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N-Glycosylation is a fundamental protein modification found in both eukaryotes and archaea. Despite lacking N-glycans, many commensal and pathogenic bacteria have developed mechanisms to degrade these isoforms for a variety of functions, including nutrient acquisition and evasion of the immune system. Although much is known about many of the enzymes responsible for N-glycan degradation, the enzymes involved in cleaving the N-glycan core have only recently been discovered. Thus, some of the structural details have yet to be characterized, and little is known about their full distribution among bacterial strains and specifically within potential Gram-positive polysaccharide utilization loci. Here, we report crystal structures for Family 5, Subfamily 18 (GH5_18) glycoside hydrolases from the gut bacterium Bifidobacterium longum (BlGH5_18) and the soil bacterium Streptomyces cattleya (ScGH5_18), which hydrolyze the core Manβ1-4GlcNAc disaccharide. Structures of these enzymes in complex with Manβ1-4GlcNAc reveal a more complete picture of the -1 subsite. They also show that a C-terminal active site cap present in BlGH5_18 is absent in ScGH5_18. Although this C-terminal cap is not widely distributed throughout the GH5_18 family, it is important for full enzyme activity. In addition, we show that GH5_18 enzymes are found in Gram-positive polysaccharide utilization loci that share common genes, likely dedicated to importing and degrading N-glycan core structures.

Paenibacillus odorifer produces a single multimodular enzyme containing a glycoside hydrolase (GH) family 74 module (AIQ73809). Recombinant production and characterization of the GH74 module (PoGH74cat) revealed a highly specific, processive endo-xyloglucanase that can hydrolyze the polysaccharide backbone at both branched and unbranched positions. X-ray crystal structures obtained for the free enzyme and oligosaccharide complexes evidenced an extensive hydrophobic binding platform — the first in GH74 extending from subsites −4 to +6 — and unique mobile active-site loops. Site-directed mutagenesis revealed that glycine-476 was uniquely responsible for the promiscuous backbone-cleaving activity of PoGH74cat; replacement with tyrosine, which is conserved in many GH74 members, resulted in exclusive hydrolysis at unbranched glucose units. Likewise, systematic replacement of the hydrophobic platform residues constituting the positive subsites indicated their relative contributions to the processive mode of action. Specifically, W347 (+3 subsite) and W348 (+5 subsite) are essential for processivity, while W406 (+2 subsite) and Y372 (+6 subsite) are not strictly essential, but aid processivity.

Bacteria that reside in the mammalian intestinal tract efficiently hydrolyze dietary carbohydrates, including starch, that escape digestion in the small intestine. Starch is an abundant dietary carbohydrate comprised of α1,4 and α1,6 linked glucose, yet mammalian intestinal glucoamylases cannot effectively hydrolyze starch that has frequent α1,6 branching as these structures hinder recognition and processing by α1,4-specific amylases. Here we present the structure of the cell surface amylase SusG from Bacteroides thetaiotaomicron complexed with a mixed linkage amylosaccharide generated from transglycosylation during crystallization. Although SusG is specific for α1,4 glucosidic bonds, binding of this new oligosaccharide at the active site demonstrates that SusG can accommodate α1,6 branch points at subsite -3 to -2, and also at subsite+1 adjacent to the site of hydrolysis, explaining how this enzyme may be able to process a wide range of limit dextrins in the intestinal environment. These data suggest that B. thetaiotaomicron and related organisms may have a selective advantage for amylosaccharide scavenging in the gut.

International audience; Glycoside hydrolase family 74 (GH74) is a historically important family of endo-␤-glucanases. On the basis of early reports of detectable activity on cellulose and soluble cellulose derivatives, GH74 was originally considered to be a "cellulase" family, although more recent studies have generally indicated a high specificity toward the ubiquitous plant cell wall matrix glycan xyloglucan. Previous studies have indicated that GH74 xyloglu-canases differ in backbone cleavage regiospecificities and can adopt three distinct hydrolytic modes of action: exo, endo-dis-sociative, and endo-processive. To improve functional predictions within GH74, here we coupled in-depth biochemical characterization of 17 recombinant proteins with structural biology-based investigations in the context of a comprehensive molecular phylogeny, including all previously characterized family members. Elucidation of four new GH74 tertiary structures , as well as one distantly related dual seven-bladed ␤-propeller protein from a marine bacterium, highlighted key structure-function relationships along protein evolutionary trajectories. We could define five phylogenetic groups, which delineated the mode of action and the regiospecificity of GH74 members. At the extremes, a major group of enzymes diverged to hydrolyze the backbone of xyloglucan nonspecifically with a dissociative mode of action and relaxed backbone regiospecific-ity. In contrast, a sister group of GH74 enzymes has evolved a large hydrophobic platform comprising 10 subsites, which facilitates processivity. Overall, the findings of our study refine our understanding of catalysis in GH74, providing a framework for future experimentation as well as for bioinformatics predictions of sequences emerging from (meta)genomic studies.

The quantitation of liberated reducing sugars by the copper-bicinchoninic acid (BCA) assay provides a highly sensitive method for the measurement of glycoside hydrolase (GH) activity, particularly on soluble polysaccharide substrates. Here, we describe a straightforward method adapted to low-volume polymerase chain reaction (PCR) tubes which enables the rapid, parallel determination of GH kinetics in applications ranging from initial activity screening and assay optimization, to precise Michaelis-Menten analysis.