In enzymology, a pyruvate oxidase (CoA-acetylating) (EC 1.2.3.6) is an enzyme that catalyzes the chemical reaction

pyruvate + CoA + O₂ acetyl-CoA + CO₂ + H₂O₂

The 3 substrates of this enzyme are pyruvate, CoA, and O₂, whereas its 3 products are acetyl-CoA, CO₂, and H₂O₂.

This enzyme belongs to the family of oxidoreductases , specifically those acting on the aldehyde or oxo group of donor with oxygen as acceptor. The systematic name of this enzyme class is pyruvate:oxygen 2 - oxidoreductase (CoA-acetylating). This enzyme participates in pyruvate metabolism. It employs one cofactor, FAD.

• Reeves RE, Warren LG, Susskind B, Lo HS (1977). "An energy-conserving pyruvate-to-acetate pathway in Entamoeba histolytica. Pyruvate synthase and a new acetate thiokinase". J. Biol. Chem. 252 (2): 726–31. doi:10.1016/S0021-9258(17)32778-3. PMID 13076.
• Takeuchi T, Weinbach EC, Diamond LS (1975). "Pyruvate oxidase (CoA acetylating) in Entamoeba histolytica". Biochem. Biophys. Res. Commun. 65 (2): 591–6. doi:10.1016/S0006-291X(75)80187-2. PMID 167776.