzymes that contain a flavin adenine dinucleotide (FAD) cofactor. A series of biochemical assays showed that AprQ is not a monooxygenase but a flavoprotein oxidase. Although molecular O-2 is strictly required for reaction turnover, four-electron oxidation can be achieved in the absence of O-2 in single turnover condition. These findings establish the detailed catalytic mechanism of AprQ and expand the growing family of flavoprotein oxidases, an increasingly important class of biocatalysts.