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.

Host Cell Factor-1 (HCF-1), a transcriptional co-regulator of human cell-cycle progression, undergoes proteolytic maturation in which any of six repeated sequences is cleaved by the nutrient-responsive glycosyltransferase, O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). We report that the tetratricopeptide-repeat domain of O-GlcNAc transferase binds the C-terminal portion of an HCF-1 proteolytic repeat such that the cleavage region lies in the glycosyltransferase active site above UDP-GlcNAc. The conformation is similar to that of a glycosylation-competent peptide substrate. Cleavage occurs between cysteine and glutamate residues and results in a pyroglutamate product. Conversion of the cleavage site glutamate into serine converts an HCF-1 proteolytic repeat into a glycosylation substrate. Thus, protein glycosylation and HCF-1 cleavage occur in the same active site.

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.

Photothermal release of DNA from gold nanoparticles either by thermolysis of the Au-S bonds used to anchor the oligonucleotides to the nanoparticle or by thermal denaturation has great therapeutic potential, however, both processes have limitations (a decreased particle stability for the former process and a prohibitively slow rate of release for the latter). Here we show that these two mechanisms are not mutually exclusive and can be controlled by adjusting laser power and ionic strength. We show this using two different double-stranded (ds)DNA-nanoparticle conjugates, in which either the anchored sense strand or the complementary antisense strand was labeled with a fluorescent marker. The amounts of release due to the two mechanisms were evaluated using fluorescence spectroscopy and capillary electrophoresis, which showed that irradiation of the decorated particles in 200 mM NaOAc containing 10 mM Mg(OAc)(2) with a pulsed 532 nm laser operating at 100 mW favors denaturation over Au-S cleavage to an extent of more than six-to-one. Due to the use of a pulsed laser, the process occurs on the order of minutes rather than hours, which is typical for continuous wave lasers. These findings encourage continued research toward developing photothermal gene therapeutics.

The retaining glycosyltransferase GalNAc-T2 is a member of a large family of human polypeptide GalNAc-transferases that is responsible for the post-translational modification of many cell-surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum-mechanics/molecular-mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic-electronic level of detail. Our study provides a detailed structural rationale for an ordered bi-bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front-face SN i-type reaction in which the substrate N-acetyl sugar substituent coordinates efficient glycosyl transfer.