Protein Engineering, Vol. 13, No. 7, 515-518,
July 2000
© 2000 Oxford University Press
Engineering the active center of the 6-phospho-ß-galactosidase from Lactococcus lactis
Arbeitsgruppe Physiologie der Mikroorganismen, Department of Biology, Ruhr-Universität Bochum, D- 44780 Bochum, Germany
Several amino acids in the active center of the 6-phospho-ß-galactosidase from Lactococcus lactis were replaced by the corresponding residues in homologous enzymes of glycosidase family 1 with different specificities. Three mutants, W429A, K435V/Y437F and S428D/ K435V/Y437F, were constructed. W429A was found to have an improved specificity for glucosides compared with the wild-type, consistent with the theory that the amino acid at this position is relevant for the distinction between galactosides and glucosides. The kcat/Km for o-nitrophenyl-ß-D-glucose-6-phosphate is 8-fold higher than for o-nitrophenyl-ß-D-galactose-6-phosphate which is the preferred substrate of the wild-type enzyme. This suggests that new hydrogen bonds are formed in the mutant between the active site residues, presumably Gln19 or Trp421 and the C-4 hydroxyl group. The two other mutants with the exchanges in the phosphate-binding loop were tested for their ability to bind phosphorylated substrates. The triple mutant is inactive. The double mutant has a dramatically decreased ability to bind o-nitrophenyl-ß-D-galactose-6-phosphate whereas the interaction with o-nitrophenyl-ß-D-galactose is barely altered. This result shows that the 6-phospho-ß-galactosidase and the related cyanogenic ß-glucosidase from Trifolium repens have different recognition mechanisms for substrates although the structures of the active sites are highly conserved.