EVOLUTION OF AN ESCHERICHIA-COLI PROTEIN WITH INCREASED RESISTANCE TOOXIDATIVE STRESS

L-1,2-Propanediol:NAD(+) 1-oxidoreductase of Escherichia coli is encod ed by the fucO gene, a member of the regulon specifying dissimilation of L-fucose, The enzyme normally fractions during fermentative growth to regenerate NAD from NADH by reducing tile metabolic intermediate L- lactaldehyde to propanediol which is excreted. During aerobic growth L -lactaldehyde is converted to L-lactate and thence to the central meta bolite pyruvate. The wasteful excretion of propanediol is minimized by oxidative inactivation of the oxidoreductase, an Fe2+-dependent enzym e which is subject to metal-catalyzed oxidation (MCO). Mutants acquiri ng the ability to grow aerobically on propanediol as sole carbon and e nergy source can be readily selected. These mutants express tile fucO gene constitutively, as a result of an IS5 insertion in the promoter r egion. In this study we show that continued selection for aerobic grow th on propanediol resulted in mutations in the oxidoreductase conferri ng increased resistance to MCO. In two independent mutants, the resist ance of the protein was respectively conferred by all Ile(7) --> Leu a nd a Leu(8) --> Val substitution near the NAD-binding consensus amino acid sequence, A site-directed mutant protein with both substitutions showed an MCO resistance greater than either mutant protein with a sin gle amino acid change.