N-linked glycans play key roles in protein folding, stability, and function. Biosynthetic modification of N-linked glycans, within the endoplasmic reticulum, features sequential trimming and readornment steps. One unusual enzyme, endo-α-mannosidase, cleaves mannoside linkages internally within an N-linked glycan chain, short circuiting the classical N-glycan biosynthetic pathway. Here, using two bacterial orthologs, we present the first structural and mechanistic dissection of endo-α-mannosidase. Structures solved at resolutions 1.7–2.1 Å reveal a ( β / α ) 8 barrel fold in which the catalytic center is present in a long substrate-binding groove, consistent with cleavage within the N-glycan chain. Enzymatic cleavage of authentic Glc 1/3 Man 9 GlcNAc 2 yields Glc 1/3 -Man. Using the bespoke substrate α-Glc-1,3-α-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-α-mannosidase inhibitor α-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, α-Glc-1,3-isofagomine, and with the reducing-end product α-1,2-mannobiose structurally define the -2 to +2 subsites of the enzyme. These structural and mechanistic data provide a foundation upon which to develop new enzyme inhibitors targeting the hijacking of N-glycan synthesis in viral disease and cancer.

Glycans are major nutrients available to the human gut microbiota. The Bacteroides are generalist glycan degraders, and this function is mediated largely by polysaccharide utilization loci (PULs). The genomes of several Bacteroides species contain a PUL, PUL1,6-β-glucan, that was predicted to target mixed linked plant 1,3;1,4-β-glucans. To test this hypothesis we characterized the proteins encoded by this locus in Bacteroides thetaiotaomicron, a member of the human gut microbiota. We show here that PUL1,6-β-glucan does not orchestrate the degradation of a plant polysaccharide but targets a fungal cell wall glycan, 1,6-β-glucan, which is a growth substrate for the bacterium. The locus is up-regulated by 1,6-β-glucan and encodes two enzymes, a surface endo-1,6-β-glucanase, BT3312, and a periplasmic β-glucosidase that targets primarily 1,6-β-glucans. The non-catalytic proteins encoded by PUL1,6-β-glucan target 1,6-β-glucans and comprise a surface glycan-binding protein and a SusD homologue that delivers glycans to the outer membrane transporter. We identified the central role of the endo-1,6-β-glucanase in 1,6-β-glucan depolymerization by deleting bt3312, which prevented the growth of B. thetaiotaomicron on 1,6-β-glucan. The crystal structure of BT3312 in complex with β-glucosyl-1,6-deoxynojirimycin revealed a TIM barrel catalytic domain that contains a deep substrate-binding cleft tailored to accommodate the hook-like structure adopted by 1,6-β-glucan. Specificity is driven by the complementarity of the enzyme active site cleft and the conformation of the substrate. We also noted that PUL1,6-β-glucan is syntenic to many PULs from other Bacteroidetes, suggesting that utilization of yeast and fungal cell wall 1,6-β-glucans is a widespread adaptation within the human microbiota.

The sulfonated carbohydrate sulfoquinovose (SQ) is produced in quantities estimated at some 10 billion tonnes annually and is thus a major participant in the global sulfur biocycle. SQ is produced by most photosynthetic organisms and incorporated into the sulfolipid sulfoquinovosyl diacylglycerol (SQDG), as well as within some archaea for incorporation into glycoprotein N-glycans. SQDG is found mainly within the thylakoid membranes of the chloroplast, where it appears to be important for membrane structure and function and for optimal activity of photosynthetic protein complexes. SQDG metabolism within the sulfur cycle involves complex biosynthetic and catabolic processes. SQDG biosynthesis is largely conserved within plants, algae and bacteria. On the other hand, two major sulfoglycolytic pathways have been discovered for SQDG degradation, the sulfo-Embden-Meyerhof-Parnas (sulfo-EMP) and sulfo-Entner-Doudoroff (sulfo-ED) pathways, which mirror the major steps in the glycolytic EMP and ED pathways. Sulfoglycolysis produces C3-sulfonates, which undergo biomineralization to inorganic sulfur species, completing the sulfur cycle. This review discusses the discovery and structural elucidation of SQDG and archaeal N-glycans, the occurrence, distribution, and speciation of SQDG, and metabolic pathways leading to the biosynthesis of SQDG and its catabolism through sulfoglycolytic and biomineralization pathways to inorganic sulfur.

Mannosidases catalyze the hydrolysis of a diverse range of polysaccharides and glycoconjugates, and the various sequence-based mannosidase families have evolved ingenious strategies to overcome the stereoelectronic challenges of mannoside chemistry. Using a combination of computational chemistry, inhibitor design and synthesis, and X-ray crystallography of inhibitor/enzyme complexes, it is demonstrated that mannoimidazole-type inhibitors are energetically poised to report faithfully on mannosidase transition-state conformation, and provide direct evidence for the conformational itinerary used by diverse mannosidases, including β-mannanases from families GH26 and GH113. Isofagomine-type inhibitors are poor mimics of transition-state conformation, owing to the high energy barriers that must be crossed to attain mechanistically relevant conformations, however, these sugar-shaped heterocycles allow the acquisition of ternary complexes that span the active site, thus providing valuable insight into active-site residues involved in substrate recognition.

Mincle is a C-type lectin receptor that has emerged as an important player in innate immunity through its capacity to recognize a wide range of lipidic species derived from damaged/altered self and foreign microorganisms. Self-ligands include sterols (e.g., cholesterol), and β-glucosylceramides, and the protein SAP130, which is released upon cell death. Foreign lipids comprise those from both microbial pathogens and commensals and include glycerol, glucose and trehalose mycolates, and glycosyl diglycerides. A large effort has focused on structural variation of these ligands to illuminate the structure–activity relationships required for the agonism of signaling though Mincle and has helped identify key differences in ligand recognition between human and rodent Mincle. These studies in turn have helped identify new Mincle ligands, further broadening our understanding of the diversity of organisms and lipidic species recognized by Mincle. Finally, progress toward the development of Mincle agonists as vaccine adjuvants providing humoral and cell-mediated immunity with reduced toxicity is discussed.

Helicobacter pylori causes gastritis, which has been attributed to the development of H. pylori–specific T cells during infection. However, the mechanism underlying innate immune detection leading to the priming of T cells is not fully understood, as H. pylori evades TLR detection. Here, we report that H. pylori metabolites modified from host cholesterol exacerbate gastritis through the interaction with C-type lectin receptors. Cholesteryl acyl α-glucoside (αCAG) and cholesteryl phosphatidyl α-glucoside (αCPG) were identified as noncanonical ligands for Mincle (Clec4e) and DCAR (Clec4b1). During chronic infection, H. pylori–specific T cell responses and gastritis were ameliorated in Mincle-deficient mice, although bacterial burdens remained unchanged. Furthermore, a mutant H. pylori strain lacking αCAG and αCPG exhibited an impaired ability to cause gastritis. Thus H. pylori–specific modification of host cholesterol plays a pathophysiological role that exacerbates gastric inflammation by triggering C-type lectin receptors.

Rhizobia are nitrogen-fixing bacteria that engage in symbiotic relationships with plant hosts but can also persist as free-living bacteria in the soil and rhizosphere. Here, we show that free-living Rhizobium leguminosarum SRDI565 can grow on the sulfosugar sulfoquinovose (SQ) or the related glycoside SQ-glycerol using a sulfoglycolytic Entner-Doudoroff (sulfo-ED) pathway, resulting in production of sulfolactate (SL) as the major metabolic end product. Comparative proteomics supports the involvement of a sulfo-ED operon encoding an ABC transporter, sulfo-ED enzymes, and an SL exporter. Consistent with an oligotrophic lifestyle, proteomics data revealed little change in expression of the sulfo-ED proteins during growth on SQ versus mannitol, a result confirmed through biochemical assay of sulfoquinovosidase activity in cell lysates. Metabolomics analysis showed that growth on SQ involves gluconeogenesis to satisfy metabolic requirements for glucose-6-phosphate and fructose-6-phosphate. Metabolomics analysis also revealed the unexpected production of small amounts of sulfofructose and 2,3-dihydroxypropanesulfonate, which are proposed to arise from promiscuous activities of the glycolytic enzyme phosphoglucose isomerase and a nonspecific aldehyde reductase, respectively. The discovery of a rhizobium isolate with the ability to degrade SQ builds our knowledge of how these important symbiotic bacteria persist within soil. IMPORTANCE Sulfonate sulfur is a major form of organic sulfur in soils but requires biomineralization before it can be utilized by plants. Very little is known about the biochemical processes used to mobilize sulfonate sulfur. We show that a rhizobial isolate from soil, Rhizobium leguminosarum SRDI565, possesses the ability to degrade the abundant phototroph-derived carbohydrate sulfonate SQ through a sulfoglycolytic Entner-Doudoroff pathway. Proteomics and metabolomics demonstrated the utilization of this pathway during growth on SQ and provided evidence for gluconeogenesis. Unexpectedly, off-cycle sulfoglycolytic species were also detected, pointing to the complexity of metabolic processes within cells under conditions of sulfoglycolysis. Thus, rhizobial metabolism of the abundant sulfosugar SQ may contribute to persistence of the bacteria in the soil and to mobilization of sulfur in the pedosphere.

Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E 3 ) conformation. Kinetic isotope effects (k cat /K M ) for anomeric-2 H and anomeric-13 C support an oxocarbenium ion-like transition state, and that for C2-18 O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.