ROLE OF FLAVIN IN ACETOIN PRODUCTION BY 2 BACTERIAL PYRUVATE OXIDASES

Escherichia coli pyruvate oxidase (POX(EC)) 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 (k(cat)/K(m)) for POX(EC) 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 (POX(PP)), which catalyzes the oxidative decarboxylation of pyruvate to CO2 and acetyl phosphate, also requires FAD for acetoin formation. POX(PP) has an oxidative rate comparable to that of POX(EC), but in comparison to POX(EC), POX(PP) catalyzes acetoin formation at a much reduced rate. Again, as was found with the POX(EC), an FAD analog incapable of undergoing facile oxidation-reduction reactions also could replace the FAD requirement in the POX(PP) 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. © 1993 Academic Press, Inc.