Glycosyltransferases (GTs) are ubiquitous enzymes that catalyze the assembly of glycoconjugates found throughout all kingdoms of nature. A longstanding problem is the rational design of probes that can be used to manipulate GT activity in cells and tissues. Here we describe the rational design and synthesis of a nucleotide sugar analogue that inhibits, with high potency both in vitro and in cells, the human GT responsible for the reversible post-translational modification of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc). We show the enzymes of the hexosamine biosynthetic pathway can transform, both in vitro and in cells, a synthetic carbohydrate precursor into the nucleotide sugar analogue. Treatment of cells with the precursor decreases O-GlcNAc in a targeted manner with a single digit micromolar EC50. This approach to inhibition of GTs should be applicable to other members of this increasingly interesting superfamily of enzymes and enable their manipulation in a biological setting.

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-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.

The design and synthesis of transition-state mimics reflects the growing need both to understand enzymatic catalysis and to influence strategies for therapeutic intervention. Iminosugars are among the most potent inhibitors of glycosidases. Here, the binding of 1-deoxynojirimycin and (+)-isofagomine to the "family GH-1" beta-glucosidase of Thermotoga maritima is investigated by kinetic analysis, isothermal titration calorimetry, and X-ray crystallography. The binding of both of these iminosugar inhibitors is driven by a large and favorable enthalpy. The greater inhibitory power of isofagomine, relative to 1-deoxynojirimycin, however, resides in its significantly more favorable entropy; indeed the differing thermodynamic signatures of these inhibitors are further highlighted by the markedly different heat capacity values for binding. The pH dependence of catalysis and of inhibition suggests that the inhibitory species are protonated inhibitors bound to enzymes whose acid/base and nucleophile are ionized, while calorimetry indicates that one proton is released from the enzyme upon binding at the pH optimum of catalysis (pH 5.8). Given that these results contradict earlier proposals that the binding of racemic isofagomine to sweet almond beta-glucosidase was entropically driven (Bülow, A. et al. J. Am. Chem. Soc. 2000, 122, 8567-8568), we reinvestigated the binding of 1-deoxynojirimycin and isofagomine to the sweet almond enzyme. Calorimetry confirms that the binding of isofagomine to sweet almond beta-glucosidases is, as observed for the T. maritima enzyme, driven by a large favorable enthalpy. The crystallographic structures of the native T. maritima beta-glucosidase, and its complexes with isofagomine and 1-deoxynojirimycin, all at approximately 2.1 A resolution, reveal that additional ordering of bound solvent may present an entropic penalty to 1-deoxynojirimycin binding that does not penalize isofagomine.

No abstract