Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target.

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2 precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole endo-acting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation.

An Epoxide Intermediate in Glycosidase Catalysis.

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 2-hydroxyl 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 ß-1,2-aziridine and ß-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-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.

Spiro-epoxyglycosides as Activity-Based Probes for Glycoside Hydrolase Family 99 Endomannosidase/Endomannanase.

N-Glycans direct protein function, stability, folding and targeting, and influence immunogenicity. While most glycosidases that process N-glycans cleave a single sugar residue at a time, enzymes from glycoside hydrolase family 99 are endo-acting enzymes that cleave within complex N-glycans. Eukaryotic Golgi endo-1,2-α-mannosidase cleaves glucose-substituted mannose within immature glucosylated high-mannose N-glycans in the secretory pathway. Certain bacteria within the human gut microbiota produce endo-1,2-α-mannanase, which cleaves related structures within fungal mannan, as part of nutrient acquisition. An unconventional mechanism of catalysis was proposed for enzymes of this family, hinted at by crystal structures of imino/azasugars complexed within the active site. Based on this mechanism, we developed the synthesis of two glycosides bearing a spiro-epoxide at C-2 as electrophilic trap, to covalently bind a mechanistically important, conserved GH99 catalytic residue. The spiro-epoxyglycosides are equipped with a fluorescent tag, and following incubation with recombinant enzyme, allow concentration, time and pH dependent visualization of the bound enzyme using gel electrophoresis.

Evidence for a boat conformation at the transition state of GH76 α-1,6-mannanases--key enzymes in bacterial and fungal mannoprotein metabolism.

α-Mannosidases and α-mannanases have attracted attention for the insight they provide into nucleophilic substitution at the hindered anomeric center of α-mannosides, and the potential of mannosidase inhibitors as cellular probes and therapeutic agents. We report the conformational itinerary of the family GH76 α-mannanases studied through structural analysis of the Michaelis complex and synthesis and evaluation of novel aza/imino sugar inhibitors. A Michaelis complex in an (O) S2 conformation, coupled with distortion of an azasugar in an inhibitor complex to a high energy B2,5 conformation are rationalized through ab initio QM/MM metadynamics that show how the enzyme surface restricts the conformational landscape of the substrate, rendering the B2,5 conformation the most energetically stable on-enzyme. We conclude that GH76 enzymes perform catalysis using an itinerary that passes through (O) S2 and B2,5 (≠) conformations, information that should inspire the development of new antifungal agents.

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism.

Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a 'selfish' model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.

Structural and kinetic dissection of the endo-α-1,2-mannanase activity of bacterial GH99 glycoside hydrolases from Bacteroides†spp.

Glycoside hydrolase family 99 (GH99) was created to categorize sequence-related glycosidases possessing endo-α-mannosidase activity: the cleavage of mannosidic linkages within eukaryotic N-glycan precursors (Glc1-3 Man9 GlcNAc2 ), releasing mono-, di- and triglucosylated-mannose (Glc1-3 -1,3-Man). GH99 family members have recently been implicated in the ability of Bacteroides spp., present within the gut microbiota, to metabolize fungal cell wall α-mannans, releasing α-1,3-mannobiose by hydrolysing αMan-1,3-αMan→1,2-αMan-1,2-αMan sequences within branches off the main α-1,6-mannan backbone. We report the development of a series of substrates and inhibitors, which we use to kinetically and structurally characterise this novel endo-α-1,2-mannanase activity of bacterial GH99 enzymes from Bacteroides thetaiotaomicron and xylanisolvens. These data reveal an approximate 5 kJ mol(-1) preference for mannose-configured substrates in the -2 subsite (relative to glucose), which inspired the development of a new inhibitor, α-mannopyranosyl-1,3-isofagomine (ManIFG), the most potent (bacterial) GH99 inhibitor reported to date. X-ray structures of ManIFG or a substrate in complex with wild-type or inactive mutants, respectively, of B. xylanisolvens GH99 reveal the structural basis for binding to D-mannose- rather than D-glucose-configured substrates.

Glycoprotein misfolding in the endoplasmic reticulum: identification of released oligosaccharides reveals a second ER-associated degradation pathway for Golgi-retrieved proteins.

Endoplasmic reticulum-associated degradation (ERAD) is a key cellular process whereby misfolded proteins are removed from the endoplasmic reticulum (ER) for subsequent degradation by the ubiquitin/proteasome system. In the present work, analysis of the released, free oligosaccharides (FOS) derived from all glycoproteins undergoing ERAD, has allowed a global estimation of the mechanisms of this pathway rather than following model proteins through degradative routes. Examining the FOS produced in endomannosidase-compromised cells following α-glucosidase inhibition has revealed a mechanism for clearing Golgi-retrieved glycoproteins that have failed to enter the ER quality control cycle. The Glc3Man7GlcNAc2 FOS species has been shown to be produced in the ER lumen by a mechanism involving a peptide: N-glycanase-like activity, and its production was sensitive to disruption of Golgi-ER trafficking. The detection of this oligosaccharide was unaffected by the overexpression of EDEM1 or cytosolic mannosidase, both of which increased the production of previously characterised cytosolically localised FOS. The lumenal FOS identified are therefore distinct in their production and regulation compared to FOS produced by the conventional route of misfolded glycoproteins directly removed from the ER. The production of such lumenal FOS is indicative of a novel degradative route for cellular glycoproteins that may exist under certain conditions.

Structural and mechanistic insight into N-glycan processing by endo-α-mannosidase.

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 (ß/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.

Synthesis of the monoterpenoid esters cypellocarpin C and cuniloside B and evidence for their widespread occurrence in Eucalyptus.

Short syntheses of cuniloside B and cypellocarpin C, (+)-(R)-oleuropeic acid-containing carbohydrates, are reported. Also disclosed are syntheses of the noreugenin glycosides, undulatoside A and corymbosins K(1) and K(2). Leaf extracts of 28 diverse eucalypts revealed cuniloside B to be present in all, and cypellocarpin C to be present in most, of the species examined. The widespread occurrence of these carbohydrate monoterpenoid esters supports their roles in essential oil biosynthesis or mobilization from sites of synthesis to secretory cavity lumena.