Engineering the active center of the 6-phospho-beta-galactosidase from Lactococcus lactis.
Several amino acids in the active center of the 6-phospho-beta-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 k(cat)/K(m) for o-nitrophenyl-beta-D-glucose-6-phosphate is 8-fold higher than for o-nitrophenyl-beta-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-beta-D-galactose-6-phosphate whereas the interaction with o-nitrophenyl-beta-D-galactose is barely altered. This result shows that the 6-phospho-beta-galactosidase and the related cyanogenic beta-glucosidase from Trifolium repens have different recognition mechanisms for substrates although the structures of the active sites are highly conserved.
Schulte
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Arbeitsgruppe Physiologie der Mikroorganismen, Department of Biology, Ruhr-Universität Bochum, D- 44780 Bochum, Germany.
Hengstenberg
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D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D004926:Escherichia coli; D005695:Galactosephosphates / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D006026:Glycoside Hydrolases; D013294:Lactococcus lactis / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009598:Nitrophenylgalactosides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics
D000596:Amino Acids; D001426:Bacterial Proteins; D005695:Galactosephosphates; D005960:Glucosides; D011993:Recombinant Fusion Proteins; C055012:2-nitrophenylgalactoside; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; C038269:galactose-6-phosphate; D006026:Glycoside Hydrolases; D001616:beta-Galactosidase; C022361:6-phospho-beta-galactosidase