Glycoside phosphorylases are a special group of carbohydrate-active enzymes, with characteristics in between those of glycoside hydrolases and glycosyl transferases. The phosphorylases from family GH-112 are exceptional because they employ galactose-1-phosphate instead of glucose-1-phosphate as glycosyl donor. Different acceptor specificities have been observed in this family, ranging from L-rhamnose to GIcNAc, GaINAc and a combination of the latter. Three new phosphorylases from previously unexplored branches of the phylogenetic tree of family GH-112 have now been characterized to shed more light on this divergence in acceptor specificity. The enzymes from Erysipelothrix rhusiopathiae and Streptobacillus moniliformis were found to prefer GaINAc as acceptor, while that from Anaerococcus prevotii displays similar activities on GaINAc and GIcNAc. These results confirm the correlation between the amino acid residue at position 162 and the enzyme's specificity, i.e. a threonine in the former group and a valine in the latter. However, mutagenesis of residue 162 did not allow the rational transformation of the substrate preference, as the substitution of valine by threonine in the enzyme from Bifidobacterium longum did not tighten its specificity towards GaINAc. Unexpectedly, introducing an isoleucine at position 162 increased the preference for GIcNAc as acceptor, which illustrates that the structure-function relationships in beta-galactoside phosphorylases are not yet completely understood. Several other positions have also been examined by mutational analysis but true determinants of the acceptor specificity in family GH-112 could not be identified. (C) 2011 Elsevier Inc. All rights reserved.