PMID:10759837
| Citation |
Hilden, I, Leggio, LL, Larsen, S and Poulsen, P (2000) Characterization and crystallization of an active N-terminally truncated form of the Escherichia coli glycogen branching enzyme. Eur. J. Biochem. 267:2150-5 |
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| Abstract |
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. |
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| Keywords |
1,4-alpha-Glucan Branching Enzyme/chemistry; 1,4-alpha-Glucan Branching Enzyme/metabolism; Amylose/metabolism; Bacterial Proteins/chemistry; Crystallization; Escherichia coli/enzymology; Evolution, Molecular; Gene Expression Regulation, Bacterial/genetics; Gene Expression Regulation, Enzymologic/genetics; Isoenzymes/chemistry; Recombinant Proteins/chemistry; Sequence Analysis; Substrate Specificity; X-Ray Diffraction |
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