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.     

The histone acetyltransferase NCOAT contains a zinc finger-like motif involved in substrate recognition

Nuclear cytoplasmic O-GlcNAcase and acetyltransferase (NCOAT) is a bifunctional enzyme with both glycoside hydrolase and alkyltransferase activity. Its O-GlcNAcase active site lies in the N terminus of the enzyme and its histone acetyltransferase (HAT) domain lies in the C terminus. Whereas the HAT domain of the enzyme is catalytically and structurally similar to other acetyltransferases across subfamilies, NCOAT has a motif resembling a zinc finger-like domain unique to the MYST family of HATs. Among the MYST family, this zinc finger, or zinc finger-like domain, is responsible for making contacts with the histone tails within nucleosomes for the HAT to catalyze its respective reaction. Here, we show that NCOAT has the ability to directly associate with both an acetylated and unacetylated histone H4 tail in vitro, and a potential zinc finger-like motif found in NCOAT is implicated in this nucleosomal contact, and is necessary for fully efficient enzymatic activity. Subsequent to the catalysis of acetyltransfer to lysine 8 of histone H4 for the enzyme, however, the substrate is released and NCOAT can no longer bind H4 in our assays. Furthermore, this finger domain by itself is sufficient to bind histone H4.     

Processing enzyme glucosidase II: proposed catalytic residues and developmental regulation during the ontogeny of the mouse mammary gland

Following the action of glucosidase I to clip the terminal alpha1,2-linked glucose, glucosidase II sequentially cleaves the two inner alpha1,3-linked glucose residues from the Glcalpha1,2Glcalpha1,3Glcalpha1,3Man(9)GlcNAc(2) oligosaccharide of the incipient glycoprotein as it undergoes folding and maturation. Glucosidase II belongs to family 31 glycosidases. These enzymes act by the acid-base catalytic mechanism. The cDNA of the wild-type and several mutant forms of the fusion protein of the enzyme in which mutations were introduced in the conserved motif D(564)MNE(567) were expressed in Sf9 cells, and the proteins were purified on Ni-NTA matrix. The catalytic activity of the purified proteins was determined with radioactive Glc(2)Man(9)GlcNAc(2) substrate. The results show that the aspartate and glutamate within the D(564)MNE(567) motif can serve for catalysis, most likely as the acid-base pair within the active site of the enzyme. The developmental regulation of glucosidase II was studied during the ontogeny of the mouse mammary gland for its growth and differentiation. The mRNA of both alpha and beta subunits of the enzyme, immunoreactive alpha and beta subunits, and enzyme activity were measured over the complete developmental cycle. The changes in all the parameters were consistent with similar fluctuations with several other enzymes of the N-glycosylation machinery reported earlier, reaching a three- to fourfold increase over the basal level in the virgin gland at the peak of lactation. Altogether it appears that there is a coordinated regulation of the enzymes involved in protein N-glycosylation during the development of the mouse mammary gland.     

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: INTRACELLULAR β-O-LINKED N-GLYCOLYLGLUCOSAMINE (GlcNGc), UDP-GlcNGc, AND THE BIOCHEMICAL AND STRUCTURAL RATIONALE FOR THE SUBSTRATE TOLERANCE OF β-O-LINKED β-N-ACETYLGLUCOSAMINIDASE

The O-GlcNAc modification involves the attachment of single β-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.     

Streptozotocin inhibits O-GlcNAcase via the production of a transition state analog

Streptozotocin (STZ) is a 2-deoxy-d-glucopyranose derivative of a class of drugs known as alkylnitrosoureas, and is an established diabetogenic agent whose cytotoxic affects on pancreatic beta-cells has been partially explained by the presence of its N-methyl-N-nitrosourea side chain, which has the ability to release nitric oxide as well as donate methyl groups to nucleotides in DNA. It has also been observed that STZ administration results in a rise in the level of O-GlcNAcylated proteins within beta-cells. Not coincidentally, STZ has also been shown to directly inhibit the O-GlcNAcase activity of the enzyme NCOAT in vitro, which is the only enzyme that possesses the ability to remove O-GlcNAc modifications on proteins in the nucleus and cytosol. Since O-GlcNAc modification plays a role on a number of proteins in a vast amount of cellular processes, this shift in whole-cell protein O-GlcNAcylation state affords another source of cell death. We set about to find the exact mechanism by which STZ inhibits O-GlcNAcase activity. Inhibition is achievable because the GlcNAc analog STZ targets the active site of the enzyme whereby it is catalyzed. During this process, the enzyme converts STZ to a compound that closely resembles the natural ligand transition state, but is distinctly more stable energetically. As a result, this analog is catalyzed to completion at a much slower rate, thereby out-competing GlcNAc substrate for the active site, and inhibiting the enzyme.     

Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants

O-GlcNAcase is a family 84 beta-N-acetylglucosaminidase catalyzing the hydrolytic cleavage of beta-O-linked 2-acetamido-2-deoxy-d-glycopyranose (O-GlcNAc) from serine and threonine residues of posttranslationally modified proteins. O-GlcNAcases use a double-displacement mechanism involving formation and breakdown of a transient bicyclic oxazoline intermediate. The key catalytic residues of any family 84 enzyme facilitating this reaction, however, are unknown. Two mutants of human O-GlcNAcase, D174A and D175A, were generated since these residues are highly conserved among family 84 glycoside hydrolases. Structure-reactivity studies of the D174A mutant enzyme reveals severely impaired catalytic activity across a broad range of substrates alongside a pH-activity profile consistent with deletion of a key catalytic residue. The D175A mutant enzyme shows a significant decrease in catalytic efficiency with substrates bearing poor leaving groups (up to 3000-fold), while for substates bearing good leading groups the difference is much smaller (7-fold). This mutant enzyme also cleaves thioglycosides with essentially the same catalytic efficiency as the wild-type enzyme. As well, addition of azide as an exogenous nucleophile increases the activity of this enzyme toward a substrate bearing an excellent leaving group. Together, these results allow unambiguous assignment of Asp(174) as the residue that polarizes the 2-acetamido group for attack on the anomeric center and Asp(175) as the residue that functions as the general acid/base catalyst. Therefore, the family 84 glycoside hydrolases use a DD catalytic pair to effect catalysis.     

Invariant Asp-1122 and Asp-1124 are essential residues for polymerization catalysis of family D DNA polymerase from Pyrococcus horikoshii

Family D DNA polymerase has recently been found in the Euryarchaeota subdomain of Archaea. Its genes are adjacent to several other genes related to DNA replication, repair, and recombination in the genome, suggesting that this enzyme may be the major DNA replicase in Euryarchaeota. Although it possesses strong polymerization and proofreading activities, the motifs common to other DNA polymerase families are absent in its sequences. Here we report the mapping of the catalytic residues in a family D DNA polymerase from Pyrococcus horikoshii. Site-directed alanine mutants for 28 conserved aspartic acid or glutamic acid residues were screened for polymerization and 3'-5' exonuclease activities. We identified the invariant aspartates Asp-1122 and Asp-1124 within the most conserved motif as the catalytic residues involved in DNA polymerization. Alanine mutation at either site caused a loss of polymerization activity, whereas the conserved mutants, D1122E, D1124N, and D1124E, had slightly reduced polymerization activity. We also found that the 3'-5' exonuclease activity remains in D1122A and D1124A, indicating that the catalytic residues of DNA polymerization are different from those of the 3'-5' exonuclease activity. Furthermore we determined the molecular mass of the recombinant enzyme by gel filtration and proposed a heterotetrameric structure for this enzyme.     

Identification of aspartic acid and histidine residues mediating the reaction mechanism and the substrate specificity of the human UDP-glucuronosyltransferases 1A

The human UDP-glucuronosyltransferase UGT1A6 is the primary phenol-metabolizing UDP-glucuronosyltransferase isoform. It catalyzes the nucleophilic attack of phenolic xenobiotics on UDP-glucuronic acid, leading to the formation of water-soluble glucuronides. The catalytic mechanism proposed for this reaction is an acid-base mechanism that involves an aspartic/glutamic acid and/or histidine residue. Here, we investigated the role of 14 highly conserved aspartic/glutamic acid residues over the entire sequence of human UGT1A6 by site-directed mutagenesis. We showed that except for aspartic residues Asp-150 and Asp-488, the substitution of carboxylic residues by alanine led to active mutants but with decreased enzyme activity and lower affinity for acceptor and/or donor substrate. Further analysis including mutation of the corresponding residue in other UGT1A isoforms suggests that Asp-150 plays a major catalytic role. In this report we also identified a single active site residue important for glucuronidation of phenols and carboxylic acid substrates by UGT1A enzyme family. Replacing Pro-40 of UGT1A4 by histidine expanded the glucuronidation activity of the enzyme to phenolic and carboxylic compounds, therefore, leading to UGT1A3-type isoform in terms of substrate specificity. Conversely, when His-40 residue of UGT1A3 was replaced with proline, the substrate specificity shifted toward that of UGT1A4 with loss of glucuronidation of phenolic substrates. Furthermore, mutation of His-39 residue of UGT1A1 (His-40 in UGT1A4) to proline led to loss of glucuronidation of phenols but not of estrogens. This study provides a step forward to better understand the glucuronidation mechanism and substrate recognition, which is invaluable for a better prediction of drug metabolism and toxicity in human.     

Importance of glycosidases in mammalian glycoprotein biosynthesis

Processing glycosidases play an important role in N-glycan biosynthesis in mammalian cells by trimming Glc(3)Man(9)GlcNAc(2) and thus providing the substrates for the formation of complex and hybrid structures by Golgi glycosyltransferases. Processing glycosidases also play a role in the folding of newly formed glycoproteins and in endoplasmic reticulum quality control. The properties and molecular nature of mammalian processing glycosidases are described in this review. Membrane-bound alpha-glucosidase I and soluble alpha-glucosidase II of the endoplasmic reticulum remove the alpha1,2-glucose and alpha1,3-glucose residues, respectively, beginning immediately following transfer of Glc(3)Man(9)GlcNAc(2) to nascent polypeptides. The alpha-glucosidases participate in glycoprotein folding mediated by calnexin and calreticulin by forming the monoglucosylated high mannose oligosaccharides required for the interaction with the chaperones. In some mammalian cells, Golgi endo alpha-mannosidase provides an alternative pathway for removal of glucose residues. Removal of alpha1,2-linked mannose residues begins in the endoplasmic reticulum where trimming of mannose residues in the endoplasmic reticulum has been implicated in the targeting of malfolded glycoproteins for degradation. Removal of mannose residues continues in the Golgi with the action of alpha1, 2-mannosidases IA and IB that can form Man(5)GlcNAc(2) and of alpha-mannosidase II that removes the alpha1,3- and alpha1,6-linked mannose from GlcNAcMan(5)GlcNAc(2) to form GlcNAcMan(3)GlcNAc(2). These membrane-bound Golgi enzymes have been cloned and shown to have very distinct patterns of tissue-specific expression. There are also broad specificity alpha-mannosidases that can trim Man(4-9)GlcNAc(2) to Man(3)GlcNAc(2), and provide an alternative pathway toward complex oligosaccharide formation. Cloning of the remaining alpha-mannosidases will be required to evaluate their specific functions in glycoprotein maturation.     

Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase.

Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.     

Crystal structure of beta1,4-galactosyltransferase complex with UDP-Gal reveals an oligosaccharide acceptor binding site

The crystal structure of the catalytic domain of bovine beta1,4-galactosyltransferase (Gal-T1) co-crystallized with UDP-Gal and MnCl(2) has been solved at 2.8 A resolution. The structure not only identifies galactose, the donor sugar binding site in Gal-T1, but also reveals an oligosaccharide acceptor binding site. The galactose moiety of UDP-Gal is found deep inside the catalytic pocket, interacting with Asp252, Gly292, Gly315, Glu317 and Asp318 residues. Compared to the native crystal structure reported earlier, the present UDP-Gal bound structure exhibits a large conformational change in residues 345-365 and a change in the side-chain orientation of Trp314. Thus, the binding of UDP-Gal induces a conformational change in Gal-T1, which not only creates the acceptor binding pocket for N-acetylglucosamine (GlcNAc) but also establishes the binding site for an extended sugar acceptor. The presence of a binding site that accommodates an extended sugar offers an explanation for the observation that an oligosaccharide with GlcNAc at the non-reducing end serves as a better acceptor than the monosaccharide, GlcNAc. Modeling studies using oligosaccharide acceptors indicate that a pentasaccharide, such as N-glycans with GlcNAc at their non-reducing ends, fits the site best. A sequence comparison of the human Gal-T family members indicates that although the binding site for the GlcNAc residue is highly conserved, the site that binds the extended sugar exhibits large variations. This is an indication that different Gal-T family members prefer different types of glycan acceptors with GlcNAc at their non-reducing ends.     

Identification and expressional analysis of two cathepsins from half-smooth tongue sole (Cynoglossus semilaevis)

Cathepsins are a family of lysosomal proteases that play an important role in protein degradation, antigen presentation, apoptosis, and inflammation. Cathepsins are divided into three groups, i.e., cysteine protease, serine protease, and aspartic protease. Cathepsin D and cathepsin L, which are aspartic protease and cysteine protease respectively, have been identified in a number of teleosts; however, the immunological relevance of fish cathepsins is largely unknown. In this study, we cloned and analyzed the expression profiles of a cathepsin D (CsCatD) and a cathepsin L (CsCatL) homologs from half-smooth tongue sole (Cynoglossus semilaevis). CsCatD is composed of 396 amino acid residues and shares 67.6-88.4% overall sequence identities with fish and human cathepsin D. Structurally CsCatD possesses an aspartic endopeptidase domain, which contains two conserved aspartic acid residues that form the catalytic site. CsCatL is 336 residues in length and shares 64.7-90.2% overall sequence identities with fish and human cathepsin L. CsCatL has an N-terminal cathepsin propeptide inhibitor domain followed by a Papain family cysteine protease domain, the latter containing four conserved catalytic residues: Gln-133, Cys-139, His-279, and Asn-303. Recombinant CsCatL purified from Escherichia coli exhibited apparent protease activity. Quantitative real time RT-PCR analysis detected constitutive expression of CsCatD and CsCatL in multiple tissues, with the lowest level found in heart and the highest level found in liver. Experimental challenge of tongue sole with the bacterial pathogen Vibrio anguillarum and megalocytivirus caused significant inductions of both CsCatD and CsCatL expression in kidney and spleen in time-dependent manners. Immunization of the fish with a subunit vaccine also enhanced CsCatD and CsCatL expression in the first week post-vaccination. These results suggest involvement of CsCatD and CsCatL in host immune reactions to bacterial and viral infections and in the process of antigen-induced immune response.     

Structure and mechanism of a bacterial beta-glucosaminidase having O-GlcNAcase activity

O-GlcNAc is an abundant post-translational modification of serine and threonine residues of nucleocytoplasmic proteins. This modification, found only within higher eukaryotes, is a dynamic modification that is often reciprocal to phosphorylation. In a manner analogous to phosphatases, a glycoside hydrolase termed O-GlcNAcase cleaves O-GlcNAc from modified proteins. Enzymes with high sequence similarity to human O-GlcNAcase are also found in human pathogens and symbionts. We report the three-dimensional structure of O-GlcNAcase from the human gut symbiont Bacteroides thetaiotaomicron both in its native form and in complex with a mimic of the reaction intermediate. Mutagenesis and kinetics studies show that the bacterial enzyme, very similarly to its human counterpart, operates via an unusual 'substrate-assisted' catalytic mechanism, which will inform the rational design of enzyme inhibitors.     

Chlorophyllase as a serine hydrolase: identification of a putative catalytic triad

Chlorophyllases (Chlases), cloned so far, contain a lipase motif with the active serine residue of the catalytic triad of triglyceride lipases. Inhibitors specific for the catalytic serine residue in serine hydrolases, which include lipases effectively inhibited the activity of the recombinant Chenopodium album Chlase (CaCLH). From this evidence we assumed that the catalytic mechanism of hydrolysis by Chlase might be similar to those of serine hydrolases that have a catalytic triad composed of serine, histidine and aspartic acid in their active site. Thus, we introduced mutations into the putative catalytic residue (Ser162) and conserved amino acid residues (histidine, aspartic acid and cysteine) to generate recombinant CaCLH mutants. The three amino acid residues (Ser162, Asp191 and His262) essential for Chlase activity were identified. These results indicate that Chlase is a serine hydrolase and, by analogy with a plausible catalytic mechanism of serine hydrolases, we proposed a mechanism for hydrolysis catalyzed by Chlase.     

Effects of Mutations in Residues near the Active Site of Human Immunodeficiency Virus Type 1 Integrase on Specific Enzyme-Substrate Interactions

The phylogenetically conserved catalytic core domain of human immunodeficiency virus type 1 (HIV-1) integrase contains elements necessary for specific recognition of viral and target DNA features. In order to identify specific amino acids that determine substrate specificity, we mutagenized phylogenetically conserved residues that were located in close proximity to the active-site residues in the crystal structure of the isolated catalytic core domain of HIV-1 integrase. Residues composing the phylogenetically conserved DD(35)E active-site motif were also mutagenized. Purified mutant proteins were evaluated for their ability to recognize the phylogenetically conserved CA/TG base pairs near the viral DNA ends and the unpaired dinucleotide at the 5′ end of the viral DNA, using disintegration substrates. Our findings suggest that specificity for the conserved A/T base pair depends on the active-site residue E152. The phenotype of IN(Q148L) suggested that Q148 may be involved in interactions with the 5′ dinucleotide of the viral DNA end. The activities of some of the proteins with mutations in residues in close proximity to the active-site aspartic and glutamic acids were salt sensitive, suggesting that these mutations disrupted interactions with DNA.     

Prediction of structure and functional residues for O-GlcNAcase,a divergent homologue of acetyltransferases

N-Acetyl-beta-D-glucosaminidase (O-GlcNAcase) is a key enzyme in the posttranslational modification of intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc). Here, we show that this protein contains two catalytic domains, one homologous to bacterial hyaluronidases and one belonging to the GCN5-related family of acetyltransferases (GNATs). Using sequence and structural information, we predict that the GNAT homologous region contains the O-GlcNAcase activity. Thus, O-GlcNAcase is the first member of the GNAT family not involved in transfer of acetyl groups, adding a new mode of evolution to this large protein family. Comparison with solved structures of different GNATs led to a reliable structure prediction and mapping of residues involved in binding of the GlcNAc-modified proteins and catalysis.     

Crystal structure of aspartic proteinase from Irpex lacteus in complex with inhibitor pepstatin

The crystal structure of Irpex lacteus aspartic proteinase (ILAP) in complex with pepstatin (a six amino acid residue peptide-like inhibitor) was determined at 1.3A resolution. ILAP is a pepsin-like enzyme, widely distributed in nature, with high milk-clotting activity relative to proteolytic activity. The overall structure was in good topological agreement with pepsin and other aspartic proteases. The structure and interaction pattern around the catalytic site were conserved, in agreement with the other aspartic proteinase/inhibitor complex structures reported previously. The high-resolution data also supported the transition state model, as proposed previously for the catalytic mechanism of aspartic proteinase. Unlike the other aspartic proteinases, ILAP was found to require hydrophobic residues either in the P(1) or P(1') site, and also in the P(4) and/or P(3) site(s) for secondary interactions. The inhibitor complex structure also revealed the substrate binding mechanism of ILAP at the P(3) and P(4) site of the substrate, where the inserted loop built up the unique hydrophobic pocket at the P(4) site.