Characterization of a new family of 6-sulfo-N-acetylglucosaminidases.

Sulfation is widespread in nature and plays an important role in modulating biological function. Among the strategies developed by microbes to access sulfated oligosaccharides as a nutrient source is the production of 6-sulfoGlcNAcases to selectively release 6-sulfoGlcNAc from target oligosaccharides. Thus far, all 6-sulfoGlcNAcases identified have belonged to the large GH20 family of β-hexosaminidases. Ηere, we identify and characterize a new, highly specific non-GH20 6-sulfoGlcNAcase from Streptococcus pneumoniae TIGR4, Sp_0475 with a greater than 110,000-fold preference toward N-acetyl-β-D-glucosamine-6-sulfate substrates over the nonsulfated version. Sp_0475 shares distant sequence homology with enzymes of GH20 and with the newly formed GH163 family. However, the sequence similarity between them is sufficiently low that Sp_0475 has been assigned as the founding member of a new glycoside hydrolase family, GH185. By combining results from site-directed mutagenesis with mechanistic studies and bioinformatics we provide insight into the substrate specificity, mechanism, and key active site residues of Sp_0475. Enzymes of the GH185 family follow a substrate-assisted mechanism, consistent with their distant homology to the GH20 family, but the catalytic residues involved are quite different. Taken together, our results highlight in more detail how microbes can degrade sulfated oligosaccharides for nutrients.