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Gideon J Davies

Publications and source records attributed to Gideon J Davies.

At least 19 recordsLinked to original sources
PubMed2007

Analysis of PUGNAc and NAG-thiazoline as transition state analogues for human O-GlcNAcase: mechanistic and structural insights into inhibitor selectivity and transition state poise.

O-GlcNAcase catalyzes the cleavage of beta-O-linked 2-acetamido-2-deoxy-beta-d-glucopyranoside (O-GlcNAc) from serine and threonine residues of post-translationally modified proteins. Two potent inhibitors of this enzyme are O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and 1,2-dideoxy-2'-methyl-alpha-d-glucopyranoso[2,1-d]-Delta2'-thiazoline (NAG-thiazoline). Derivatives of these inhibitors differ in their selectivity for human O-GlcNAcase over the functionally related human lysosomal beta-hexosamindases, with PUGNAc derivatives showing modest selectivities and NAG-thiazoline derivatives showing high selectivities. The molecular basis for this difference in selectivities is addressed as is how well these inhibitors mimic the O-GlcNAcase-stabilized transition state (TS). Using a series of substrates, ground state (GS) inhibitors, and transition state mimics having analogous structural variations, we describe linear free energy relationships of log(KM/kcat) versus log(KI) for PUGNAc and NAG-thiazoline. These relationships suggest that PUGNAc is a poor transition state analogue, while NAG-thiazoline is revealed as a transition state mimic. Comparative X-ray crystallographic analyses of enzyme-inhibitor complexes reveal subtle molecular differences accounting for the differences in selectivities between these two inhibitors and illustrate key molecular interactions. Computational modeling of species along the reaction coordinate, as well as PUGNAc and NAG-thiazoline, provide insight into the features of NAG-thiazoline that resemble the transition state and reveal where PUGNAc fails to capture significant binding energy. These studies also point to late transition state poise for the O-GlcNAcase catalyzed reaction with significant nucleophilic participation and little involvement of the leaving group. The potency of NAG-thiazoline, its transition state mimicry, and its lack of traditional transition state-like design features suggest that potent rationally designed glycosidase inhibitors can be developed that exploit variation in transition state poise.

Acetylglucosamine↗
PubMed2006

Functional analysis of a group A streptococcal glycoside hydrolase Spy1600 from family 84 reveals it is a beta-N-acetylglucosaminidase and not a hyaluronidase.

Group A streptococcus (Streptococcus pyogenes) is the causative agent of severe invasive infections such as necrotizing fasciitis (the so-called 'flesh eating disease') and toxic-shock syndrome. Spy1600, a glycoside hydrolase from family 84 of the large superfamily of glycoside hydrolases, has been proposed to be a virulence factor. In the present study we show that Spy1600 has no activity toward galactosaminides or hyaluronan, but does remove beta-O-linked N-acetylglucosamine from mammalian glycoproteins--an observation consistent with the inclusion of eukaryotic O-glycoprotein 2-acetamido-2-deoxy-beta-D-glucopyranosidases within glycoside hydrolase family 84. Proton NMR studies, structure-reactivity studies for a series of fluorinated analogues and analysis of 1,2-dideoxy-2'-methyl-alpha-D-glucopyranoso-[2,1-d]-Delta2'-thiazoline as a competitive inhibitor reveals that Spy1600 uses a double-displacement mechanism involving substrate-assisted catalysis. Family 84 glycoside hydrolases are therefore comprised of both prokaryotic and eukaryotic beta-N-acetylglucosaminidases using a conserved catalytic mechanism involving substrate-assisted catalysis. Since these enzymes do not have detectable hyaluronidase activity, many family 84 glycoside hydrolases are most likely incorrectly annotated as hyaluronidases.

Acetylglucosamine↗
PubMed2006

Structural, kinetic, and thermodynamic analysis of glucoimidazole-derived glycosidase inhibitors.

Inhibition of glycosidases has great potential in the quest for highly potent and specific drugs to treat diseases such as diabetes, cancer, and viral infections. One of the most effective ways of designing such compounds is by mimicking the transition state. Here we describe the structural, kinetic, and thermodynamic dissection of binding of two glucoimidazole-derived compounds, which are among the most potent glycosidase inhibitors reported to date, with two family 1 beta-glycosidases. Provocatively, while inclusion of the phenethyl moiety improves binding by a factor of 20-80-fold, this does not appear to result from better noncovalent interactions with the enzyme; instead, improved affinity may be derived from significantly better entropic contributions to binding displayed by the phenethyl-substituted imidazole compound.

Archaeal Proteins↗
PubMed2006

Crystal structures of Clostridium thermocellum xyloglucanase, XGH74A, reveal the structural basis for xyloglucan recognition and degradation.

The enzymatic degradation of the plant cell wall is central both to the natural carbon cycle and, increasingly, to environmentally friendly routes to biomass conversion, including the production of biofuels. The plant cell wall is a complex composite of cellulose microfibrils embedded in diverse polysaccharides collectively termed hemicelluloses. Xyloglucan is one such polysaccharide whose hydrolysis is catalyzed by diverse xyloglucanases. Here we present the structure of the Clostridium thermocellum xyloglucanase Xgh74A in both apo and ligand-complexed forms. The structures, in combination with mutagenesis data on the catalytic residues and the kinetics and specificity of xyloglucan hydrolysis reveal a complex subsite specificity accommodating seventeen monosaccharide moieties of the multibranched substrate in an open substrate binding terrain.

Amino Acid Sequence↗
PubMed2006

Family 6 carbohydrate binding modules in beta-agarases display exquisite selectivity for the non-reducing termini of agarose chains.

Carbohydrate recognition is central to the biological and industrial exploitation of plant structural polysaccharides. These insoluble polymers are recalcitrant to microbial degradation, and enzymes that catalyze this process generally contain non-catalytic carbohydrate binding modules (CBMs) that potentiate activity by increasing substrate binding. Agarose, a repeat of the disaccharide 3,6-anhydro-alpha-L-galactose-(1,3)-beta-D-galactopyranose-(1,4), is the dominant matrix polysaccharide in marine algae, yet the role of CBMs in the hydrolysis of this important polymer has not previously been explored. Here we show that family 6 CBMs, present in two different beta-agarases, bind specifically to the non-reducing end of agarose chains, recognizing only the first repeat of the disaccharide. The crystal structure of one of these modules Aga16B-CBM6-2, in complex with neoagarohexaose, reveals the mechanism by which the protein displays exquisite specificity, targeting the equatorial O4 and the axial O3 of the anhydro-L-galactose. Targeting of the CBM6 to the non-reducing end of agarose chains may direct the appended catalytic modules to areas of the plant cell wall attacked by beta-agarases where the matrix polysaccharide is likely to be more amenable to further enzymic hydrolysis.

Amino Acid Sequence↗
PubMed2006

Structural insight into the ligand specificity of a thermostable family 51 arabinofuranosidase, Araf51, from Clostridium thermocellum.

The digestion of the plant cell wall requires the concerted action of a diverse repertoire of enzyme activities. An important component of these hydrolase consortia are arabinofuranosidases, which release L-arabinofuranose moieties from a range of plant structural polysaccharides. The anaerobic bacterium Clostridium thermocellum, a highly efficient plant cell wall degrader, possesses a single alpha-L-arabinofuranosidase (EC 3.2.1.55), CtAraf51A, located in GH51 (glycoside hydrolase family 51). The crystal structure of the enzyme has been solved in native form and in 'Michaelis' complexes with both alpha-1,5-linked arabinotriose and alpha-1,3 arabinoxylobiose, both forming a hexamer in the asymmetric unit. Kinetic studies reveal that CtAraf51A, in contrast with well-characterized GH51 enzymes including the Cellvibrio japonicus enzyme [Beylot, McKie, Voragen, Doeswijk-Voragen and Gilbert (2001) Biochem. J. 358, 607-614], catalyses the hydrolysis of alpha-1,5-linked arabino-oligosaccharides and the alpha-1,3 arabinosyl side chain decorations of xylan with equal efficiency. The paucity of direct hydrogen bonds with the aglycone moiety and the flexible conformation adopted by Trp(178), which stacks against the sugar at the +1 subsite, provide a structural explanation for the plasticity in substrate specificity displayed by the clostridial arabinofuranosidase.

Binding Sites↗
PubMed2006

Structure and mechanism of a bacterial beta-glucosaminidase having O-GlcNAcase activity.

O-GlcNAc is an abundant post-translational modification of serine and threonine residues of nucleocytoplasmic proteins. This modification, found only within higher eukaryotes, is a dynamic modification that is often reciprocal to phosphorylation. In a manner analogous to phosphatases, a glycoside hydrolase termed O-GlcNAcase cleaves O-GlcNAc from modified proteins. Enzymes with high sequence similarity to human O-GlcNAcase are also found in human pathogens and symbionts. We report the three-dimensional structure of O-GlcNAcase from the human gut symbiont Bacteroides thetaiotaomicron both in its native form and in complex with a mimic of the reaction intermediate. Mutagenesis and kinetics studies show that the bacterial enzyme, very similarly to its human counterpart, operates via an unusual 'substrate-assisted' catalytic mechanism, which will inform the rational design of enzyme inhibitors.

Acetylglucosaminidase↗
PubMed2006

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.

Crystallography, X-Ray↗
PubMed2006

Structure and activity of two metal ion-dependent acetylxylan esterases involved in plant cell wall degradation reveals a close similarity to peptidoglycan deacetylases.

The enzymatic degradation of plant cell wall xylan requires the concerted action of a diverse enzymatic syndicate. Among these enzymes are xylan esterases, which hydrolyze the O-acetyl substituents, primarily at the O-2 position of the xylan backbone. All acetylxylan esterase structures described previously display a alpha/beta hydrolase fold with a "Ser-His-Asp" catalytic triad. Here we report the structures of two distinct acetylxylan esterases, those from Streptomyces lividans and Clostridium thermocellum, in native and complex forms, with x-ray data to between 1.6 and 1.0 A resolution. We show, using a novel linked assay system with PNP-2-O-acetylxyloside and a beta-xylosidase, that the enzymes are sugar-specific and metal ion-dependent and possess a single metal center with a chemical preference for Co2+. Asp and His side chains complete the catalytic machinery. Different metal ion preferences for the two enzymes may reflect the surprising diversity with which the metal ion coordinates residues and ligands in the active center environment of the S. lividans and C. thermocellum enzymes. These "CE4" esterases involved in plant cell wall degradation are shown to be closely related to the de-N-acetylases involved in chitin and peptidoglycan degradation (Blair, D. E., Schuettelkopf, A. W., MacRae, J. I., and Aalten, D. M. (2005) Proc. Natl. Acad. Sci. U. S. A., 102, 15429-15434), which form the NodB deacetylase "superfamily."

Acetylesterase↗
PubMed2006

Insights into the synthesis of lipopolysaccharide and antibiotics through the structures of two retaining glycosyltransferases from family GT4.

Glycosyltransferases (GTs) catalyze the synthesis of the myriad glycoconjugates that are central to life. One of the largest families is GT4, which contains several enzymes of therapeutic significance, exemplified by WaaG and AviGT4. WaaG catalyses a key step in lipopolysaccharide synthesis, while AviGT4, produced by Streptomyces viridochromogenes, contributes to the synthesis of the antibiotic avilamycin A. Here we present the crystal structure of both WaaG and AviGT4. The two enzymes contain two "Rossmann-like" (beta/alpha/beta) domains characteristic of the GT-B fold. Both recognition of the donor substrate and the catalytic machinery is similar to other retaining GTs that display the GT-B fold. Structural information is discussed with respect to the evolution of GTs and the therapeutic significance of the two enzymes.

Anti-Bacterial Agents↗
PubMed2005

Structure of a group A streptococcal phage-encoded virulence factor reveals a catalytically active triple-stranded beta-helix.

Streptococcus pyogenes (group A Streptococcus) causes severe invasive infections including scarlet fever, pharyngitis (streptococcal sore throat), skin infections, necrotizing fasciitis (flesh-eating disease), septicemia, erysipelas, cellulitis, acute rheumatic fever, and toxic shock. The conversion from nonpathogenic to toxigenic strains of S. pyogenes is frequently mediated by bacteriophage infection. One of the key bacteriophage-encoded virulence factors is a putative "hyaluronidase," HylP1, a phage tail-fiber protein responsible for the digestion of the S. pyogenes hyaluronan capsule during phage infection. Here we demonstrate that HylP1 is a hyaluronate lyase. The 3D structure, at 1.8-angstroms resolution, reveals an unusual triple-stranded beta-helical structure and provides insight into the structural basis for phage tail assembly and the role of phage tail proteins in virulence. Unlike the triple-stranded beta-helix assemblies of the bacteriophage T4 injection machinery and the tailspike endosialidase of the Escherichia coli K1 bacteriophage K1F, HylP1 possesses three copies of the active center on the triple-helical fiber itself without the need for an accessory catalytic domain. The triple-stranded beta-helix is not simply a structural scaffold, as previously envisaged; it is harnessed to provide a 200-angstroms-long substrate-binding groove for the optimal reduction in hyaluronan viscosity to aid phage penetration of the capsule.

Catalysis↗
PubMed2005

Recent structural insights into the expanding world of carbohydrate-active enzymes.

Enzymes that catalyse the synthesis and breakdown of glycosidic bonds account for 1-3% of the proteins encoded by the genomes of most organisms. At the current rate, over 12 000 glycosyltransferase and glycoside hydrolase open reading frames will appear during 2006. Recent advances in the study of the structure and mechanism of these carbohydrate-active enzymes reveal that glycoside hydrolases continue to display a wide variety of scaffolds, whereas nucleotide-sugar-dependent glycosyltransferases tend to be grafted onto just two protein folds. The past two years have seen significant advances, including the discovery of a novel NAD+-dependent glycosidase mechanism, the dissection of the reaction coordinate of sialidases and a better understanding of the expanding roles of auxiliary carbohydrate-binding domains.

Binding Sites↗
PubMed2005

Crystal structure of levansucrase from the Gram-negative bacterium Gluconacetobacter diazotrophicus.

The endophytic Gram-negative bacterium Gluconacetobacter diazotrophicus SRT4 secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10), which converts sucrose into fructooligosaccharides and levan. The enzyme is included in GH (glycoside hydrolase) family 68 of the sequence-based classification of glycosidases. The three-dimensional structure of LsdA has been determined by X-ray crystallography at a resolution of 2.5 A (1 A=0.1 nm). The structure was solved by molecular replacement using the homologous Bacillus subtilis (Bs) levansucrase (Protein Data Bank accession code 1OYG) as a search model. LsdA displays a five-bladed beta-propeller architecture, where the catalytic residues that are responsible for sucrose hydrolysis are perfectly superimposable with the equivalent residues of the Bs homologue. The comparison of both structures, the mutagenesis data and the analysis of GH68 family multiple sequences alignment show a strong conservation of the sucrose hydrolytic machinery among levansucrases and also a structural equivalence of the Bs levansucrase Ca2+-binding site to the LsdA Cys339-Cys395 disulphide bridge, suggesting similar fold-stabilizing roles. Despite the strong conservation of the sucrose-recognition site observed in LsdA, Bs levansucrase and GH32 family Thermotoga maritima invertase, structural differences appear around residues involved in the transfructosylation reaction.

Amino Acid Sequence↗
PubMed2005

How family 26 glycoside hydrolases orchestrate catalysis on different polysaccharides: structure and activity of a Clostridium thermocellum lichenase, CtLic26A.

One of the most intriguing features of the 90 glycoside hydrolase families (GHs) is the range of specificities displayed by different members of the same family, whereas the catalytic apparatus and mechanism are often invariant. Family GH26 predominantly comprises beta-1,4 mannanases; however, a bifunctional Clostridium thermocellum GH26 member (hereafter CtLic26A) displays a markedly different specificity. We show that CtLic26A is a lichenase, specific for mixed (Glcbeta1,4Glcbeta1,4Glcbeta1,3)n oligo- and polysaccharides, and displays no activity on manno-configured substrates or beta-1,4-linked homopolymers of glucose or xylose. The three-dimensional structure of the native form of CtLic26A has been solved at 1.50-A resolution, revealing a characteristic (beta/alpha)8 barrel with Glu-109 and Glu-222 acting as the catalytic acid/base and nucleophile in a double-displacement mechanism. The complex with the competitive inhibitor, Glc-beta-1,3-isofagomine (Ki 1 microm), at 1.60 A sheds light on substrate recognition in the -2 and -1 subsites and illuminates why the enzyme is specific for lichenan-based substrates. Hydrolysis of beta-mannosides by GH26 members is thought to proceed through transition states in the B2,5 (boat) conformation in which structural distinction of glucosides versus mannosides reflects not the configuration at C2 but the recognition of the pseudoaxial O3 of the B2,5 conformation. We suggest a different conformational itinerary for the GH26 enzymes active on gluco-configured substrates.

Binding, Competitive↗