The first crystal structure of a family 129 glycoside hydrolase from a probiotic bacterium reveals critical residues and metal cofactors.

The α-N-acetylgalactosaminidase from the probiotic bacterium Bifidobacterium bifidum (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 O-glycans on mucin glycoproteins by B. bifidum 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 Ca2+, is involved in the recognition of the GalNAc N-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-N-acetylgalactosaminidases, but several critical differences in substrate recognition and reaction mechanism account for the different activities of these two enzymes.

Molecular Insight into Evolution of Symbiosis between Breast-Fed Infants and a Member of the Human Gut Microbiome Bifidobacterium longum.

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.

Synergistic activity of fosfomycin, ß-lactams and peptidoglycan recycling inhibition against Pseudomonas aeruginosa.

OBJECTIVES: To evaluate the interconnection between peptidoglycan (PG) recycling, fosfomycin susceptibility and synergy between fosfomycin and ß-lactams in Pseudomonas aeruginosa METHODS: Fosfomycin MICs were determined by broth microdilution and Etest for a panel of 47 PAO1 mutants defective in several components of PG recycling and/or AmpC induction pathways. PAO1 fosfomycin MICs were also determined in the presence of a 5 mM concentration of the NagZ inhibitor PUGNAc. Population analysis of fosfomycin susceptibility and characterization of the resistant mutants that emerged was also performed for selected strains. Finally, fosfomycin, imipenem and fosfomycin + imipenem killing curves were assessed. RESULTS: Mutants defective in AmpG, NagZ or all three AmpD amidases showed a marked increase in fosfomycin susceptibility (at least two 2-fold dilutions with respect to WT PAO1). Moreover, PAO1 fosfomycin MICs were consistently reduced from 48 to 24 mg/L in the presence of a 5 mM concentration of PUGNAc. Fosfomycin hypersusceptibility of the ampG, nagZ and triple ampD mutants was also clearly confirmed in the performed population analysis, although the emergence of resistant mutants, through GlpT mutations, was not avoided. Synergy between fosfomycin and imipenem was evidenced for the WT strain, the AmpC-hyperproducing strain (triple AmpD mutant) and the NagZ and AmpG mutants in killing curves. Moreover, regrowth of resistant mutants was not evidenced for the combination. CONCLUSIONS: PG recycling inhibitors are envisaged as useful adjuvants in the treatment of P. aeruginosa infections with ß-lactams and fosfomycin and therefore further development of these molecules is encouraged.

Modifying the phenyl group of PUGNAc: reactivity tuning to deliver selective inhibitors for N-acetyl-D-glucosaminidases.

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.

Gaining insight into the catalysis by GH20 lacto-N-biosidase using small molecule inhibitors and structural analysis.

The synthesis of potent inhibitors for lacto-N-biosidases and X-ray structural characterization of these compounds in complex with BbLNBase is described.

Inhibition of the family 20 glycoside hydrolase catalytic modules in the Streptococcus pneumoniae exo-ß-D-N-acetylglucosaminidase, StrH.

Streptococcus pneumoniae produces a cell-surface attached ß-N-acetylglucosaminidase called StrH that is used by this pathogen to process the termini of host complex N-linked glycans. N-Acetyl-D-glucosamine-thiazoline (NAG-Thiazoline, NGT) and O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl carbamate (PUGNAc) are inhibitors of the two family 20 glycoside hydrolase catalytic modules within StrH and these inhibitors have proven useful in modulating the activity of StrH in assays that model aspects of the host-bacterium interaction. Here we explore the molecular basis of StrH inhibition through structural, kinetic, thermodynamic and site-directed mutagenic analyses using the recombinantly produced independent catalytic modules of StrH (GH20A and GH20B) and the inhibitors NGT and PUGNAc. The results reveal a similar binding mode of the sugar moiety of these inhibitors at the -1 subsite in the active sites of GH20A and GH20B. The lower affinity of NGT as compared to PUGNAc for these catalytic modules can be attributed to the hydrophobic phenylcarbamate moiety of PUGNAc that is absent in NGT. This moiety also displayed variations in its interactions with the active sites of GH20A and GH20B that provide a rationale for the 400-fold difference observed in the Ki values of this compound for these two ß-N-acetylglucosaminidase catalytic modules.

Crystal structures of a glycoside hydrolase family 20 lacto-N-biosidase from Bifidobacterium bifidum.

Human milk oligosaccharides contain a large variety of oligosaccharides, of which lacto-N-biose I (Gal-ß1,3-GlcNAc; LNB) predominates as a major core structure. A unique metabolic pathway specific for LNB has recently been identified in the human commensal bifidobacteria. Several strains of infant gut-associated bifidobacteria possess lacto-N-biosidase, a membrane-anchored extracellular enzyme, that liberates LNB from the nonreducing end of human milk oligosaccharides and plays a key role in the metabolic pathway of these compounds. Lacto-N-biosidase belongs to the glycoside hydrolase family 20, and its reaction proceeds via a substrate-assisted catalytic mechanism. Several crystal structures of GH20 ß-N-acetylhexosaminidases, which release monosaccharide GlcNAc from its substrate, have been determined, but to date, a structure of lacto-N-biosidase is unknown. Here, we have determined the first three-dimensional structures of lacto-N-biosidase from Bifidobacterium bifidum JCM1254 in complex with LNB and LNB-thiazoline (Gal-ß1,3-GlcNAc-thiazoline) at 1.8-Å resolution. Lacto-N-biosidase consists of three domains, and the C-terminal domain has a unique ß-trefoil-like fold. Compared with other ß-N-acetylhexosaminidases, lacto-N-biosidase has a wide substrate-binding pocket with a -2 subsite specific for ß-1,3-linked Gal, and the residues responsible for Gal recognition were identified. The bound ligands are recognized by extensive hydrogen bonds at all of their hydroxyls consistent with the enzyme's strict substrate specificity for the LNB moiety. The GlcNAc sugar ring of LNB is in a distorted conformation near (4)E, whereas that of LNB-thiazoline is in a (4)C1 conformation. A possible conformational pathway for the lacto-N-biosidase reaction is discussed.

Development of tools to study lacto-N-biosidase: an important enzyme involved in the breakdown of human milk oligosaccharides.

Milk and sugar? The elucidation of the catalytic mechanism and the development of the first known inhibitor for lacto-N-biosidases, which are important enzymes involved in the breakdown of human milk oligosaccharides, are described.