Stability and reconstitution of pyruvate oxidase from lactobacillus plantarum: Dissection of the stabilizing effects of coenzyme binding and subunit interaction

Abstract

Pyruvate oxidase from Lactobacillus plantarum is a homotetrameric flavoprotein with strong binding sites for FAD, TPP, and a divalent cation. Treatment with acid ammonium sulfate in the presence of 1.5 M KBr leads to the release of the cofactors, yielding the stable apoenzyme. In the present study, the effects of FAD, TPP, and Mn²⁺ on the structural properties of the apoenzyme and the reconstitution of the active holoenzyme from its constituents have been investigated. As shown by circular dichroism and fluorescence emission, as well as by Nile red binding, the secondary and tertiary structures of the apoenzyme and the holoenzyme do not exhibit marked differences. The quaternary structure is stabilized significantly in the presence of the cofactors. Size‐exclusion high‐performance liquid chromatography and analytical ultracentrifugation demonstrate that the holoenzyme retains its tetrameric state down to 20 μg/mL, whereas the apoenzyme shows stepwise tetramer‐dimer‐monomer dissociation, with the monomer as the major component, at a protein concentration of 2+/TPP) is characterized by a dimer‐tetramer equilibrium transition with an association constant of K_a = 2 × 10⁷ M⁻¹. The apoenzyme TPP complex dimer associates with the tetrameric holoenzyme in the presence of 10 μM FAD. This association step obeys second‐order kinetics with an association rate constant k = 7.4 × 10³ M⁻¹ s⁻¹ at 20 °C. FAD binding to the tetrameric binary TPP complex is too fast to be resolved by manual mixing.