Understanding functional dynamics and conformational stability of beta-glycosidases

Due to their central physiological roles in living organisms, retaining beta-glycosidases have been the subject of tremendous research efforts to examine their structure/function relation using numerous biophysical and biochemical approaches. Since the proposition of the hydrolysis mechanism in the late fifties by Koshland1, the fundamental research on retaining b-glycosidases has been revolutionized by the discovery of multiple reversible and irreversible inhibitors. One of the most successful class of inhibitors are mechanism based inactivators, which were extensively used to identify the nucleophilic catalytic residues and to comprehend the catalytic mechanism and substrate itinerary. Subsequently, covalent inhibitors were used as warheads to synthesize chromogenic activity based probes (ABPs), which were widely used to selectively label and discover new retaining beta-glycosidases in complex biological samples. The organic synthesis and biological applications of these ABPs has become routine. Nevertheless, their binding mechanism and influences on protein conformation and dynamics remained unexplored. Therefore, this work is aimed to establish a bridge between the two research disciplines, using ABP technology to understand functional dynamics and conformational stability of retaining bglycosidases in solution and in vivo. The research relied on standard biochemistry and advanced NMR spectroscopy research approaches.NWOMacromolecular Biochemistr

The architects of bacterial DNA bridges: a structurally and functionally conserved family of proteins

Every organism across the tree of life compacts and organizes its genome with architectural chromatin proteins. While eukaryotes and archaea express histone proteins, the organization of bacterial chromosomes is dependent on nucleoid-associated proteins. In Escherichia coli and other proteobacteria, the histone-like nucleoid structuring protein (H-NS) acts as a global genome organizer and gene regulator. Functional analogues of H-NS have been found in other bacterial species: MvaT in Pseudomonas species, Lsr2 in actinomycetes and Rok in Bacillus species. These proteins complement hns− phenotypes and have similar DNA-binding properties, despite their lack of sequence homology. In this review, we focus on the structural and functional characteristics of these four architectural proteins. They are able to bridge DNA duplexes, which is key to genome compaction, gene regulation and their response to changing conditions in the environment. Structurally the domain organization and charge distribution of these proteins are conserved, which we suggest is at the basis of their conserved environment responsive behaviour. These observations could be used to find and validate new members of this protein family and to predict their response to environmental changes.Macromolecular Biochemistr

Tuning the transglycosylation reaction of a GH11 xylanase by a delicate enhancement of its thumb flexibility

Glycoside hydrolases (GHs) are attractive tools for multiple biotechnological applications. In conjunction with their hydrolytic function, GHs can perform transglycosylation under specific conditions. In nature, oligosaccharide synthesis is performed by glycosyltransferases (GTs); however, the industrial use of GTs is limited by their instability in solution. A key difference between GTs and GHs is the flexibility of their binding site architecture. We have used the xylanase from Bacillus circulans (BCX) to study the interplay between active-site flexibility and transglycosylation. Residues of the BCX "thumb" were substituted to increase the flexibility of the enzyme binding site. Replacement of the highly conserved residue P116 with glycine shifted the balance of the BCX enzymatic reaction toward transglycosylation. The effects of this point mutation on the structure and dynamics of BCX were investigated by NMR spectroscopy. The P116G mutation induces subtle changes in the configuration of the thumb and enhances the millisecond dynamics of the active site. Based on our findings, we propose the remodelling of the GH enzymes glycon site flexibility as a strategy to improve the transglycosylation efficiency of these biotechnologically important catalysts.Macromolecular BiochemistryBio-organic SynthesisMedical Biochemistr

Novel anti-repression mechanism of H-NS proteins by a phage protein

Macromolecular Biochemistr

Dynamics of ligand binding to a rigid glycosidase

The single-domain GH11 glycosidase from  Bacillus circulans  (BCX) is involved in the degradation of hemicellulose, one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, attributed to an enzyme induced substrate distortion. A model is proposed in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates the acyl formation step of the hydrolysis reaction.Medical BiochemistryMacromolecular BiochemistryBio-organic Synthesi

Stabilization of Glucocerebrosidase by Active Site Occupancy

Macromolecular Biochemistr

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Understanding functional dynamics and conformational stability of beta-glycosidases

Due to their central physiological roles in living organisms, retaining beta-glycosidases have been the subject of tremendous research efforts to examine their structure/function relation using numerous biophysical and biochemical approaches. Since the proposition of the hydrolysis mechanism in the late fifties by Koshland1, the fundamental research on retaining b-glycosidases has been revolutionized by the discovery of multiple reversible and irreversible inhibitors. One of the most successful class of inhibitors are mechanism based inactivators, which were extensively used to identify the nucleophilic catalytic residues and to comprehend the catalytic mechanism and substrate itinerary. Subsequently, covalent inhibitors were used as warheads to synthesize chromogenic activity based probes (ABPs), which were widely used to selectively label and discover new retaining beta-glycosidases in complex biological samples. The organic synthesis and biological applications of these ABPs has become routine. Nevertheless, their binding mechanism and influences on protein conformation and dynamics remained unexplored. Therefore, this work is aimed to establish a bridge between the two research disciplines, using ABP technology to understand functional dynamics and conformational stability of retaining bglycosidases in solution and in vivo. The research relied on standard biochemistry and advanced NMR spectroscopy research approaches.</p

Distinguishing the differences in beta-glycosylceramidase folds, dynamics, and actions informs therapeutic uses

Glycosyl hydrolases (GHs) are carbohydrate-active enzymes that hydrolyze a specific β-glycosidic bond in glycoconjugate substrates; β-glucosidases degrade glucosylceramide, a ubiquitous glycosphingolipid. GHs are grouped into structurally similar families that themselves can be grouped into clans. GH1, GH5, and GH30 glycosidases belong to clan A hydrolases with a catalytic (β/α)8 TIM barrel domain, whereas GH116 belongs to clan O with a catalytic (α/α)6 domain. In humans, GH abnormalities underlie metabolic diseases. The lysosomal enzyme glucocerebrosidase (family GH30), deficient in Gaucher disease and implicated in Parkinson disease etiology, and the cytosol-facing membrane-bound glucosylceramidase (family GH116) remove the terminal glucose from the ceramide lipid moiety. Here, we compare enzyme differences in fold, action, dynamics, and catalytic domain stabilization by binding site occupancy. We also explore other glycosidases with reported glycosylceramidase activity, including human cytosolic β-glucosidase, intestinal lactase-phlorizin hydrolase, and lysosomal galactosylceramidase. Last, we describe the successful translation of research to practice: recombinant glycosidases and glucosylceramide metabolism modulators are approved drug products (enzyme replacement therapies). Activity-based probes now facilitate the diagnosis of enzyme deficiency and screening for compounds that interact with the catalytic pocket of glycosidases. Future research may deepen the understanding of the functional variety of these enzymes and their therapeutic potential. </p