36 research outputs found
O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry
The
attachment of the sugar N-acetyl-D-glucosamine (GlcNAc) to
specific serine and threonine residues on proteins is referred to
as protein O-GlcNAcylation. O-GlcNAc transferase (OGT) is the enzyme
responsible for carrying out the modification, while O-GlcNAcase (OGA)
reverses it. Protein O-GlcNAcylation has been implicated in a wide
range of cellular processes including transcription, proteostasis,
and stress response. Dysregulation of O-GlcNAc has been linked to
diabetes, cancer, and neurodegenerative and cardiovascular disease.
OGA has been proposed to be a drug target for the treatment of Alzheimerâs
and cardiovascular disease given that increased O-GlcNAc levels appear
to exert a protective effect. The search for specific, potent, and
drug-like OGA inhibitors with bioavailability in the brain is therefore
a field of active research, requiring orthogonal high-throughput assay
platforms. Here, we describe the synthesis of a novel probe for use
in a fluorescence polarization based assay for the discovery of inhibitors
of OGA. We show that the probe is suitable for use with both human
OGA, as well as the orthologous bacterial counterpart from <i>Clostridium perfringens</i>, <i>Cp</i>OGA, and the
lysosomal hexosaminidases HexA/B. We structurally characterize <i>Cp</i>OGA in complex with a ligand identified from a fragment
library screen using this assay. The versatile synthesis procedure
could be adapted for making fluorescent probes for the assay of other
glycoside hydrolases
Inhibitors against Fungal Cell Wall Remodeling Enzymes
Fungal ÎČ-1,3-glucan glucanosyltransferases are glucan-remodeling enzymes that play important roles in cell wall integrity, and are essential for the viability of pathogenic fungi and yeasts. As such, they are considered possible drug targets, although inhibitors of this class of enzymes have not yet been reported. Herein we report a multidisciplinary approach based on a structure-guided design using a highly conserved transglycosylase from Sacharomyces cerevisiae, that leads to carbohydrate derivatives with high affinity for Aspergillus fumigatus Gel4. We demonstrate by X-ray crystallography that the compounds bind in the active site of Gas2/Gel4 and interact with the catalytic machinery. The topological analysis of noncovalent interactions demonstrates that the combination of a triazole with positively charged aromatic moieties are important for optimal interactions with Gas2/Gel4 through unusual pyridinium cationâÏ and face-to-face ÏâÏ interactions. The lead compound is capable of inhibiting AfGel4 with an IC value of 42 ÎŒm.This work was supported by Spanish MINECO Contracts (CTQ2016â76155âR to P.M., and BFU2016â75633âP to R.H.âG.), and an MRC Programme Grant (M004139) to D.M.F.v.A. We also acknowledge the Government of AragĂłn (Spain) (Bioorganic Chemistry group Eâ10 and Protein Targets group Bâ89) for financial support. The European Commission is gratefully acknowledged (BioStructâX grant agreement no. 283570 and BIOSTRUCTX_5186).Peer Reviewe
A structural and biochemical model of processive chitin synthesis
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
Evidence for substrate assisted catalysis in <i>N</i>-acetylphosphoglucosamine mutase
11 pags, 4 figs, 2 tabs . -- Supplementary data is available at the Publisher's web pageN-acetylphosphoglucosamine mutase (AGM1) is a key component of the hexosamine biosynthetic pathway that produces UDP-GlcNAc, an essential precursor for a wide range of glycans in eukaryotes. AGM belongs to the α-D-phosphohexomutase metalloenzyme superfamily and catalyzes the interconversion of N-acetylglucosamine-6-phosphate (GlcNAc-6P) to N-acetylglucosamine-1-phosphate (GlcNAc-1P) through N-acetylglucosa-mine-1,6-bisphosphate (GlcNAc-1,6-bisP) as the catalytic intermediate. Although there is an understanding of the phosphoserine-dependent catalytic mechanism at enzymatic and structural level, the identity of the requisite catalytic base in AGM1/phosphoglucomu-tases is as yet unknown. Here, we present crystal structures of a Michaelis complex of AGM1 with GlcNAc-6P and Mg, and a complex of the inactive Ser69Ala mutant together with glucose-1,6-bisphosphate (Glc-1,6-bisP) that represents key snapshots along the reaction co-ordinate. Together with mutagenesis, these structures reveal that the phosphate group of the hexose-1,6-bisP intermediate may act as the catalytic base.This work was funded by the MRC Programme Grant M004139
Screening-based discovery of <em>Aspergillus fumigatus </em>plant-type chitinase inhibitors
AbstractA limited therapeutic arsenal against increasing clinical disease due to Aspergillus spp. necessitates urgent characterisation of new antifungal targets. Here we describe the discovery of novel, low micromolar chemical inhibitors of Aspergillus fumigatus family 18 plant-type chitinase A1 (AfChiA1) by high-throughput screening (HTS). Analysis of the binding mode by X-ray crystallography confirmed competitive inhibition and kinetic studies revealed two compounds with selectivity towards fungal plant-type chitinases. These inhibitors provide new chemical tools to probe the effects of chitinase inhibition on A. fumigatus growth and virulence, presenting attractive starting points for the development of further potent drug-like molecules
Nucleocytoplasmic human O-GlcNAc transferase is sufficient for O-GlcNAcylation of mitochondrial proteins
O-linked N-acetylglucosamine modification (O-GlcNAcylation) is a nutrient-dependent protein post-translational modification (PTM), dynamically and reversibly driven by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that catalyse the addition and the removal of the O-GlcNAc moieties to/from serine and threonine residues of target proteins respectively. Increasing evidence suggests involvement of O-GlcNAcylation in many biological processes, including transcription, signalling, neuronal development and mitochondrial function. The presence of a mitochondrial O-GlcNAc proteome and a mitochondrial OGT (mOGT) isoform has been reported. We explored the presence of mOGT in human cell lines and mouse tissues. Surprisingly, analysis of genomic sequences indicates that this isoform cannot be expressed in most of the species analysed, except some primates. In addition, we were not able to detect endogenous mOGT in a range of human cell lines. Knockdown experiments and Western blot analysis of all the predicted OGT isoforms suggested the expression of only a single OGT isoform. In agreement with this, we demonstrate that overexpression of the nucleocytoplasmic OGT (ncOGT) isoform leads to increased O-GlcNAcylation of mitochondrial proteins, suggesting that ncOGT is necessary and sufficient for the generation of the O-GlcNAc mitochondrial proteome
The structural basis of acyl coenzyme A-dependent regulation of the transcription factor FadR
FadR is an acyl-CoA-responsive transcription factor, regulating fatty acid biosynthetic and degradation genes in Escherichia coli. The apo-protein binds DNA as a homodimer, an interaction that is disrupted by binding of acyl-CoA. The recently described structure of apo-FadR shows a DNA binding domain coupled to an acyl-CoA binding domain with a novel fold, but does not explain how binding of the acyl-CoA effector molecule >30Â â« away from the DNA binding site affects transcriptional regulation. Here, we describe the structures of the FadRâoperator and FadRâ myristoyl-CoA binary complexes. The FadRâDNA complex reveals a novel winged helixâturnâhelix proteinâDNA interaction, involving sequence-specific contacts from the wing to the minor groove. Binding of acyl-CoA results in dramatic conformational changes throughout the protein, with backbone shifts up to 4.5Â â«. The net effect is a rearrangement of the DNA binding domains in the dimer, resulting in a change of 7.2Â â« in separation of the DNA recognition helices and the loss of DNA binding, revealing the molecular basis of acyl-CoA-responsive regulation