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

The prokaryotic glycogen branching enzymes (GBE) can be divided into two gr oups on the basis of their primary structures: the first group of enzymes, which includes GBE from Escherichia coli, is characterized by a long N-term inal extension that is absent in the enzymes of the second group. The exten sion consists of approximately 100 amino-acid residues with unknown functio n. 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 w ere purified to homogeneity by a simple purification procedure involving am monium sulphate precipitation, ion-exchange chromatography, and a second am monium sulphate precipitation. Purified full-length enzyme was poorly solub le 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 concentr ation. The ability to overexpress nGBE in a highly soluble form has allowed us to produce diffracting crystals of a branching enzyme for the first tim e. A comparison of the specific activities of purified GBE and nGBE in assa ys where amylose was used as substrate demonstrated that nGBE retained appr oximately half of the branching activity of full-length GBE and is therefor e a suitable model for the study of the enzymes' catalytic mechanism.