Structural, mechanistic and functional characterization of glycoside hydrolases of family GH99

Glycosylation is a very common post-translational modification and the glycans can be attached to oxygen (O-linked), nitrogen (N-linked) or carbon (C-linked). N-linked glycosylation has implications for protein folding and is also essential in viral infectivity and cell-cell signalling. Endo-α-1,2-mannosidase from family GH99 is a unique enzyme within the N-glycosylation pathway as it is the only one which does not cleave the terminal sugar from the reducing end of the glycan, but instead releases an α-Glc-1,3-Man disaccharide, with overall retention of stereochemistry at the anomeric carbon. Previously it was proposed that GH99 endo-acting mannosidases and mannanases proceed through a neighbouring group participation mechanism with a 1,2-anhydrosugar as a reaction intermediate. This Thesis contains evidence supporting this hypothesis. Chapter 2 presents structures of the bacterial GH99 with its substrate, with mimics of the reaction intermediate and with the products of the reaction. Kinetic and structural data on various intermediate mimics show that the compound whose structure is the closest to the intermediate is turned over by the enzyme. In Chapter 3, analysis of different designs of GH99 inhibitors and their conformation on-enzyme is presented. Chapter 4 presents purification and solution of the crystal structure of the catalytic domain of the human endomannosidase (MANEA). Multiple crystal forms were obtained, which made it possible to look at the conformation of a feature present in the eukaryotic but not bacterial GH99: a loop spanning residues 191–201. This loop was disordered when no ligand was present in the –2/–1 sites, and ordered when these sites were occupied. Chapter 5 explores attempts at producing MANEAL, a paralog of MANEA which is found in bony vertebrates. The Thesis concludes with an analysis of the phylogeny of endomannosidase genes and perspectives for future research: studies of endomannosidase in mammalian model organisms are needed to understand its significance

ChtVis-Tomato, a genetic reporter for in vivo visualization of chitin deposition in Drosophila

Chitin is a polymer of N-acetylglucosamine that is abundant and widely found in the biological world. It is an important constituent of the cuticular exoskeleton that plays a key role in the insect life cycle. To date, the study of chitin deposition during cuticle formation has been limited by the lack of a method to detect it in living organisms. To overcome this limitation, we have developed ChtVis-Tomato, an in vivo reporter for chitin in Drosophila. ChtVis-Tomato encodes a fusion protein that contains an apical secretion signal, a chitin-binding domain (CBD), a fluorescent protein and a cleavage site to release it from the plasma membrane. The chitin reporter allowed us to study chitin deposition in time lapse experiments and by using it we have identified unexpected deposits of chitin fibers in Drosophila pupae. ChtVis-Tomato should facilitate future studies on chitin in Drosophila and other insects

Exploration of Strategies for Mechanism-Based Inhibitor Design for Family GH99 endo-α-1,2-Mannanases

endo-α-1,2-Mannosidases and -mannanases, members of glycoside hydrolase family 99 (GH99), cleave α-Glc/Man-1,3-α-Man-OR structures within mammalian N-linked glycans and fungal α-mannan, respectively. They are proposed to act through a two-step mechanism involving a 1,2-anhydrosugar "epoxide" intermediate incorporating two conserved catalytic carboxylates. In the first step, one carboxylate acts as a general base to deprotonate the 2-hydroxy group adjacent to the fissile glycosidic bond, and the other provides general acid assistance to the departure of the aglycon. We report herein the synthesis of two inhibitors designed to interact with either the general base (α-mannosyl-1,3-(2-aminodeoxymannojirimycin), Man2NH2 DMJ) or the general acid (α-mannosyl-1,3-mannoimidazole, ManManIm). Modest affinities were observed for an endo-α-1,2-mannanase from Bacteroides thetaiotaomicron. Structural studies revealed that Man2NH2 DMJ binds like other iminosugar inhibitors, which suggests that the poor inhibition shown by this compound is not a result of a failure to achieve the expected interaction with the general base, but rather the reduction in basicity of the endocyclic nitrogen caused by introduction of a vicinal, protonated amine at C2. ManManIm binds with the imidazole headgroup distorted downwards, a result of an unfavourable interaction with a conserved active site tyrosine. This study has identified important limitations associated with mechanism-inspired inhibitor design for GH99 enzymes

From 1,4-Disaccharide to 1,3-Glycosyl Carbasugar : Synthesis of a Bespoke Inhibitor of Family GH99 Endo-α-mannosidase

Understanding the enzyme reaction mechanism can lead to the design of enzyme inhibitors. A Claisen rearrangement was used to allow conversion of an α-1,4-disaccharide into an α-1,3-linked glycosyl carbasugar to target the endo-α-mannosidase from the GH99 glycosidase family, which, unusually, is believed to act through a 1,2-anhydrosugar "epoxide" intermediate. Using NMR and X-ray crystallography, it is shown that glucosyl carbasugar α-aziridines can act as reasonably potent endo-α-mannosidase inhibitors, likely by virtue of their shape mimicry and the interactions of the aziridine nitrogen with the conserved catalytic acid/base of the enzyme active site

Contribution of shape and charge to the inhibition of a family GH99 endo-α-1,2-mannanase

[Image: see text] Inhibitor design incorporating features of the reaction coordinate and transition-state structure has emerged as a powerful approach for the development of enzyme inhibitors. Such inhibitors find use as mechanistic probes, chemical biology tools, and therapeutics. Endo-α-1,2-mannosidases and endo-α-1,2-mannanases, members of glycoside hydrolase family 99 (GH99), are interesting targets for inhibitor development as they play key roles in N-glycan maturation and microbiotal yeast mannan degradation, respectively. These enzymes are proposed to act via a 1,2-anhydrosugar “epoxide” mechanism that proceeds through an unusual conformational itinerary. Here, we explore how shape and charge contribute to binding of diverse inhibitors of these enzymes. We report the synthesis of neutral dideoxy, glucal and cyclohexenyl disaccharide inhibitors, their binding to GH99 endo-α-1,2-mannanases, and their structural analysis by X-ray crystallography. Quantum mechanical calculations of the free energy landscapes reveal how the neutral inhibitors provide shape but not charge mimicry of the proposed intermediate and transition state structures. Building upon the knowledge of shape and charge contributions to inhibition of family GH99 enzymes, we design and synthesize α-Man-1,3-noeuromycin, which is revealed to be the most potent inhibitor (K(D) 13 nM for Bacteroides xylanisolvens GH99 enzyme) of these enzymes yet reported. This work reveals how shape and charge mimicry of transition state features can enable the rational design of potent inhibitors

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

A family of dual-activity glycosyltransferasesphosphorylases mediates mannogen turnover and virulence in Leishmania parasites

Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of β-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches

Phenotypes associated with 42 hr genes.

<p>A and B show the dorsal notum of Ore-R and <i>ap>CG8213</i>. <i>apterous-Gal4</i> drives expression in the cells that form the dorsal surface of the wing but not those that form the ventral surface. It also drives expression in the dorsal thorax (notum). Panels C-H show adult flies where a 42 hr gene was knocked down using <i>ap-Gal4</i>. Ore-R is shown (C) for comparison. I and J show a thoracic macrochaete from Ore-R and from <i>ap>CG8213</i>. Panels K-N show unmounted wings from Ore-R or knocked down 42 hr genes. Note the curved kd wings.</p

Heat maps for 4 groups of related genes.

<p>A. Annotated cuticle proteins. B. Genes with a known function in cuticle deposition or maturation. C. ZP domain protein encoding genes. D. Genes that regulate transcription.</p