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Characterization and crystallization of an active N‐terminally truncated form of the Escherichia coli glycogen branching enzyme

Hilden, Ida, Leggio, Leila L., Larsen, Sine, Poulsen, Peter
European journal of biochemistry 2000 v.267 no.8 pp. 2150-2155
1,4-alpha-glucan branching enzyme, Escherichia coli, ammonium sulfate, amylose, crystallization, crystals, glycogen, ion exchange chromatography, models
The prokaryotic glycogen branching enzymes (GBE) can be divided into two groups on the basis of their primary structures: the first group of enzymes, which includes GBE from Escherichia coli, is characterized by a long N‐terminal extension that is absent in the enzymes of the second group. The extension consists of approximately 100 amino‐acid residues with unknown function. In order to characterize the function of this region, the 728 amino‐acid residue, full‐length E. coli GBE, and a truncated form (nGBE) missing the first 107 amino‐acid residues were overexpressed in E. coli. Both enzymes were purified to homogeneity by a simple purification procedure involving ammonium sulphate precipitation, ion‐exchange chromatography, and a second ammonium sulphate precipitation. Purified full‐length enzyme was poorly soluble and formed aggregates, which were inactive, at concentrations above 1 mg·mL−1. In contrast, the truncated form could be concentrated to 6 mg·mL−1 without any visible signs of aggregation or loss of activity on concentration. The ability to overexpress nGBE in a highly soluble form has allowed us to produce diffracting crystals of a branching enzyme for the first time. A comparison of the specific activities of purified GBE and nGBE in assays where amylose was used as substrate demonstrated that nGBE retained approximately half of the branching activity of full‐length GBE and is therefore a suitable model for the study of the enzymes’ catalytic mechanism.