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J Biol Chem, Vol. 273, Issue 14, 8308-8316, April 3, 1998

Evolution of an Escherichia coli Protein with Increased Resistance to Oxidative Stress

Zhe Lu, Elisa Cabiscol^¶, Nuria Obradors, Jordi Tamarit^¶, Joaquim Ros^¶, Juan Aguilar, and E. C. C. Lin

From the  Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, the ^¶ Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, 25198 Lleida, Spain, and the  Departament de Bioquímica, Facultat de Farmacia, Universitat de Barcelona, 08028 Barcelona, Spain

L-1,2-Propanediol:NAD⁺ 1-oxidoreductase of Escherichia coli is encoded by the fucO gene, a member of the regulon specifying dissimilation of L-fucose. The enzyme normally functions during fermentative growth to regenerate NAD from NADH by reducing the metabolic intermediate L-lactaldehyde to propanediol which is excreted. During aerobic growth L-lactaldehyde is converted to L-lactate and thence to the central metabolite pyruvate. The wasteful excretion of propanediol is minimized by oxidative inactivation of the oxidoreductase, an Fe²⁺-dependent enzyme which is subject to metal-catalyzed oxidation (MCO). Mutants acquiring the ability to grow aerobically on propanediol as sole carbon and energy source can be readily selected. These mutants express the fucO gene constitutively, as a result of an IS5 insertion in the promoter region. In this study we show that continued selection for aerobic growth on propanediol resulted in mutations in the oxidoreductase conferring increased resistance to MCO. In two independent mutants, the resistance of the protein was respectively conferred by an Ile⁷  Leu and a Leu⁸  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 single amino acid change.

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