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 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-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

Structure of human endo-a-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 Glc3Man9GlcNAc2precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-a-1,2-mannosidase (MANEA) is the sole endoacting 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

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

Design and synthesis of chemical tools for the study of family GH99 and GH76 glycoside hydrolases that process n-linked glycans

© 2015 Dr. Zalihe HakkiN-linked glycans have significant roles in protein folding, stability, and function. Their aberrant expression is implicated in diseases including cancer and viral infection. Considerable effort has been expended over the years in the search for inhibitors that may interfere with the enzymes involved in their biosynthesis. The focus of many studies has been on inhibiting the enzymes involved in the early processing steps of N-linked glycans, and in particular the ER resident glycosidases. However targeting these enzymes has yielded limited success largely due to the existence of a unique enzyme, a Golgi-resident glycoside hydrolase termed endo-α-1,2-mannosidase. This family GH99 enzyme provides a ‘back-up’ pathway to the biosynthesis of mature N-linked glycans. The first part of this thesis investigates the fundamental features of this poorly understood enzyme using rationally-designed chemical tools and bacterial orthologs of endo-α-1,2-mannosidase from Bacteroides spp. Using the activated substrate α-Glc-1,3-Man-F the enzyme is determined to act with retention of stereochemistry. The first X-ray structure of any GH99 enzyme is solved and complexes with the established endo-α-1,2-mannosidase inhibitor α-Glc-1,3-Man-DMJ and herein synthesized α-Glc-1,3-IFG are used to structurally define the active site of the enzyme. The inhibitor α-Glc-1,3-IFG is instrumental in revealing the absence of a candidate nucleophilic residue and forms the basis for the proposal of an unusual mechanism for family GH99, which is proposed to proceed through a 1,2-anhydro intermediate. Preliminary studies with α-Glc-1,3-IFG also demonstrate that endo-α-1,2-mannosidase is a viable anti-viral target. The subsequent chapters detail the development of a ‘blocked’ derivative of α-Glc-1,3-IFG as a potential selective inhibitor of endo-α-1,2-mannosidase for use in cellular studies. Some work is also detailed towards the development of a dye-labelled α-Glc-1,3-IFG derivative for diagnostic purposes. The thesis proceeds to examine the role of bacterial endo-α-1,2-mannosidase orthologs in bacteria. Mammalian N-linked glycans are structurally-related to yeast α-mannans and it is speculated that bacterial endo-α-1,2-mannosidase may have a role in the degradation of yeast α-mannans. A representative yeast mannan fragment α-Man-1,3-α-Man-1,2-α-Man-1,2-α-Man-OMe is synthesized and shown to be a substrate of bacterial endo-α-1,2-mannosidase. Further the epimerically-related substrates α-Glc-1,3-α-Man-methylumbelliferone and α-Man-1,3-α-Man-methylumbelliferone reveal a ten-fold preference of the bacterial enzyme to process D-manno configured substrates. On the basis of these results, an improved inhibitor, α-Man-1,3-IFG is developed. Comparison of X-ray crystal structures of α-Glc-1,3-IFG and α-Man-1,3-IFG reveal a Trp residue that confers favourable binding of D-manno configured substrates and inhibitors. The final part of this thesis entails efforts to better understand GH76 endo-α-1,6-mannanases, another poorly understood mannosidase family that is present exclusively in bacteria and fungi. In fungi, these enzymes are speculated to be involved in the cross-linking of the cell glycophosphatidylinositol (GPI)-anchored glycoproteins into the fungal cell wall and in bacteria they are proposed to act as hydrolytic enzymes facilitating the degradation of yeast α-mannans. Although previous structures of GH76 have been reported, nothing was known about the catalytic mechanism and conformational itinerary of the enzyme due to a lack of substrates and inhibitors. Our X-ray structures of Bacillus circulans GH76 in complex with substrates and rationally-designed inhibitors proved instrumental in obtaining insights into the active site, mechanism and conformational itinerary of a representative GH76 enzyme for the first time. Through the use of p-nitrophenyl α-1,6-mannobioside, GH76 is shown to be retaining enzyme, supporting its role as a transglycosidase in cross-linking GPI anchors to fungal cell walls. The complex of BcGH76 with the inhibitor α-Man-1,6-IFG reveals two catalytic Asp residues that are likely to facilitate a conventional double displacement retaining mechanism. GH76 is also shown to be the first mannosidase to distort the IFG moiety of α-Man-1,6-IFG to an energetically unfavourable boat conformation, suggesting this conformation has special significance. X-ray structures and QM/MM simulations suggest that GH76 likely operates through a OS2 ↔ B2,5‡ ↔ 1S5 conformational itinerary

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

A Single Glycosidase Harnesses Different Pyranoside Ring Transition State Conformations for Hydrolysis of Mannosides and Glucosides

Hydrolysis of β-d-mannosides by β-mannosidases typically proceeds via a <i>B</i>_2,5 transition state conformation for the pyranoside ring, while that of β-d-glucosides by β-glucosidases proceeds through a distinct ⁴<i>H</i>₃ transition state conformation. However, rice Os7BGlu26 β-glycosidase hydrolyzes 4-nitrophenyl β-d-glucoside and β-d-mannoside with similar efficiencies. The origin of this dual substrate specificity was investigated by kinetic, structural, and computational approaches. The glycosidase inhibitors glucoimidazole and mannoimidazole inhibited Os7BGlu26 with <i>K</i>_i values of 2.7 nM and 10.4 μM, respectively. In X-ray crystal structures of complexes with Os7BGlu26, glucoimidazole bound to the active site in a ⁴<i>E</i> conformation, while mannoimidazole bound in a <i>B</i>_2,5 conformation, suggesting different transition state conformations. Moreover, calculation of quantum mechanics/molecular mechanics (QM/MM) free energy landscapes showed that 4-nitrophenyl β-d-glucoside adopts a ¹<i>S</i>₃/⁴<i>E</i> conformation in the Michaelis complex, while 4-nitrophenyl β-d-mannoside adopts a ¹<i>S</i>₅/<i>B</i>_2,5 conformation. The QM/MM simulations of Os7BGlu26 catalysis of hydrolysis also supported the itineraries of ¹<i>S</i>₃ → ⁴<i>E</i>/⁴<i>H</i>₃^⧧ → ⁴<i>C</i>₁ for β-d-glucosides and ¹<i>S</i>₅ → <i>B</i>_2,5^⧧ → ^O<i>S</i>₂ for β-d-mannosides, thereby revealing that a single glycoside hydrolase can hydrolyze glycosides of different configurations via distinct transition state pyranoside conformations

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, 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 modelling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unprecedented flattened, envelope (E3) conformation. Kinetic isotope effects 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.</div