Improving solubility of catalytic domain of human beta-1,4-galactosyltransferase 1 through rationally designed amino acid replacements.
beta-1,4-galactosyltransferase 1 (beta4gal-T1, EC 2.4.1.38) transfers galactose from UDP-galactose to free N-acetyl-D-glucosamine or bound N-acetyl-D-glucosamine-R. Soluble beta4gal-T1, purified from human milk has been refractory to structural studies by X-ray or NMR. In a previous study (Malissard et al. 1996, Eur. J. Biochem. 239, 340-348) we produced in the yeast Saccaromyces cerevisiae an N-deglycosylated form of soluble beta4gal-T1 that was much more homogeneous than the human enzyme, as it displayed only two isoforms when analysed by IEF as compared to 13 isoforms for the native beta4gal-T1. The propensity of recombinant beta4gal-T1 to aggregate at concentrations > 1 mg.mL(-1) prevented structural and biophysical studies. In an attempt to produce a beta4gal-T1 form suitable for structural studies, we combined site-directed mutagenesis and heterologous expression in Escherichia coli. We produced a mutated form of the catalytic domain of beta4gal-T1 (sfbeta4gal-T1mut) in which seven mutations were introduced at nonconserved sites (A155E, N160K, M163T, A168T, T242N, N255D and A259T). Sfbeta4gal-T1mut was shown to be much more soluble than beta4gal-T1 expressed in S. cerevisiae (8.5 mg.mL(-1) vs. 1 mg.mL(-1)). Catalytic activity and kinetic parameters of sfbeta4gal-T1mut produced in E. coli were shown not to differ to any significant extent from those of the native enzyme.
Malissard
M
M
Institute of Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Berger
E G
EG
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005700:Galactosyltransferases / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012995:Solubility
D000596:Amino Acids; D020033:Protein Isoforms; D011994:Recombinant Proteins; D005700:Galactosyltransferases; C119701:beta-1,4-galactosyltransferase I