Publications

• Role of flavin in acetoin production by two bacterial pyruvate oxidases.

  Bertagnolli B.L., Hager L.P.

  Escherichia coli pyruvate oxidase (POXEC) requires FAD both for the oxidative decarboxylation of pyruvate to acetate and CO2 and for the formation of acetoin from pyruvate and acetaldehyde. Prior work has shown that the catalytic activity (kcat/Km) for POXEC in the oxidative reaction is stimulated ... >> More

  Escherichia coli pyruvate oxidase (POXEC) requires FAD both for the oxidative decarboxylation of pyruvate to acetate and CO2 and for the formation of acetoin from pyruvate and acetaldehyde. Prior work has shown that the catalytic activity (kcat/Km) for POXEC in the oxidative reaction is stimulated approximately 450-fold by amphiphilic activators. This paper shows that the acetoin reaction does not respond to activation. The FAD requirement for acetoin formation can be replaced by 5-deaza-FAD and 6-hydroxy-FAD, FAD analogs which form kinetically stable oxidized and reduced enzyme species, respectively. As would be expected, the 5-deaza- and 6-hydroxy-FAD enzymes are not active in the oxidative reaction. A second flavin pyruvate oxidase from Pediococcus pseudomonas (POXPP), which catalyzes the oxidative decarboxylation of pyruvate to CO2 and acetyl phosphate, also requires FAD for acetoin formation. POXPP has an oxidative rate comparable to that of POXEC, but in comparison to POXEC, POXPP catalyzes acetoin formation at a much reduced rate. Again, as was found with the POXEC, an FAD analog incapable of undergoing facile oxidation-reduction reactions also could replace the FAD requirement in the POXPP acetoin reaction. The results indicate that the role for FAD in acetoin formation with both enzymes is based on a structural requirement and that FAD does not participate in a redox function in the acetoin reaction. << Less

  Arch Biochem Biophys 300:364-371(1993) [PubMed] [EuropePMC]

• Reconstitution of native Escherichia coli pyruvate oxidase from apoenzyme monomers and FAD.

  Recny M.A., Hager L.P.

  Pyruvate oxidase, a tetrameric enzyme consisting of 4 identical subunits, dissociates into apoenzyme monomers and free FAD when treated with acid ammonium sulfate in the presence of high concentrations of potassium bromide. Reconstitution of the native enzymatically active protein can be accomplis ... >> More

  Pyruvate oxidase, a tetrameric enzyme consisting of 4 identical subunits, dissociates into apoenzyme monomers and free FAD when treated with acid ammonium sulfate in the presence of high concentrations of potassium bromide. Reconstitution of the native enzymatically active protein can be accomplished by incubating equimolar concentrations of apomonomers and FAD at pH 6.5. The kinetics of the reconstitution reaction have been measured by 1) enzyme activity assays, 2) spectrophotometric assays to measure FAD binding, and 3) high performance liquid chromatography analysis measuring the distribution of monomeric, dimeric, and tetrameric species during reconstitution. The kinetic analysis indicates that the second order reaction of apomonomers with FAD to form an initial monomer-FAD complex is fast. The rate-limiting step for enzymatic reactivation appears to be the folding of the polypeptide chain in the monomer-FAD complex to reconstitute the three-dimensional FAD binding site prior to subunit reassociation. The subsequent formation of native tetramers appears to proceed via an essentially irreversible dimer assembly pathway. << Less

  J. Biol. Chem. 257:12878-12886(1982) [PubMed] [EuropePMC]

• Role of the tetrameric structure of Escherichia coli pyruvate oxidase in enzyme activation and lipid binding.

  Wang A.Y., Chang Y.Y., Cronan J.E. Jr.

  Pyruvate oxidase of Escherichia coli, an enzyme greatly activated by phospholipids, is a tetramer of a Mr 62,000 subunit. We have utilized the differing electrophoretic mobilities of several mutant oxidases on native polyacrylamide gels to study the role of the quaternary structure of the enzyme i ... >> More

  Pyruvate oxidase of Escherichia coli, an enzyme greatly activated by phospholipids, is a tetramer of a Mr 62,000 subunit. We have utilized the differing electrophoretic mobilities of several mutant oxidases on native polyacrylamide gels to study the role of the quaternary structure of the enzyme in the activation process. We found that when two poxB gene alleles coexisted in cells, heterotetrameric species were formed in addition to homotetramers. The concentration of each tetrameric species varied according to the concentration of the different subunits present, and the distribution seemed virtually identical to those expected from random mixing. We showed that the intrinsic activity of pyruvate oxidase was not affected by interactions among the four subunits. However, binding of the enzyme to lipids, a property required for function in vivo, required that a tetramer contain at least two subunits capable of lipid binding. Our data fit the model proposed previously (Grabau, C., Chang, Y.-Y., and Cronan, J. E., Jr. (1989) J. Biol. Chem. 264, 12510-12519) in which the carboxyl termini of two subunits interact to form a functional lipid-binding domain. We also have detected oxidase activity in a form of oxidase of unusually high electrophoretic mobility. This form seems to be either a monomeric or a dimeric form (more probably the former) of the oxidase subunit. << Less

  J Biol Chem 266:10959-10966(1991) [PubMed] [EuropePMC]