Background: Infant gut-associated bifidobacteria possess lacto-N-biosidase, which releases lacto-N-biose I (LNB) from human milk oligosaccharides. Results: The crystal structures of lacto-N-biosidase complexed with LNB and LNB-thiazoline were determined. Conclusion: The intermediate analog complex allows the proposal of a conformational reaction coordinate. Significance: The structures of a key enzyme in the colonization of human commensal bacteria provided its structural basis and insight into the development of inhibitors.

Breast-fed infants generally have a bifidobacteria-rich microbiota with recent studies indicating that human milk oligosaccharides (HMOs) selectively promote bifidobacterial growth. Bifidobacterium bifidum possesses a glycoside hydrolase family 20 lacto-N-biosidase for liberating lacto-N-biose I from lacto-N-tetraose, an abundant HMO unique to human milk, while Bifidobacterium longum subsp. longum has a non-classified enzyme (LnbX). Here, we determined the crystal structure of the catalytic domain of LnbX and provide evidence for creation of a novel glycoside hydrolase family, GH136. The structure, in combination with inhibition and mutation studies, provides insight into the molecular mechanism and broader substrate specificity of this enzyme. Moreover, through genetic studies, we show that lnbX is indispensable for B. longum growth on lacto-N-tetraose and is a key genetic factor for persistence in the gut of breast-fed infants. Overall, this study reveals possible evolutionary routes for the emergence of symbiosis between humans and bifidobacterial species in the infant gut.

The α--acetylgalactosaminidase from the probiotic bacterium (NagBb) belongs to the glycoside hydrolase family 129 and hydrolyzes the glycosidic bond of Tn-antigen (GalNAcα1-Ser/Thr). NagBb is involved in assimilation of-glycans on mucin glycoproteins by in the human gastrointestinal tract, but its catalytic mechanism has remained elusive because of a lack of sequence homology around putative catalytic residues and of other structural information. Here we report the X-ray crystal structure of NagBb, representing the first GH129 family structure, solved by the single-wavelength anomalous dispersion method based on sulfur atoms of the native protein. We determined ligand-free, GalNAc, and inhibitor complex forms of NagBb and found that Asp-435 and Glu-478 are located in the catalytic domain at appropriate positions for direct nucleophilic attack at the anomeric carbon and proton donation for the glycosidic bond oxygen, respectively. A highly conserved Asp-330 forms a hydrogen bond with the O4 hydroxyl of GalNAc in the -1 subsite, and Trp-398 provides a stacking platform for the GalNAc pyranose ring. Interestingly, a metal ion, presumably Ca, is involved in the recognition of the GalNAc -acetyl group. Mutations at Asp-435, Glu-478, Asp-330, and Trp-398 and residues involved in metal coordination (including an all-Ala quadruple mutant) significantly reduced the activity, indicating that these residues and the metal ion play important roles in substrate recognition and catalysis. Interestingly, NagBb exhibited some structural similarities to the GH101 endo-α--acetylgalactosaminidases, but several critical differences in substrate recognition and reaction mechanism account for the different activities of these two enzymes.

PG recycling inhibitors are envisaged as useful adjuvants in the treatment of P. aeruginosa infections with β-lactams and fosfomycin and therefore further development of these molecules is encouraged.

The synthesis of analogues of the potent N-acetylhexosaminidase inhibitor, PUGNAc, are described. These compounds were assayed against a set of biologically important N-acetyl-d-glucosaminidases and were found to vary in both potency and selectivity.