Chris A. Tarling

Structure of a Flavonoid Glucosyltransferase Reveals the Basis for Plant Natural Product Modification.

Glycosylation is a key mechanism for orchestrating the bioactivity, metabolism and location of small molecules in living cells. In plants, a large multigene family of glycosyltransferases is involved in these processes, conjugating hormones, secondary metabolites, biotic and abiotic environmental toxins, to impact directly on cellular homeostasis. The red grape enzyme UDP‐glucose:flavonoid 3‐O‐glycosyltransferase (VvGT1) is responsible for the formation of anthocyanins, the health‐promoting compounds which, in planta, function as colourants determining flower and fruit colour and are precursors for the formation of pigmented polymers in red wine. We show that VvGT1 is active, in vitro, on a range of flavonoids. VvGT1 is somewhat promiscuous with respect to donor sugar specificity as dissected through full kinetics on a panel of nine sugar donors. The three‐dimensional structure of VvGT1 has also been determined, both in its ‘Michaelis’ complex with a UDP‐glucose‐derived donor and the acceptor kaempferol and in complex with UDP and quercetin. These structures, in tandem with kinetic dissection of activity, provide the foundation for understanding the mechanism of these enzymes in small molecule homeostasis.

The Search for Novel Human Pancreatic α‐Amylase Inhibitors: High‐Throughput Screening of Terrestrial and Marine Natural Product Extracts

Specific inhibitors of human pancreatic α‐amylase (HPA) have potential as oral agents for the control of blood glucose levels in the treatment of diabetes and obesity. In a search for novel inhibitors, a library of 30 000 crude biological extracts of terrestrial and marine origin has been screened. A number of inhibitory extracts were identified, of which the most potent was subjected to bioassay‐guided purification. A family of three glycosylated acyl flavonols, montbretins A–C, was thereby identified and characterized as competitive amylase inhibitors, with Ki values ranging from 8.1–6100 nM. Competitive inhibition by myricetin, which corresponds to the flavone core, and noncompetitive inhibition by a second fragment, ethyl caffeiate, suggest a binding mode for these inhibitors.

Crystal Structure of Thermotoga Maritima Alpha-L-Fucosidase. Insights Into the Catalytic Mechanism and the Molecular Basis for Fucosidosis.

Fucosylated glycoconjugates are involved in numerous biological events, and α-l-fucosidases, the enzymes responsible for their processing, are therefore of crucial importance. Deficiency in α-l-fucosidase activity is associated with fucosidosis, a lysosomal storage disorder characterized by rapid neurodegeneration, resulting in severe mental and motor deterioration. To gain insight into α-l-fucosidase function at the molecular level, we have determined the crystal structure of Thermotoga maritima α-l-fucosidase. This enzyme assembles as a hexamer and displays a two-domain fold, composed of a catalytic (β/α)8-like domain and a C-terminal β-sandwich domain. The structures of an enzyme-product complex and of a covalent glycosyl-enzyme intermediate, coupled with kinetic and mutagenesis studies, allowed us to identify the catalytic nucleophile, Asp244, and the Brønsted acid/base, Glu266. Because T. maritima α-l-fucosidase occupies a unique evolutionary position, being far more closely related to the mammalian enzymes than to any other prokaryotic homolog, a structural model of the human enzyme was built to document the structural consequences of the genetic mutations associated with fucosidosis.

The Donor Subsite of Trehalose-6-Phosphate Synthase: Binary Complexes with Udp-Glucose and Udp-2-Deoxy-2-Fluoro-Glucose at 2 A Resolution

Trehalose is an unusual non-reducing disaccharide that plays a variety of biological roles, from food storage to cellular protection from environmental stresses such as desiccation, pressure, heat-shock, extreme cold, and oxygen radicals. It is also an integral component of the cell-wall glycolipids of mycobacteria. The primary enzymatic route to trehalose first involves the transfer of glucose from a UDP-glucose donor to glucose-6-phosphate to form α,α-1,1 trehalose-6-phosphate. This reaction, in which the configurations of two glycosidic bonds are set simultaneously, is catalyzed by the glycosyltransferase trehalose-6-phosphate synthase (OtsA), which acts with retention of the anomeric configuration of the UDP-sugar donor. The classification of activated sugar-dependent glycosyltransferases into approximately 70 distinct families based upon amino acid sequence similarities places OtsA in glycosyltransferase family 20 (see afmb.cnrs-mrs.fr/CAZY/). The recent 2.4 Å structure of Escherichia coli OtsA revealed a two-domain enzyme with catalysis occurring at the interface of the twin β/α/β domains. Here we present the 2.0 Å structures of the E. coli OtsA in complex with either UDP-Glc or the non-transferable analogue UDP-2-deoxy-2-fluoroglucose. Both complexes unveil the donor subsite interactions, confirming a strong similarity to glycogen phosphorylases, and reveal substantial conformational differences to the previously reported complex with UDP and glucose 6-phosphate. Both the relative orientation of the two domains and substantial (up to 10 Å) movements of an N-terminal loop (residues 9–22) characterize the more open “relaxed” conformation of the binary UDP-sugar complexes reported here.

Identification of the Catalytic Nucleophile of the Family 29 α-L-Fucosidase from Thermotoga maritima through Trapping of a Covalent Glycosyl-Enzyme Intermediate and Mutagenesis

Fucose-containing glycoconjugates are key antigenic determinants in many biological processes. A change in expression levels of the enzymes responsible for tailoring these glycoconjugates has been associated with many pathological conditions and it is therefore surprising that little information is known regarding the mechanism of action of these important catabolic enzymes. Thermotoga maritima, a thermophilic bacterium, produces a wide range of carbohydrate-processing enzymes including a 52-kDa α-l-fucosidase that has 38% sequence identity and 56% similarity to human fucosidases. The catalytic nucleophile of this enzyme was identified to be Asp-224 within the peptide sequence 222WNDMGWPEKGKEDL235 using the mechanism-based covalent inactivator 2-deoxy-2-fluoro-α-l-fucosyl fluoride. The 104-fold lower activity (kcat/Km) of the site-directed mutant D224A, and the subsequent rescue of activity upon addition of exogenous nucleophiles, conclusively confirms this assignment. This article presents the first direct identification of the catalytic nucleophile of an α-l-fucosidase, a key step in the understanding of these important enzymes.

Fluorous iminoalditols: a new family of glycosidase inhibitors and pharmacological chaperones.

A collection of new reversible glycosidase inhibitors of the iminoalditol type featuring N‐substituents containing perfluorinated regions has been prepared for evaluation of physicochemical, biochemical and diagnostic properties. The vast variety of feasible oligofluoro moieties allows for modular approaches to customised structures according to the intended applications, which are influenced by the fluorine content as well as the distance of the fluorous moiety from the ring nitrogen. The first examples, in particular in the D‐galacto series, exhibited excellent inhibitory activities. A preliminary screen with two human cell lines showed that, at subinhibitory concentrations, they are powerful pharmacological chaperones enhancing the activities of the catalytically handicapped lysosomal D‐galactosidase mutants associated with GM1 gangliosidosis and Morquio B disease.

Structural and Mechanistic Analyses of endo-Glycoceramidase II, a Membrane-associated Family 5 Glycosidase in the Apo and GM3 Ganglioside-bound Forms.

endo-Glycoceramidase, a membrane-associated family 5 glycosidase, deviates from the typical polysaccharide substrate specificity of other soluble members of the family, preferentially hydrolyzing glycosidic linkages between the oligosaccharide and ceramide moieties of gangliosides. Here we report the first x-ray crystal structures of an endo-glycoceramidase from Rhodococcus sp., in the apo form, in complex with the ganglioside GM3 (Svennerholm ganglioside nomenclature (Svennerholm, L. (1964) J. Lipid Res. 5, 145-155)), and trapped as a glycosyl-enzyme intermediate. These snapshots provide the first molecular insight into enzyme recognition and association with gangliosides, revealing the structural adaptations necessary for glycosidase-catalyzed hydrolysis and detailing a novel ganglioside binding topology. Consistent with the chemical duality of the substrate, the active site of endo-glycoceramidase is split into a wide, polar cavity to bind the polyhydroxylated oligosaccharide moiety and a narrow, hydrophobic tunnel to bind the ceramide lipid chains. The specific interactions with the ceramide polar head group manifest a surprising aglycone specificity, an observation substantiated by our kinetic analyses. Collectively, the reported structural and kinetic data provide insight toward rational redesign of the synthetic glycosynthase mutant of endo-glycoceramidase to enable facile synthesis of nonnatural, therapeutically useful gangliosides.

Structural analysis of Golgi alpha-mannosidase II inhibitors identified from a focused glycosidase inhibitor screen.

The N-glycosylation pathway is a target for pharmaceutical intervention in a number of pathological conditions including cancer. Golgi alpha-mannosidase II (GMII) is the final glycoside hydrolase in the pathway and has been the target for a number of synthetic efforts aimed at providing more selective and effective inhibitors. Drosophila GMII (dGMII) has been extensively studied due to the ease of obtaining high resolution structural data, allowing the observation of substrate distortion upon binding and after formation of a trapped covalent reaction intermediate. However, attempts to find new inhibitor leads by high-throughput screening of large commercial libraries or through in silico docking were unsuccessful. In this paper we provide a kinetic and structural analysis of five inhibitors derived from a small glycosidase-focused library. Surprisingly, four of these were known inhibitors of beta-glucosidases. X-ray crystallographic analysis of the dGMII:inhibitor complexes highlights the ability of the zinc-containing GMII active site to deform compounds, even ones designed as conformationally restricted transition-state mimics of beta-glucosidases, into binding entities that have inhibitory activity. Although these deformed conformations do not appear to be on the expected conformational itinerary of the enzyme, and are thus not transition-state mimics of GMII, they allow positioning of the three vicinal hydroxyls of the bound gluco-inhibitors into similar locations to those found with mannose-containing substrates, underlining the importance of these hydrogen bonds for binding. Further, these studies show the utility of targeting the acid-base catalyst using appropriately positioned positively charged nitrogen atoms, as well as the challenges associated with aglycon substitutions.

Synthesis of lipophilic 1-deoxygalactonojirimycin derivatives as D-galactosidase inhibitors

Summary N-Alkylation at the ring nitrogen of the D-galactosidase inhibitor 1-deoxygalactonojirimycin with a functionalised C6 alkyl chain followed by modification with different aromatic substituents provided lipophilic 1-deoxygalactonojirimycin derivatives which exhibit inhibitory properties against β-glycosidases from E. coli and Agrobacterium sp. as well as green coffee bean α-galactosidase. In preliminary studies, these compounds also showed potential as chemical chaperones for GM1-gangliosidosis related β-galactosidase mutants.

Chitinase Inhibition by Chitobiose and Chitotriose Thiazolines

A good imitation: Di- and trisaccharide analogues of the oxazoline intermediate formed during enzymatic hydrolysis of chitin were found to be potent inhibitors of chitinase A. The high affinity and enzymatic stability of a readily synthesized thioamide trisaccharide (two molecules of which are shown in the enzyme active site), and the mechanism-based mode of inhibition, make this analogue a promising candidate for broad-spectrum chitinase inhibition.