In vitro determined kinetic properties of mutant phosphoglucomutases and their effects on sugar catabolism in Escherichia coli.
Based on primary amino acid sequence comparisons with other phosphoglucomutases, 12 conserved residues in the Acetobacter xylinum phosphoglucomutase (CelB) were substituted by site-directed mutagenesis, resulting in mutant enzymes with Kcat values [glucose-1-phosphate (G-1-P) to glucose-6-phosphate] ranging from 0 to 46% relative to that of the wild-type enzyme. In combination with a versatile set of plasmid expression vectors these proteins were used in a metabolic engineering study on sugar catabolism in Escherichia coli. Mutants of E. coli deficient in phosphoglucomutase synthesize intracellular amylose when grown on galactose, due to accumulation of G-1-P. Wild-type celB can complement this lesion, and we show here that the ability of the mutant enzymes to complement is sensitive to variations in their respective in vitro determined Kcat and Km G-1-P values. Reduced catalytic efficiencies could be compensated by increasing the CelB expression level, and in this way a mutant protein (substitution of Thr-45 to Ala) displaying a 7600-fold reduced catalytic efficiency could be used to eliminate the amylose accumulation. Complementation experiments with the homologous phosphoglucomutase indicated that a Km G-1-P value significantly below that of CelB is not critical for the in vivo conversion of the substrate.
Brautaset
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UNIGEN Center for Molecular Biology, Department of Biotechnology, Norwegian University of Science and Technology, Trondheim.
Petersen
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Valla
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D000091:Acetobacter / Q000201:enzymology / Q000235:genetics; D000595:Amino Acid Sequence; D050260:Carbohydrate Metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005818:Genetic Engineering; D007700:Kinetics; D008969:Molecular Sequence Data; D010733:Phosphoglucomutase / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D016415:Sequence Alignment; D013379:Substrate Specificity
D010733:Phosphoglucomutase