L-Lactate oxidase (LOX) from Aerococcus viridans is a member of the alpha-hydroxyacid-oxidase flavoenzyme family. We have determined the three-dimensional structure of LOX and revealed the mechanism of substrate recognition. The LOX monomer structure has a typical alpha(8)/beta(8) motif commonly found in other flavin family proteins. A related enzyme, glycolate oxidase, catalyzes the oxidation of glycolate rather than lactate. Comparison of the two enzyme structures highlights the importance of five residues around the FMN prosthetic group of LOX. which act synergistically to discriminate between the L/D configurations of lactate. X-ray crystallography of LOX gave a space group 1422 of unit-cell parameters a = b = 191,096 angstrom, c = 194.497 angstrom and alpha = beta = gamma = 90 degrees with four monomers per asymmetric unit. The four independent monomers display slight structural differences around the active site. Diffraction data were collected, under cryogenic conditions to 2.1 angstrom resolution at the synchrotron facilities in Japan. (c) 2006 Elsevier Inc. All rights reserved.