Catalyzes the oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate (DHAP), with a reduction of O2 to H2O2. The formation of hydrogen peroxide by this enzyme is crucial for cytotoxic effects of M.pneumoniae on host cells. Is involved in the metabolism of glycerol and is essential for glycerol utilization; glycerol is one of the few carbon sources that can be utilized by M.pneumoniae for growth (PubMed:19028882). To a lesser extent, is also able to use glyceraldehyde 3-phosphate (GAP), an intermediate in the glycolysis pathway, as a substrate (but the structure of the product has not been elucidated). Therefore, in the absence of glycerol, GAP may serve as a substrate in the GlpO reaction to supply H2O2 during mycoplasma infection (PubMed:25712468). Does not show any dehydrogenase activity with NAD(+) (PubMed:19028882). O2 + sn-glycerol 3-phosphate = dihydroxyacetone phosphate + H2O2 kcat is 4.2 sec(-1) with sn-glycerol 3-phosphate as substrate (at pH 7 and 4 degrees Celsius). kcat is 60.1 sec(-1) with sn-glycerol 3-phosphate as substrate (at pH 7 and 25 degrees Celsius). kcat is 0.6 sec(-1) with sn-glycerol 3-phosphate as substrate (at pH 7 and 25 degrees Celsius). Polyol metabolism; glycerol degradation via glycerol kinase pathway; glycerone phosphate from sn-glycerol 3-phosphate (aerobic route): step 1/1. Monomer. Is present predominantly in the cytoplasm, but a few proteins can also be detected at the cell surface. Constitutively expressed. Cells lacking this gene do not grow at all in glycerol-containing medium, and exhibit a significantly reduced formation of hydrogen peroxide and a severely reduced cytotoxicity. The reaction mechanism follows a ping-pong model; after flavin-mediated substrate oxidation, DHAP leaves rapidly prior to the oxygen reaction that reoxidizes the flavin and produces H2O2.