Conserved residues Arg188 and Asp302 are critical for active site organization and catalysis in human ABO(H) blood group A and B glycosyltransferases.

Homologous glycosyltransferases GTA and GTB perform the final step in human ABO(H) blood group A and B antigen synthesis by transferring the sugar moiety from donor UDP-GalNAc/UDP-Gal to the terminal H antigen disaccharide acceptor. Like other GT-A fold family 6 glycosyltransferases, GTA and GTB undergo major conformational changes in two mobile regions, the C-terminal tail and internal loop, to achieve the closed, catalytic state. These changes are known to establish a salt bridge network among conserved active site residues Arg188, Asp211 and Asp302, which move to accommodate a series of discrete donor conformations while promoting loop ordering and formation of the closed enzyme state. However, the individual significance of these residues in linking these processes remains unclear. Here, we report the kinetics and high-resolution structures of GTA/GTB mutants of residues 188 and 302. The structural data support a conserved salt bridge network critical to mobile polypeptide loop organization and stabilization of the catalytically competent donor conformation. Consistent with the X-ray crystal structures, the kinetic data suggest that disruption of this salt bridge network has a destabilizing effect on the transition state, emphasizing the importance of Arg188 and Asp302 in the glycosyltransfer reaction mechanism. The salt bridge network observed in GTA/GTB structures during substrate binding appears to be conserved not only among other Carbohydrate Active EnZyme family 6 glycosyltransferases but also within both retaining and inverting GT-A fold glycosyltransferases. Our findings augment recently published crystal structures, which have identified a correlation between donor substrate conformational changes and mobile loop ordering.

Molecular Imaging of Hydrolytic Enzymes Using PET and SPECT.

Hydrolytic enzymes are a large class of biological catalysts that play a vital role in a plethora of critical biochemical processes required to maintain human health. However, the expression and/or activity of these important enzymes can change in many different diseases and therefore represent exciting targets for the development of positron emission tomography (PET) and single-photon emission computed tomography (SPECT) radiotracers. This review focuses on recently reported radiolabeled substrates, reversible inhibitors, and irreversible inhibitors investigated as PET and SPECT tracers for imaging hydrolytic enzymes. By learning from the most successful examples of tracer development for hydrolytic enzymes, it appears that an early focus on careful enzyme kinetics and cell-based studies are key factors for identifying potentially useful new molecular imaging agents.

Imaging of enzyme replacement therapy using PET.

Direct enzyme replacement therapy (ERT) has been introduced as a means to treat a number of rare, complex genetic conditions associated with lysosomal dysfunction. Gaucher disease was the first for which this therapy was applied and remains the prototypical example. Although ERT using recombinant lysosomal enzymes has been shown to be effective in altering the clinical course of Gaucher disease, Fabry disease, Hurler syndrome, Hunter syndrome, Maroteaux-Lamy syndrome, and Pompe disease, the recalcitrance of certain disease manifestations underscores important unanswered questions related to dosing regimes, tissue half-life of the recombinant enzyme and the ability of intravenously administered enzyme to reach critical sites of known disease pathology. We have developed an innovative method for tagging acid beta-glucocerebrosidase (GCase), the recombinant enzyme formulated in Cerezyme(R) used to treat Gaucher disease, using an (18)F-labeled substrate analogue that becomes trapped within the active site of the enzyme. Using micro-PET we show that the tissue distribution of injected enzyme can be imaged in a murine model and that the PET data correlate with tissue (18)F counts. Further we show that PET imaging readily monitors pharmacokinetic changes effected by receptor blocking. The ability to (18)F-label GCase to monitor the enzyme distribution and tissue half-life in vivo by PET provides a powerful research tool with an immediate clinical application to Gaucher disease and a clear path for application to other ERTs.

OGA inhibition by GlcNAc-selenazoline.

The title compound, which differs from the powerful O-GlcNAcase (OGA) inhibitor GlcNAc-thiazoline only at the chalcogen atom (Se for S), is a much weaker inhibitor in a direct OGA assay. In human cells, however, the selenazoline shows comparable ability to induce hyper-O-GlcNAc-ylation, and the two show similar reduction of insulin-stimulated translocation of glucose transporter 4 in differentiated 3T3 adipocytes.

Directed "in situ" inhibitor elongation as a strategy to structurally characterize the covalent glycosyl-enzyme intermediate of human pancreatic alpha-amylase.

While covalent catalytic intermediates of retaining alpha-transglycosylases have been structurally characterized previously, no such information for a hydrolytic alpha-amylase has been obtained. This study presents a new "in situ" enzymatic elongation methodology that, for the first time, has allowed the isolation and structural characterization of a catalytically competent covalent glycosyl-enzyme intermediate with human pancreatic alpha-amylase. This has been achieved by the use of a 5-fluoro-beta-l-idosyl fluoride "warhead" in conjunction with either alpha-maltotriosyl fluoride or 4'-O-methyl-alpha-maltosyl fluoride as elongation agents. This generates an oligosaccharyl-5-fluoroglycosyl fluoride that then reacts with the free enzyme. The resultant covalent intermediates are extremely stable, with hydrolytic half-lives on the order of 240 h for the trisaccharide complex. In the presence of maltose, however, they undergo turnover via transglycosylation according to a half-life of less than 1 h. Structural studies of intermediate complexes unambiguously show the covalent attachment of a 5-fluoro-alpha-l-idosyl moiety in the chair conformation to the side chain of the catalytic nucleophile D197. The elongated portions of the intermediate complexes are found to bind in the high-affinity -2 and -3 binding subsites, forming extensive hydrogen-bonding interactions. Comparative structural analyses with the related noncovalent complex formed by acarbose highlight the structural rigidity of the enzyme surface during catalysis and the key role that substrate conformational flexibility must play in this process. Taken together, the structural data provide atomic details of several key catalytic steps. The scope of this elongation approach to probe the active sites and catalytic mechanisms of alpha-amylases is further demonstrated through preliminary experiments with porcine pancreatic alpha-amylase.

Covalent inhibitors of glycosidases and their applications in biochemistry and biology.

Glycoside hydrolases are important enzymes in a number of essential biological processes. Irreversible inhibitors of this class of enzyme have attracted interest as probes of both structure and function. In this review we discuss some of the compounds used to covalently modify glycosidases, their use in residue identification, structural and mechanistic investigations, and finally their applications, both in vitro and in vivo, to complex biological systems.

N-Acetylhexosaminidase inhibitory properties of C-1 homologated GlcNAc- and GalNAc-thiazolines.

Several C-1 homologated GlcNAc- and GalNAc-thiazolines, as well as a related GalNAc-thiazole, have been prepared. The compounds are analogues of GlcNAc-thiazoline, a potent transition-state-mimicking inhibitor of retaining beta-N-acetylglycosaminidases. Kinetic evaluation of these fused pyranose-heterocycles against the bacterial N-acetylhexosaminidase SpHex suggests active site steric restrictions around the substrate anomeric carbon.

A homology model for human alpha-l-iduronidase: insights into human disease.

Genotype-phenotype correlations in genetic diseases for which missense mutations lead to disease remain a challenge. This is particularly true for diseases caused by alterations of proteins for which no three-dimensional structure is available. One such disease is Mucopolysaccharidosis type I, a disorder arising from a lack of activity of the lysosomal enzyme alpha-l-iduronidase (IDUA, EC 3.2.1.76). This deficiency compromises the degradation pathway of glycosaminoglycans such as heparan sulfate and dermatan sulfate, leading to substrate accumulation, which ultimately results in a multisystem disorder. Patients with IDUA deficiency have a wide spectrum of disease ranging from an early onset, rapidly progressive form leading to death in the first decade of life, to an attenuated disease which manifests in adolescence and leads to progressive joint and cardiac disease but is associated with a normal life span. Many patients fit into a disease phenotype intermediate to these extremes. While a number of point mutations have been described as leading to varying degrees of disease severity, a structural basis for these genotype-phenotype correlations has not been available owing to the lack of a three-dimensional structure for IDUA. A homology model for the IDUA enzyme was constructed based on the recently solved crystal structure of the beta-xylosidase from Thermoanaerobacterium saccharolyticum (XyTS, EC 3.2.1.37), both of which belong to the same sequence-related family (CAZY family 39). This model provides insights into why certain point mutations produce severely misfolded proteins and thus lead to severe disease, and why other mutations produce proteins with only minor structural perturbations and therefore the attenuated form of the disease.

Synthesis and kinetic analysis of the N-acetylhexosaminidase inhibitor XylNAc-isofagomine.

[reaction: see text] An efficient 10-step preparation from 4-methoxypyridine of (2R,3R,4R)-2-acetamido-3,4-dihydroxypiperidine ("XylNAc-isofagomine") in optically active form is described. Key steps include an enantioselective reduction with catecholborane/(S)-2-methyl-CBS-oxazaborolidine, and a stereoselective pseudo-glycosylation of lithium azide by a cyclic sulfite ester. The title compound showed a Ki = 21 microM when evaluated against the N-acetyl-beta-hexosaminidase from Streptomyces plicatus.

Characterization of the recognition of blood group B trisaccharide derivatives by the lectin from Marasmius oreades using frontal affinity chromatography-mass spectrometry.

A novel lectin from the mushroom Marasmius oreades (MOA) has been shown to bind to human blood group B oligosaccharides [1]. In the present work we examine the binding of a series of analogues of the blood group B-trisaccharide, alphaGal(1-3)[alphaFuc(1-2)]betaGal-OR (1, R = (CH2)8COOMe). MOA was biotinylated and immobilized on a micro column (9.8 microL) for evaluation by Frontal Affinity Chromatography-Mass Spectrometry (FAC-MS) [2]. The trisaccharide 1 was found to be the epitope needed for maximum recognition (Kd = 3.6 microM). A series of synthetic deoxygenated and O-methylated analogues of the B-trisaccharide (R = OMe) were then screened against the lectin, and the key structural elements for binding were determined. OH-4 of the beta-Gal residue and OH-2 of the alpha-Gal residue were found to be critical for recognition. The FAC-MS technique also proved powerful in evaluating mixtures of compounds. Since the solution NMR structure and crystal structure of the B-trisaccharide are known [3], we propose the specific surface of the trisaccharide that is recognized by the lectin.