In Vitro Determined Kinetic Properties of Mutant Phosphoglucomutases and Their Effects on Sugar Catabolism in Escherichia coli

✍ Scribed by Trygve Brautaset; Steffen B. Petersen; Svein Valla

• Publisher: Elsevier Science
• Year: 2000
• Tongue: English
• Weight: 254 KB
• Volume: 2
• Category: Article
• ISSN: 1096-7176
• DOI: 10.1006/mben.1999.0145

✦ Synopsis

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 K cat values [glucose-1phosphate (G-1-P) to glucose-6-phosphate] ranging from 0 to 46 0 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 K cat and K m 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 K m G-1-P value significantly below that of CelB is not critical for the in vivo conversion of the substrate.