title: Characterization of β-galactoside phosphorylases with diverging acceptor specificities.
abstract: 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 GlcNAc, GalNAc 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 GalNAc as acceptor, while that from Anaerococcus prevotii displays similar activities on GalNAc and GlcNAc. 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 GalNAc. Unexpectedly, introducing an isoleucine at position 162 increased the preference for GlcNAc as acceptor, which illustrates that the structure-function relationships in β-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.
authors: Centre for Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium. chao.chen@ugent.be
authors: Chao
authors: C
authors: Chen
authors: Chen
authors: C
authors: Chao
authors: Wim
authors: W
authors: Soetaert
authors: Soetaert
authors: S
authors: Wim
authors: Tom
authors: T
authors: Desmet
authors: Desmet
authors: D
authors: Tom
mesh_terms: D000116:Acetylgalactosamine / Q000378:metabolism; D000117:Acetylglucosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001644:Bifidobacterium / Q000201:enzymology / Q000235:genetics; D003013:Clostridium / Q000201:enzymology / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004888:Erysipelothrix / Q000201:enzymology / Q000235:genetics; D005697:Galactosides / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D006005:Phosphorylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012210:Rhamnose / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013288:Streptobacillus / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005697:Galactosides; D011994:Recombinant Proteins; C007816:beta-galactoside; D006005:Phosphorylases; D000116:Acetylgalactosamine; D012210:Rhamnose; D000117:Acetylglucosamine