Improving the solubility of the catalytic domain of human beta-1,4-galactosyltransferase 1 through rationally designed amino-acid replacements

beta -1,4-galactosyltransferase 1 (beta 4gal-T1, EC2.4.1.38) transfers gala ctose from UDP-galactose to free N-acetyl-D-glucosamine or bound N-acetyl-D -glucosamine-R. Soluble beta 4gal-T1, purified from human milk has been ref ractory to structural studies by X-ray or NMR. In a previous study (Malissa rd et al. 1996, Eur J. Biochem. 239, 340-348) we produced in the yeast Sacc haromyces cerevisiae an N-deglycosylated form of soluble beta 4gal-T1 that was much more homogeneous than the human enzyme, as it displayed only two i soforms when analysed by IEF as compared to 13 isoforms for the native beta 4gal-T1. The propensity of recombinant beta 4gal-T1 to aggregate at concen trations > 1 mg.mL(-1) prevented structural and biophysical studies. In an attempt to produce a beta 4gal-T1 form suitable for structural studies, we combined site-directed mutagenesis and heterologous expression in Escherich ia coli. We produced a mutated form of the catalytic domain of beta 4gal-T1 (sf beta 4gal-T1mut) in which seven mutations were introduced at nonconser ved sites (A155E, N160K, M163T, A168T, T242N, N255D and A259T). Sf beta 4ga l-T1mut was shown to be much more soluble than beta 4gal-T1 expressed in S. cerevisiae (8.5 mg.mL(-1) vs. 1 mg.mL(-1)). Catalytic activity and kinetic parameters of sf beta 4gal-T1mut produced in E. coli were shown not to dif fer to any significant extent from those of the native enzyme.