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 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.
Hilden
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Danisco Cultor Innovation, Copenhagen, Denmark; Centre for Crystallographic Studies, University of Copenhagen, Denmark. G71DH@danisco.dk
Leggio
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Larsen
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Poulsen
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D015061:1,4-alpha-Glucan Branching Enzyme / Q000737:chemistry* / Q000378:metabolism; D000688:Amylose / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology*; D019143:Evolution, Molecular; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017421:Sequence Analysis; D013379:Substrate Specificity; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D000688:Amylose; D015061:1,4-alpha-Glucan Branching Enzyme