Jump to Main Content
Characterization and crystallization of an active Nâterminally 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 Nâterminal extension that is absent in the enzymes of the second group. The extension consists of approximately 100 aminoâacid residues with unknown function. In order to characterize the function of this region, the 728 aminoâacid residue, fullâlength E.âcoli GBE, and a truncated form (nGBE) missing the first 107 aminoâacid residues were overexpressed in E.âcoli. Both enzymes were purified to homogeneity by a simple purification procedure involving ammonium sulphate precipitation, ionâexchange chromatography, and a second ammonium sulphate precipitation. Purified fullâlength enzyme was poorly soluble and formed aggregates, which were inactive, at concentrations above 1âmgÂ·mLâ1. In contrast, the truncated form could be concentrated to 6âmgÂ·mLâ1 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 fullâlength GBE and is therefore a suitable model for the study of the enzymesâ catalytic mechanism.