Selective biochemical labeling of Campylobacter jejuni cell-surface glycoconjugates.

The display of cell-surface glycolipids and glycoproteins is essential for the motility, adhesion and colonization of pathogenic bacteria such as Campylobacter jejuni. Recently, the cell-surface display of C. jejuni glycoconjugates has been the focus of considerable attention; however, our understanding of the roles that glycosylation plays in bacteria still pales in comparison with our understanding of mammalian glycosylation. One of the reasons for this is that carbohydrate metabolic labeling, a powerful tool for studying mammalian glycans, is difficult to establish in bacterial systems and has a significantly more limited scope. Herein, we report the development of an alternative strategy that can be used to study bacterial cell-surface glycoconjugates. Galactose oxidase (GalO) is used to generate an aldehyde at C-6 of terminal GalNAc residues of C. jejuni glycans. This newly generated aldehyde can be conjugated with aminooxy-functionalized purification tags or fluorophores. The label can be targeted towards specific glycoconjugates using C. jejuni mutant strains with N-glycan or lipo-oligosaccharides (LOS) assembly defects. GalO-catalyzed labeling of cell-surface glycoproteins with biotin, allowed for the purification and identification of known extracellular N-linked glycoproteins as well as a recently identified O-linked glycan modifying PorA. To expand the scope of the GalO reaction, live-cell fluorescent labeling of C. jejuni was used to compare the levels of surface-exposed LOS to the levels of N-glycosylated, cell-surface proteins. While this study focuses on the GalO-catalyzed labeling of C. jejuni, it can in principle be used to evaluate glycosylation patterns and identify glycoproteins of interest in any bacteria.

Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis.

Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro analysis of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine, which contrasts with the polyprenyl diphosphate intermediates that feature in both eukaryotes and bacteria. Notably, AglK has high sequence homology to dolichyl phosphate ß-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked dolichyl monophosphate glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide what is to our knowledge the first biochemical evidence revealing that, despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life.

Structural snapshots of the reaction coordinate for O-GlcNAc transferase.

Visualization of the reaction coordinate undertaken by glycosyltransferases has remained elusive but is critical for understanding this important class of enzyme. Using substrates and substrate mimics, we describe structural snapshots of all species along the kinetic pathway for human O-linked ß-N-acetylglucosamine transferase (O-GlcNAc transferase), an intracellular enzyme that catalyzes installation of a dynamic post-translational modification. The structures reveal key features of the mechanism and show that substrate participation is important during catalysis.

Metabolism of vertebrate amino sugars with N-glycolyl groups: intracellular ß-O-linked N-glycolylglucosamine (GlcNGc), UDP-GlcNGc, and the biochemical and structural rationale for the substrate tolerance of ß-O-linked ß-N-acetylglucosaminidase.

The O-GlcNAc modification involves the attachment of single ß-O-linked N-acetylglucosamine residues to serine and threonine residues of nucleocytoplasmic proteins. Interestingly, previous biochemical and structural studies have shown that O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from proteins, has an active site pocket that tolerates various N-acyl groups in addition to the N-acetyl group of GlcNAc. The remarkable sequence and structural conservation of residues comprising this pocket suggest functional importance. We hypothesized this pocket enables processing of metabolic variants of O-GlcNAc that could be formed due to inaccuracy within the metabolic machinery of the hexosamine biosynthetic pathway. In the accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, 28865-28881), N-glycolylglucosamine (GlcNGc) was shown to be a catabolite of NeuNGc. Here, we show that the hexosamine salvage pathway can convert GlcNGc to UDP-GlcNGc, which is then used to modify proteins with O-GlcNGc. The kinetics of incorporation and removal of O-GlcNGc in cells occur in a dynamic manner on a time frame similar to that of O-GlcNAc. Enzymatic activity of O-GlcNAcase (OGA) toward a GlcNGc glycoside reveals OGA can process glycolyl-containing substrates fairly efficiently. A bacterial homolog (BtGH84) of OGA, from a human gut symbiont, also processes O-GlcNGc substrates, and the structure of this enzyme bound to a GlcNGc-derived species reveals the molecular basis for tolerance and binding of GlcNGc. Together, these results demonstrate that analogs of GlcNAc, such as GlcNGc, are metabolically viable species and that the conserved active site pocket of OGA likely evolved to enable processing of mis-incorporated analogs of O-GlcNAc and thereby prevent their accumulation. Such plasticity in carbohydrate processing enzymes may be a general feature arising from inaccuracy in hexosamine metabolic pathways.

The development of selective inhibitors of NagZ: increased susceptibility of Gram-negative bacteria to ß-lactams.

The increasing incidence of inducible chromosomal AmpC ß-lactamases within the clinic is a growing concern because these enzymes deactivate a broad range of even the most recently developed ß-lactam antibiotics. As a result, new strategies are needed to block the action of this antibiotic resistance enzyme. Presented here is a strategy to combat the action of inducible AmpC by inhibiting the ß-glucosaminidase NagZ, which is an enzyme involved in regulating the induction of AmpC expression. A divergent route facilitating the rapid synthesis of a series of N-acyl analogues of 2-acetamido-2-deoxynojirimycin is reported here. Among these compounds are potent NagZ inhibitors that are selective against functionally related human enzymes. These compounds reduce minimum inhibitory concentration values for ß-lactams against a clinically relevant Gram-negative bacterium bearing inducible chromosomal AmpC ß-lactamase, Pseudomonas aeruginosa. The structure of a NagZ-inhibitor complex provides insight into the molecular basis for inhibition by these compounds.

Analysis of a new family of widely distributed metal-independent alpha-mannosidases provides unique insight into the processing of N-linked glycans.

The modification of N-glycans by α-mannosidases is a process that is relevant to a large number of biologically important processes, including infection by microbial pathogens and colonization by microbial symbionts. At present, the described mannosidases specific for α1,6-mannose linkages are very limited in number. Through structural and functional analysis of two sequence-related enzymes, one from Streptococcus pneumoniae (SpGH125) and one from Clostridium perfringens (CpGH125), a new glycoside hydrolase family, GH125, is identified and characterized. Analysis of SpGH125 and CpGH125 reveal them to have exo-α1,6-mannosidase activity consistent with specificity for N-linked glycans having their α1,3-mannose branches removed. The x-ray crystal structures of SpGH125 and CpGH125 obtained in apo-, inhibitor-bound, and substrate-bound forms provide both mechanistic and molecular insight into how these proteins, which adopt an (α/α)(6)-fold, recognize and hydrolyze the α1,6-mannosidic bond by an inverting, metal-independent catalytic mechanism. A phylogenetic analysis of GH125 proteins reveals this to be a relatively large and widespread family found frequently in bacterial pathogens, bacterial human gut symbionts, and a variety of fungi. Based on these studies we predict this family of enzymes will primarily comprise such exo-α1,6-mannosidases.

Molecular basis of 1,6-anhydro bond cleavage and phosphoryl transfer by Pseudomonas aeruginosa 1,6-anhydro-N-acetylmuramic acid kinase.

Anhydro-N-acetylmuramic acid kinase (AnmK) catalyzes the ATP-dependent conversion of the Gram-negative peptidoglycan (PG) recycling intermediate 1,6-anhydro-N-acetylmuramic acid (anhMurNAc) to N-acetylmuramic acid-6-phosphate (MurNAc-6-P). Here we present crystal structures of Pseudomonas aeruginosa AnmK in complex with its natural substrate, anhMurNAc, and a product of the reaction, ADP. AnmK is homodimeric, with each subunit comprised of two subdomains that are separated by a deep active site cleft, which bears similarity to the ATPase core of proteins belonging to the hexokinase-hsp70-actin superfamily of proteins. The conversion of anhMurNAc to MurNAc-6-P involves both cleavage of the 1,6-anhydro ring of anhMurNAc along with addition of a phosphoryl group to O6 of the sugar, and thus represents an unusual enzymatic mechanism involving the formal addition of H3PO4 to anhMurNAc. The structural complexes and NMR analysis of the reaction suggest that a water molecule, activated by Asp-182, attacks the anomeric carbon of anhMurNAc, aiding cleavage of the 1,6-anhydro bond and facilitating the capture of the γ phosphate of ATP by O6 via an in-line phosphoryl transfer. AnmK is active only against anhMurNAc and not the metabolically related 1,6-anhydro-N-acetylmuramyl peptides, suggesting that the cytosolic N-acetyl-anhydromuramyl-l-alanine amidase AmpD must first remove the stem peptide from these PG muropeptide catabolites before anhMurNAc can be acted upon by AnmK. Our studies provide the foundation for a mechanistic model for the dual activities of AnmK as a hydrolase and a kinase of an unusual heterocyclic monosaccharide.

Differential recognition and hydrolysis of host carbohydrate antigens by Streptococcus pneumoniae family 98 glycoside hydrolases.

The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-beta-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-beta-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

Mammalian Notch is modified by D-Xyl-alpha1-3-D-Xyl-alpha1-3-D-Glc-beta1-O-Ser: implementation of a method to study O-glucosylation.

Notch is a key cell surface protein receptor that is a vital component of intercellular signaling occurring during development. The O-glucosylation of the extracellular Notch epidermal growth factor-like (EGF) repeats has recently been found to play an important role in the proper functioning of Notch in Drosophila. Previous efforts to identify the fine structure of the O-glucose-containing glycan of mammalian Notch have been hindered by limitations associated with approaches used to date. Here, we report the development of an alternative strategy that can be used to study this modification from a range of different tissues. To implement this approach, we have generated standards of the D-Xyl-alpha1-3-D-Xyl-alpha1-3-D-Glc trisaccharide, isomers of this structure, as well as the d-Xyl-alpha1-3-d-Glc disaccharide found previously on secreted EGF-containing proteins of the blood coagulation cascade. Following derivatization with 8-aminopyrene-1,3,6-trisulfonate (APTS), we use these standards in capillary electrophoretic analyses of O-glycans released from Notch1 EGF repeats in conjunction with exo-alpha-xylosidase digestion. These studies collectively reveal that the O-glucose-containing glycan decorating mammalian Notch is the D-Xyl-alpha1-3-D-Xyl-alpha1-3-D-Glc trisaccharide; an assignment in accord with previous predictions. Given the demonstrated importance of this modification in the function of Notch in Drosophila, we expect that the identification of this glycan decorating mammalian Notch1 should aid studies into the functional role of O-glycosylation of mammalian Notch isoforms. Wider application of this approach should facilitate identification of other EGF-containing proteins bearing this O-glycan and aid in their study.

A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo.

Pathological hyperphosphorylation of the microtubule-associated protein tau is characteristic of Alzheimer's disease (AD) and the associated tauopathies. The reciprocal relationship between phosphorylation and O-GlcNAc modification of tau and reductions in O-GlcNAc levels on tau in AD brain offers motivation for the generation of potent and selective inhibitors that can effectively enhance O-GlcNAc in vertebrate brain. We describe the rational design and synthesis of such an inhibitor (thiamet-G, K(i) = 21 nM; 1) of human O-GlcNAcase. Thiamet-G decreased phosphorylation of tau in PC-12 cells at pathologically relevant sites including Thr231 and Ser396. Thiamet-G also efficiently reduced phosphorylation of tau at Thr231, Ser396 and Ser422 in both rat cortex and hippocampus, which reveals the rapid and dynamic relationship between O-GlcNAc and phosphorylation of tau in vivo. We anticipate that thiamet-G will find wide use in probing the functional role of O-GlcNAc in vertebrate brain, and it may also offer a route to blocking pathological hyperphosphorylation of tau in AD.

Active site plasticity within the glycoside hydrolase NagZ underlies a dynamic mechanism of substrate distortion.

NagZ is a glycoside hydrolase that participates in peptidoglycan (PG) recycling by removing ß-N-acetylglucosamine from PG fragments that are excised from the bacterial cell wall during growth. Notably, the products formed by NagZ, 1,6-anhydroMurNAc-peptides, activate ß-lactam resistance in many Gram-negative bacteria, making this enzyme of interest as a potential therapeutic target. Crystal structure determinations of NagZ from Salmonella typhimurium and Bacillus subtilis in complex with natural substrate, trapped as a glycosyl-enzyme intermediate, and bound to product, define the reaction coordinate of the NagZ family of enzymes. The structures, combined with kinetic studies, reveal an uncommon degree of structural plasticity within the active site of a glycoside hydrolase, and unveil how NagZ drives substrate distortion using a highly mobile loop that contains a conserved histidine that has been proposed as the general acid/base.

Analysis of PUGNAc and NAG-thiazoline as transition state analogues for human O-GlcNAcase: mechanistic and structural insights into inhibitor selectivity and transition state poise.

O-GlcNAcase catalyzes the cleavage of beta-O-linked 2-acetamido-2-deoxy-beta-d-glucopyranoside (O-GlcNAc) from serine and threonine residues of post-translationally modified proteins. Two potent inhibitors of this enzyme are O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and 1,2-dideoxy-2'-methyl-alpha-d-glucopyranoso[2,1-d]-Delta2'-thiazoline (NAG-thiazoline). Derivatives of these inhibitors differ in their selectivity for human O-GlcNAcase over the functionally related human lysosomal beta-hexosamindases, with PUGNAc derivatives showing modest selectivities and NAG-thiazoline derivatives showing high selectivities. The molecular basis for this difference in selectivities is addressed as is how well these inhibitors mimic the O-GlcNAcase-stabilized transition state (TS). Using a series of substrates, ground state (GS) inhibitors, and transition state mimics having analogous structural variations, we describe linear free energy relationships of log(KM/kcat) versus log(KI) for PUGNAc and NAG-thiazoline. These relationships suggest that PUGNAc is a poor transition state analogue, while NAG-thiazoline is revealed as a transition state mimic. Comparative X-ray crystallographic analyses of enzyme-inhibitor complexes reveal subtle molecular differences accounting for the differences in selectivities between these two inhibitors and illustrate key molecular interactions. Computational modeling of species along the reaction coordinate, as well as PUGNAc and NAG-thiazoline, provide insight into the features of NAG-thiazoline that resemble the transition state and reveal where PUGNAc fails to capture significant binding energy. These studies also point to late transition state poise for the O-GlcNAcase catalyzed reaction with significant nucleophilic participation and little involvement of the leaving group. The potency of NAG-thiazoline, its transition state mimicry, and its lack of traditional transition state-like design features suggest that potent rationally designed glycosidase inhibitors can be developed that exploit variation in transition state poise.

O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitors.

The post-translational modification of serine and threonine residues of nucleocytoplasmic proteins with 2-acetamido-2-deoxy-d-glucopyranose (GlcNAc) is a reversible process implicated in multiple cellular processes. The enzyme O-GlcNAcase catalyzes the cleavage of beta-O-linked GlcNAc (O-GlcNAc) from modified proteins and is a member of the family 84 glycoside hydrolases. The family 20 beta-hexosaminidases bear no apparent sequence similarity yet are functionally related to O-GlcNAcase because both enzymes cleave terminal GlcNAc residues from glycoconjugates. Lysosomal beta-hexosaminidase is known to use substrate-assisted catalysis involving the 2-acetamido group of the substrate; however, the catalytic mechanism of human O-GlcNAcase is unknown. By using a series of 4-methylumbelliferyl 2-deoxy-2-N-fluoroacetyl-beta-D-glucopyranoside substrates, Taft-like linear free energy analyses of these enzymes indicates that O-GlcNAcase uses a catalytic mechanism involving anchimeric assistance. Consistent with this proposal, 1,2-dideoxy-2'-methyl-alpha-D-glucopyranoso-[2,1-d]-Delta2'-thiazoline, an inhibitor that mimics the oxazoline intermediate proposed in the catalytic mechanism of family 20 glycoside hydrolases, is shown to act as a potent competitive inhibitor of both O-GlcNAcase (K(I) = 0.070 microm) and beta-hexosaminidase (K = 0.070 microm). A series of 1,2-dideoxy-2'-methyl-alpha-D-glucopyranoso-[2,1-d]-Delta2'-thiazoline analogues were prepared, and one inhibitor demonstrated a remarkable 1500-fold selectivity for O-GlcNAcase (K(I) = 0.230 microm) over beta-hexosaminidase (K(I) = 340 microm). These inhibitors are cell permeable and modulate the activity of O-GlcNAcase in tissue culture. Because both enzymes have vital roles in organismal health, these potent and selective inhibitors of O-GlcNAcase should prove useful in studying the role of this enzyme at the organismal level without generating a complex chemical phenotype stemming from concomitant inhibition of beta-hexosaminidase.