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 pg/mL, whereas the apoenzyme shows stepwise tetramer-dimer-monomer dissociation, with the monomer as the major component, at a protein concentration of <20 pg/mL.In the presence of divalent cations, the coenzymes FAD and TPP bind to the apoenzyme, forming the inactive binary FAD or TPP complexes. Both FAD and TPP affect the quaternary structure by shifting the equilibrium of association toward the dimer or tetramer. High FAD concentrations exert significant stabilization against urea and heat denaturation, whereas excess TPP has no effect.Reconstitution of the holoenzyme from its components yields full reactivation. The kinetic analysis reveals a compulsory sequential mechanism of cofactor binding and quaternary structure formation, with TPP binding as the first step. The binary TPP complex (in the presence of 1 mM Mn2+/TPP) is characterized by a dimer-tetramer equilibrium transition with an association constant of K, = 2 x lo7 M" . The apoenzyme TPP complex dimer associates with the tetrameric holoenzyme in the presence of 10 pM FAD. This association step obeys second-order kinetics with an association rate constant k = 7.4 x lo3 M" s-' at 20 "C. FAD binding to the tetrameric binary TPP complex is too fast to be resolved by manual mixing.

Characterization of the stabilizing effect of point mutations of pyruvate oxidase from lactobacillus plantarum: Protection of the native state by modulating coenzyme binding and subunit interaction

“…Instead, the various apoenzymes exhibit equal, or even decreased, stability compared to the wild-type apoenzyme. The first (local) unfolding transition, indicative for a structured intermediate, is characterized by a decrease in protein fluorescence (Risse et al, 1992). The wild-type protein and mutant proteins 201 and 202 do not exhibit significant differences in the formation of this intermediate (Fig.…”

“…In the companion paper it was shown that the tertiary structures of apo-and holo-pyruvate oxidase are indistinguishable (Risse et al, 1992). Therefore, the apoenzyme may be used to determine the effect of the amino acid exchanges on the stability of the tertiary interactions.…”

“…As pointed out in the companion paper (Risse et al, 1992), both FAD release and dissociation of the holoenzyme into its subunits coincide. However, deactivation precedes FAD release and the concomitant dissociation of the tetramers (Risse et al, 1992). The slight difference in the deactivation and dissociation profiles was explained by the recombination of FAD with apoenzyme in the course of the urea-induced dissociation equilibrium, leading to inactive, tetrameric binary FAD complexes (Risse et al, 1992).…”

“…In the companion paper (Risse et al, 1992), the stability of the wild-type enzyme was characterized in detail. As has been demonstrated by the enhanced stability at high protein and FAD concentrations, quaternary contacts play a significant role in the stabilization of the enzyme.…”

Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli

“…In their denaturation pathways, loss of catalytic activity usually belongs to the steps that occur early, in which either the oligomeric structure is lost because of subunit dissociation, or the active-site integrity is destroyed by other often small conformational changes [e.g. [8][9][10][11][12]. For α-glucan phosphorylases, the interactions at the dimer interface are quite well known [3,[13][14][15][16][17][18].…”