Characterization of β-galactoside phosphorylases with diverging acceptor specificities

Area of Science:

• Biochemistry
• Enzymology
• Carbohydrate Chemistry

Background:

• Glycoside phosphorylases are carbohydrate-active enzymes with unique biochemical properties.
• Family GH-112 glycoside phosphorylases uniquely utilize galactose-1-phosphate as a glycosyl donor.
• This family exhibits diverse acceptor specificities, including L-rhamnose, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc).

Purpose of the Study:

• To characterize novel phosphorylases from unexplored phylogenetic branches of family GH-112.
• To investigate the divergence in acceptor specificity within this enzyme family.
• To elucidate the structure-function relationships governing acceptor specificity in GH-112 phosphorylases.

Main Methods:

• Phylogenetic analysis to identify new enzyme candidates.
• Biochemical characterization of enzyme activity and acceptor preference.
• Site-directed mutagenesis to probe amino acid residue roles in specificity.

Main Results:

• Three new GH-112 phosphorylases were characterized from Erysipelothrix rhusiopathiae, Streptobacillus moniliformis, and Anaerococcus prevotii.
• Enzymes from E. rhusiopathiae and S. moniliformis preferred GalNAc, while A. prevotii showed activity on both GalNAc and GlcNAc.
• A correlation between residue 162 (threonine/valine) and specificity was confirmed, but mutagenesis yielded unexpected results, indicating complex structure-function relationships.

Conclusions:

• The study expands the understanding of GH-112 phosphorylase diversity and acceptor specificity.
• Amino acid residue at position 162 is a key determinant, but not the sole factor, in acceptor specificity.
• Further research is needed to fully comprehend the intricate structure-function relationships in β-galactoside phosphorylases.