Identification of amino acid residues that determine the substrate preference of 1,3-β-galactosyl-N-acetylhexosamine phosphorylase

Three amino acid residues of 1,3-β-galactosyl-N-acetylhexosamine phosphorylase (GalHexNAcP) were assigned as the determinants of substrate preference for galacto-N-biose (GNB) and lacto-N-biose I (LNB) based on the three-dimensional structure of the protein. Mutants of GalHexNAcP from Bifidobacterium longum, which acts similarly on both GNB and LNB, were constructed and characterized. V162T mutation led to an increase in the selectivity on GNB. P161S and S336A mutations independently enhanced the selectivity on LNB. The alignment of amino acid sequences suggests that the activities of most homologous sequences are predictable by comparing the corresponding three residues. .