Broad substrate specificity (S)-2-hydroxy-acid oxidase that preferentially oxidizes glycolate. The glyoxylate produced by the oxidation of glycolate can then be utilized by alanine-glyoxylate aminotransferase for the peroxisomal synthesis of glycine; this pathway appears to be an important step for the detoxification of glyoxylate which, if allowed to accumulate, may be metabolized to oxalate with formation of kidney stones. Can also catalyze the oxidation of glyoxylate, and long chain hydroxyacids such as 2-hydroxyhexadecanoate and 2-hydroxyoctanoate. Active in vitro with the artificial electron acceptor 2,6-dichlorophenolindophenol (DCIP), but O2 is believed to be the physiological electron acceptor, leading to the production of H2O2. a (2S)-2-hydroxycarboxylate + O2 = a 2-oxocarboxylate + H2O2 glycolate + O2 = glyoxylate + H2O2 glyoxylate + H2O + O2 = H(+) + H2O2 + oxalate 2-hydroxyhexadecanoate + O2 = 2-oxohexadecanoate + H2O2 2-hydroxyoctanoate + O2 = 2-oxooctanoate + H2O2 Amino-acid biosynthesis; glycine biosynthesis. Homotetramer. Belongs to the FMN-dependent alpha-hydroxy acid dehydrogenase family.