L-LACTATE OXIDASE AND L-LACTATE MONOOXYGENASE - MECHANISTIC VARIATIONS ON A COMMON STRUCTURAL THEME

Properties of L-lactate oxidase from Aerococcus viridans are described . The gene encoding the enzyme has been isolated. From its cDNA sequen ce the amino acid sequence has been derived and shown to have high sim ilarity with those of other enzymes catalyzing oxidation of L-alpha-hy droxy acids, including flavocytochrome b(2), lactate monooxygenase, gl ycolate oxidase, mandelate dehydrogenases and a long chain alpha-hydro xy acid oxidase. The enzyme is expressed in Escherichia coli, and is a flavoprotein containing FMN as prosthetic group. It shares many prope rties of other alpha-hydroxy acid oxidizing enzymes, eg stabilization of the anionic semiquinone form of the flavin, facile formation of fla vin-N(5)-sulfite adducts and a set of conserved amino acid residues ar ound the bound flavin. Steady-state- and rapid reaction kinetics of th e enzyme have been studied and found to share many characteristics wit h those of L-lactate monooxygenase, but to differ from the latter in q uantitative aspects. It is these quantitative differences between the two enzymes which account for the differences in the overall reactions catalyzed. These differences arise from different stabilities of a co mmon intermediate of reduced flavin enzyme and pyruvate. In the case o f the monooxygenase this complex is very stable, and is the form that reacts with O-2 to give a complex in which the oxidative decarboxylati on occurs, yielding the products, acetate, CO2, and H2O (Lockridge O, Massey V, Sullivan PA (1972) J Biol Chem 247, 8097-8106). With lactate oxidase, the complex dissociates rapidly, with the result that it is the free reduced flavin form of the enzyme that reacts with O-2, to gi ve the observed products, pyruvate and H2O2.