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1.  A Structural and Biochemical Model of Processive Chitin Synthesis* 
The Journal of Biological Chemistry  2014;289(33):23020-23028.
Background: Chitin synthesis is an attractive drug target in a range of organisms but is not understood at the molecular level.
Results: The chitooligosaccharide synthase NodC can be assayed with a novel HTS assay, and the mechanism/fold can be probed by site-directed mutagenesis and topology mapping.
Conclusion: NodC is a model system to probe chitin synthesis.
Significance: This work enables the exploitation of chitin synthesis as a drug target.
Chitin synthases (CHS) produce chitin, an essential component of the fungal cell wall. The molecular mechanism of processive chitin synthesis is not understood, limiting the discovery of new inhibitors of this enzyme class. We identified the bacterial glycosyltransferase NodC as an appropriate model system to study the general structure and reaction mechanism of CHS. A high throughput screening-compatible novel assay demonstrates that a known inhibitor of fungal CHS also inhibit NodC. A structural model of NodC, on the basis of the recently published BcsA cellulose synthase structure, enabled probing of the catalytic mechanism by mutagenesis, demonstrating the essential roles of the DD and QXXRW catalytic motifs. The NodC membrane topology was mapped, validating the structural model. Together, these approaches give insight into the CHS structure and mechanism and provide a platform for the discovery of inhibitors for this antifungal target.
doi:10.1074/jbc.M114.563353
PMCID: PMC4132801  PMID: 24942743
Carbohydrate Biosynthesis; Enzyme Inhibitor; Enzyme Mechanism; Glycosyltransferase; Protein Structure
2.  Bisubstrate UDP–peptide conjugates as human O-GlcNAc transferase inhibitors 
Biochemical Journal  2014;457(Pt 3):497-502.
Inhibitors of OGT (O-GlcNAc transferase) are valuable tools to study the cell biology of protein O-GlcNAcylation. We report OGT bisubstrate-linked inhibitors (goblins) in which the acceptor serine in the peptide VTPVSTA is covalently linked to UDP, eliminating the GlcNAc pyranoside ring. Goblin1 co-crystallizes with OGT, revealing an ordered C3 linker and retained substrate-binding modes, and binds the enzyme with micromolar affinity, inhibiting glycosyltransfer on to protein and peptide substrates.
Inhibitors of OGT (O-GlcNAc transferase) are valuable tools to study the cell biology of protein O-GlcNAcylation. We report OGT bisubstrate-linked inhibitors (goblins) in which the acceptor serine in the peptide VTPVSTA is covalently linked to UDP, inhibiting glycosyltransfer on to protein and peptide substrates.
doi:10.1042/BJ20131272
PMCID: PMC3927924  PMID: 24256146
bisubstrate analogue inhibitor; glycosyltransferase; O-GlcNAc; rational drug design; DIPEA, N,N-di-isopropylethylamine; DMF, dimethylformamide; goblin, OGT bisubstrate-linked inhibitor; h, human; HRMS, high-resolution MS; MP, p-methoxyphenyl; OGA, O-GlcNAc hydrolase; OGT, O-GlcNAc:polypeptidyl transferase; TAB1, TGF (transforming growth factor)-β-activated kinase-binding protein 1
3.  Structure of a bacterial putative acetyltransferase defines the fold of the human O-GlcNAcase C-terminal domain 
Open Biology  2013;3(10):130021.
The dynamic modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an essential posttranslational modification present in higher eukaryotes. Removal of O-GlcNAc is catalysed by O-GlcNAcase, a multi-domain enzyme that has been reported to be bifunctional, possessing both glycoside hydrolase and histone acetyltransferase (AT) activity. Insights into the mechanism, protein substrate recognition and inhibition of the hydrolase domain of human OGA (hOGA) have been obtained via the use of the structures of bacterial homologues. However, the molecular basis of AT activity of OGA, which has only been reported in vitro, is not presently understood. Here, we describe the crystal structure of a putative acetyltransferase (OgpAT) that we identified in the genome of the marine bacterium Oceanicola granulosus, showing homology to the hOGA C-terminal AT domain (hOGA-AT). The structure of OgpAT in complex with acetyl coenzyme A (AcCoA) reveals that, by homology modelling, hOGA-AT adopts a variant AT fold with a unique loop creating a deep tunnel. The structures, together with mutagenesis and surface plasmon resonance data, reveal that while the bacterial OgpAT binds AcCoA, the hOGA-AT does not, as explained by the lack of key residues normally required to bind AcCoA. Thus, the C-terminal domain of hOGA is a catalytically incompetent ‘pseudo’-AT.
doi:10.1098/rsob.130021
PMCID: PMC3814719  PMID: 24088714
signalling; O-GlcNAc; glycobiology; protein structure
4.  Structural and biochemical characterization of a trapped coenzyme A adduct of Caenorhabditis elegans glucosamine-6-phosphate N-acetyltransferase 1 
Glucosamine-6-phosphate N-acetyltransferase is an essential enzyme of the eukaryotic UDP-GlcNAc biosynthetic pathway. A crystal structure at 1.55 Å resolution revealed a highly unusual covalent product complex and biochemical studies investigated the function of a fully conserved active-site cysteine.
Glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) produces GlcNAc-6-phosphate from GlcN-6-phosphate and acetyl coenzyme A. Early mercury-labelling experiments implicated a conserved cysteine in the reaction mechanism, whereas recent structural data appear to support a mechanism in which this cysteine plays no role. Here, two crystal structures of Caenorhabditis elegans GNA1 are reported, revealing an unusual covalent complex between this cysteine and the coenzyme A product. Mass-spectrometric and reduction studies showed that this inactive covalent complex can be reactivated through reduction, yet mutagenesis of the cysteine supports a previously reported bi-bi mechanism. The data unify the apparently contradictory earlier reports on the role of a cysteine in the GNA1 active site.
doi:10.1107/S0907444912019592
PMCID: PMC3413214  PMID: 22868768
carbohydrates; glycobiology; Caenorhabditis elegans; glucosamine-6-phosphate N-acetyltransferase; coenzyme A adduct; mechanism
5.  Cell-Penetrant, Nanomolar O-GlcNAcase Inhibitors Selective against Lysosomal Hexosaminidases 
Chemistry & Biology  2010;17(11):1250-1255.
Summary
Posttranslational modification of metazoan nucleocytoplasmic proteins with N-acetylglucosamine (O-GlcNAc) is essential, dynamic, and inducible and can compete with protein phosphorylation in signal transduction. Inhibitors of O-GlcNAcase, the enzyme removing O-GlcNAc, are useful tools for studying the role of O-GlcNAc in a range of cellular processes. We report the discovery of nanomolar OGA inhibitors that are up to 900,000-fold selective over the related lysosomal hexosaminidases. When applied at nanomolar concentrations on live cells, these cell-penetrant molecules shift the O-GlcNAc equilibrium toward hyper-O-GlcNAcylation with EC50 values down to 3 nM and are thus invaluable tools for the study of O-GlcNAc cell biology.
Highlights
► Structure-guided design of human O-GlcNAcase inhibitors, GlcNAcstatins ► The GlcNAcstatins are competitive, nanomolar inhibitors ► The molecular basis of the exquisite selectivity revealed by crystallography ► First direct evidence of O-GlcNAcase inhibitors penetrating cells
doi:10.1016/j.chembiol.2010.09.014
PMCID: PMC3032886  PMID: 21095575
6.  Substrate and product analogues as human O-GlcNAc transferase inhibitors 
Amino Acids  2010;40(3):781-792.
Protein glycosylation on serine/threonine residues with N-acetylglucosamine (O-GlcNAc) is a dynamic, inducible and abundant post-translational modification. It is thought to regulate many cellular processes and there are examples of interplay between O-GlcNAc and protein phosphorylation. In metazoa, a single, highly conserved and essential gene encodes the O-GlcNAc transferase (OGT) that transfers GlcNAc onto substrate proteins using UDP–GlcNAc as the sugar donor. Specific inhibitors of human OGT would be useful tools to probe the role of this post-translational modification in regulating processes in the living cell. Here, we describe the synthesis of novel UDP–GlcNAc/UDP analogues and evaluate their inhibitory properties and structural binding modes in vitro alongside alloxan, a previously reported weak OGT inhibitor. While the novel analogues are not active on living cells, they inhibit the enzyme in the micromolar range and together with the structural data provide useful templates for further optimisation.
Electronic supplementary material
The online version of this article (doi:10.1007/s00726-010-0688-y) contains supplementary material, which is available to authorized users.
doi:10.1007/s00726-010-0688-y
PMCID: PMC3040809  PMID: 20640461
O-GlcNAc; Post-translational modification; Inhibitor; Signalling; Crystal structure
7.  Screening-based discovery of drug-like O-GlcNAcase inhibitor scaffolds 
Febs Letters  2010;584(4):694-700.
O-GlcNAcylation is an essential posttranslational modification in metazoa. Modulation of O-GlcNAc levels with small molecule inhibitors of O-GlcNAc hydrolase (OGA) is a useful strategy to probe the role of this modification in a range of cellular processes. Here we report the discovery of novel, low molecular weight and drug-like O-GlcNAcase inhibitor scaffolds by high-throughput screening. Kinetic and X-ray crystallographic analyses of the binding modes with human/bacterial O-GlcNAcases identify some of these as competitive inhibitors. Comparative kinetic experiments with the mechanistically related human lysosomal hexosaminidases reveal that three of the inhibitor scaffolds show selectivity towards human OGA. These scaffolds provide attractive starting points for the development of non-carbohydrate, drug-like OGA inhibitors.
doi:10.1016/j.febslet.2009.12.020
PMCID: PMC2828546  PMID: 20026047
O-GlcNAc; Posttranslational modification; Inhibitor; Crystal structure
8.  GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation 
Biochemical Journal  2009;420(Pt 2):221-227.
O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.
doi:10.1042/BJ20090110
PMCID: PMC2691177  PMID: 19275764
GlcNAcstatin; inhibition; O-GlcNAc; O-GlcNAcase; GST, glutathione transferase; HEK, human embryonic kidney; Hex, hexosaminidase; 4MU-NAG, 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide; OGA, O-GlcNAcase; CpOGA, Clostridium perfringens OGA; hOGA, human OGA; OGT, O-GlcNAc transferase; PUGNAc, O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenylcarbamate
9.  Chemical Dissection of the Link between Streptozotocin, O-GlcNAc, and Pancreatic Cell Death 
Chemistry & Biology  2008;15(8):799-807.
Summary
Streptozotocin is a natural product that selectively kills insulin-secreting β cells, and is widely used to generate mouse models of diabetes or treat pancreatic tumors. Several studies suggest that streptozotocin toxicity stems from its N-nitrosourea moiety releasing nitric oxide and possessing DNA alkylating activity. However, it has also been proposed that streptozotocin induces apoptosis by inhibiting O-GlcNAcase, an enzyme that, together with O-GlcNAc transferase, is important for dynamic intracellular protein O-glycosylation. We have used galacto-streptozotocin to chemically dissect the link between O-GlcNAcase inhibition and apoptosis. Using X-ray crystallography, enzymology, and cell biological studies on an insulinoma cell line, we show that, whereas streptozotocin competitively inhibits O-GlcNAcase and induces apoptosis, its galacto-configured derivative no longer inhibits O-GlcNAcase, yet still induces apoptosis. This supports a general chemical poison mode of action for streptozotocin, suggesting the need for using more specific inhibitors to study protein O-GlcNAcylation.
doi:10.1016/j.chembiol.2008.06.010
PMCID: PMC2568864  PMID: 18721751
CHEMBIO; SIGNALING

Results 1-9 (9)