RESUMO
Acid ß-galactosidase (GLB1) and galactocerebrosidase (GALC) are retaining exo-ß-galactosidases involved in lysosomal glycoconjugate metabolism. Deficiency of GLB1 may result in the lysosomal storage disorders GM1 gangliosidosis, Morquio B syndrome, and galactosialidosis, and deficiency of GALC may result in Krabbe disease. Activity-based protein profiling (ABPP) is a powerful technique to assess the activity of retaining glycosidases in relation to health and disease. This work describes the use of fluorescent and biotin-carrying activity-based probes (ABPs) to assess the activity of both GLB1 and GALC in cell lysates, culture media, and tissue extracts. The reported ABPs, which complement the growing list of retaining glycosidase ABPs based on configurational isomers of cyclophellitol, should assist in fundamental and clinical research on various ß-galactosidases, whose inherited deficiencies cause debilitating lysosomal storage disorders.
Assuntos
Gangliosidose GM1 , Leucodistrofia de Células Globoides , Doenças por Armazenamento dos Lisossomos , Mucopolissacaridose IV , Humanos , beta-Galactosidase/metabolismo , GalactosilceramidaseRESUMO
Bacteria and yeasts grow on biomass polysaccharides by expressing and excreting a complex array of glycoside hydrolase (GH) enzymes. Identification and annotation of such GH pools, which are valuable commodities for sustainable energy and chemistries, by conventional means (genomics, proteomics) are complicated, as primary sequence or secondary structure alignment with known active enzymes is not always predictive for new ones. Here we report a "low-tech", easy-to-use, and sensitive multiplexing activity-based protein-profiling platform to characterize the xyloglucan-degrading GH system excreted by the soil saprophyte, Cellvibrio japonicus, when grown on xyloglucan. A suite of activity-based probes bearing orthogonal fluorophores allows for the visualization of accessory exo-acting glycosidases, which are then identified using biotin-bearing probes. Substrate specificity of xyloglucanases is directly revealed by imbuing xyloglucan structural elements into bespoke activity-based probes. Our ABPP platform provides a highly useful tool to dissect xyloglucan-degrading systems from various sources and to rapidly select potentially useful ones. The observed specificity of the probes moreover bodes well for the study of other biomass polysaccharide-degrading systems, by modeling probe structures to those of desired substrates.
RESUMO
Exo-ß-mannosidases are a broad class of stereochemically retaining hydrolases that are essential for the breakdown of complex carbohydrate substrates found in all kingdoms of life. Yet the detection of exo-ß-mannosidases in complex biological samples remains challenging, necessitating the development of new methodologies. Cyclophellitol and its analogues selectively label the catalytic nucleophiles of retaining glycoside hydrolases, making them valuable tool compounds. Furthermore, cyclophellitol can be readily redesigned to enable the incorporation of a detection tag, generating activity-based probes (ABPs) that can be used to detect and identify specific glycosidases in complex biological samples. Towards the development of ABPs for exo-ß-mannosidases, we present a concise synthesis of ß-manno-configured cyclophellitol, cyclophellitol aziridine, and N-alkyl cyclophellitol aziridines. We show that these probes covalently label exo-ß-mannosidases from GH families 2, 5, and 164. Structural studies of the resulting complexes support a canonical mechanism-based mode of action in which the active site nucleophile attacks the pseudoanomeric centre to form a stable ester linkage, mimicking the glycosyl enzyme intermediate. Furthermore, we demonstrate activity-based protein profiling using an N-alkyl aziridine derivative by specifically labelling MANBA in mouse kidney tissue. Together, these results show that synthetic manno-configured cyclophellitol analogues hold promise for detecting exo-ß-mannosidases in biological and biomedical research.
Assuntos
Cicloexanóis/química , Sondas Moleculares/química , beta-Manosidase/análise , Cicloexanóis/síntese química , Conformação Molecular , Sondas Moleculares/síntese química , beta-Manosidase/metabolismoRESUMO
Gaucher disease (GD) is a lysosomal storage disorder caused by inherited deficiencies in ß-glucocerebrosidase (GBA). Current treatments require rapid disease diagnosis and a means of monitoring therapeutic efficacy, both of which may be supported by the use of GBA-targeting activity-based probes (ABPs). Here, we report the synthesis and structural analysis of a range of cyclophellitol epoxide and aziridine inhibitors and ABPs for GBA. We demonstrate their covalent mechanism-based mode of action and uncover binding of the new N-functionalised aziridines to the ligand binding cleft. These inhibitors became scaffolds for the development of ABPs; the O6-fluorescent tags of which bind in an allosteric site at the dimer interface. Considering GBA's preference for O6- and N-functionalised reagents, a bi-functional aziridine ABP was synthesized as a potentially more powerful imaging agent. Whilst this ABP binds to two unique active site clefts of GBA, no further benefit in potency was achieved over our first generation ABPs. Nevertheless, such ABPs should serve useful in the study of GBA in relation to GD and inform the design of future probes.
Assuntos
Corantes Fluorescentes , Glucosilceramidase , Domínio Catalítico , Glucosilceramidase/metabolismoRESUMO
There is a vast genomic resource for enzymes active on carbohydrates. Lagging far behind, however, are functional chemical tools for the rapid characterization of carbohydrate-active enzymes. Activity-based probes (ABPs) offer one chemical solution to these issues with ABPs based upon cyclophellitol epoxide and aziridine covalent and irreversible inhibitors representing a potent and widespread approach. Such inhibitors for enzymes active on polysaccharides are potentially limited by the requirement for several glycosidic bonds, themselves substrates for the enzyme targets. Here, it is shown that non-hydrolysable trisaccharide can be synthesized and applied even to enzymes with challenging subsite requirements. It was found that incorporation of carbasugar moieties, which was accomplished by cuprate-assisted regioselective trans-diaxial epoxide opening of carba-mannal synthesised for this purpose, yields inactivators that act as powerful activity-based inhibitors for α-1,6 endo-mannanases. 3-D structures at 1.35-1.47â Å resolutions confirm the design rationale and binding to the enzymatic nucleophile. Carbasugar oligosaccharide cyclophellitols offer a powerful new approach for the design of robust endoglycosidase inhibitors, while the synthesis procedures presented here should allow adaptation towards activity-based endoglycosidase probes as well as configurational isosteres targeting other endoglycosidase families.
Assuntos
Carbaçúcares , Glicosídeo Hidrolases , Oligossacarídeos , Compostos de EpóxiRESUMO
Glycosidase inhibitors have shown great potential as pharmacological chaperones for lysosomal storage diseases. In light of this, a series of new cyclopentanoid ß-galactosidase inhibitors were prepared and their inhibitory and pharmacological chaperoning activities determined and compared with those of lipophilic analogs of the potent ß-d-galactosidase inhibitor 4-epi-isofagomine. Structure-activity relationships were investigated by X-ray crystallography as well as by alterations in the cyclopentane moiety such as deoxygenation and replacement by fluorine of a "strategic" hydroxyl group. New compounds have revealed highly promising activities with a range of ß-galactosidase-compromised human cell lines and may serve as leads towards new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.
Assuntos
Ciclopentanos/farmacologia , Galactosidases/metabolismo , Imino Piranoses/farmacologia , Lisossomos/enzimologia , Chaperonas Moleculares/metabolismo , Cristalização , Ciclopentanos/síntese química , Ciclopentanos/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Galactosidases/antagonistas & inibidores , Humanos , Imino Piranoses/síntese química , Imino Piranoses/química , Ligantes , Lisossomos/efeitos dos fármacos , Conformação Molecular , Proteínas Mutantes/metabolismoRESUMO
Correction for 'Synthesis and application of a highly branched, mechanism-based 2-deoxy-2-fluoro-oligosaccharide inhibitor of endo-xyloglucanases' by Namrata Jain et al., Org. Biomol. Chem., 2018, 16, 8732-8741.
RESUMO
Xyloglucan (XyG) is a complex polysaccharide that is ubiquitous and often abundant in the cell walls of terrestrial plants. XyG metabolism is therefore a key component of the global carbon cycle, and hence XyG enzymology is of significant fundamental and applied importance in biomass conversion. To facilitate structure-function analyses of XyG-specific endo-glucanases, we have synthesized a 2',4'-dinitrophenyl 2-deoxy-2-fluoro-ß-glycoside mechanism-based inhibitor based on the highly branched XyG repeating motif XXXG (Xyl3Glc4: ([α-d-Xylp-(1â6)]-ß-d-Glcp-(1â4)-[α-d-Xylp-(1â6)]-ß-d-Glcp-(1â4)-[α-d-Xylp-(1â6)]-ß-d-Glcp-(1â4)-d-Glcp. Key steps in the chemo-enzymatic synthesis included selective enzyme hydrolysis of XyG polysaccharide to produce the core heptasaccharide, per-O-acetylation, α-bromination, reductive glycal formation, electrophilic fluorination, SNAr glycosylation, and Zemplen deprotection. The resulting compound, XXXG(2F)-ß-DNP, specifically labelled the active sites of several endo-(xylo)glucanases by accumulation of a covalent glycosyl-enzyme intermediate, as revealed by intact protein mass spectrometry. Crystallography of a complex with a Cellvibrio japonicus Glycoside Hydrolase Family 5 (GH5) endo-xyloglucanase corroborated the covalent nature of the intermediate, and further revealed the anticipated specificity for the catalytic nucleophile of this anomeric-configuration-retaining glycosidase. This specificity complements that of an analogous XXXG N-bromoacetylglycosylamine inhibitor, which labelled the catalytic acid-base sidechain in the same enzyme [Attia, et al., Biotechnol. Biofuels, 2018, 11, 45]. We anticipate that these inhibitors may find continued use in mechanistic analyses of endo-(xylo)glucanases from diverse GH families.
RESUMO
The elucidation and prediction of how changes in a protein result in altered activities and selectivities remain a major challenge in chemistry. Two hurdles have prevented accurate family-wide models: obtaining (i) diverse datasets and (ii) suitable parameter frameworks that encapsulate activities in large sets. Here, we show that a relatively small but broad activity dataset is sufficient to train algorithms for functional prediction over the entire glycosyltransferase superfamily 1 (GT1) of the plant Arabidopsis thaliana. Whereas sequence analysis alone failed for GT1 substrate utilization patterns, our chemical-bioinformatic model, GT-Predict, succeeded by coupling physicochemical features with isozyme-recognition patterns over the family. GT-Predict identified GT1 biocatalysts for novel substrates and enabled functional annotation of uncharacterized GT1s. Finally, analyses of GT-Predict decision pathways revealed structural modulators of substrate recognition, thus providing information on mechanisms. This multifaceted approach to enzyme prediction may guide the streamlined utilization (and design) of biocatalysts and the discovery of other family-wide protein functions.
Assuntos
Proteínas de Arabidopsis/metabolismo , Biologia Computacional/métodos , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Relação Estrutura-Atividade , Algoritmos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Domínio Catalítico , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Mutagênese Sítio-Dirigida , Novobiocina/metabolismo , Filogenia , Resveratrol/metabolismoRESUMO
Gluco-azoles competitively inhibit glucosidases by transition-state mimicry and their ability to interact with catalytic acid residues in glucosidase active sites. We noted that no azole-type inhibitors described, to date, possess a protic nitrogen characteristic for 1 H-imidazoles. Here, we present gluco-1 H-imidazole, a gluco-azole bearing a 1 H-imidazole fused to a glucopyranose-configured cyclitol core, and three close analogues as new glucosidase inhibitors. All compounds inhibit human retaining ß-glucosidase, GBA1, with the most potent ones inhibiting this enzyme (deficient in Gaucher disease) on a par with glucoimidazole. None inhibit glucosylceramide synthase, cytosolic ß-glucosidase GBA2 or α-glucosidase GAA. Structural, physical and computational studies provide first insights into the binding mode of this conceptually new class of retaining ß-glucosidase inhibitors.
Assuntos
Azóis/farmacologia , Inibidores Enzimáticos/farmacologia , beta-Glucosidase/antagonistas & inibidores , Azóis/síntese química , Azóis/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Conformação Molecular , beta-Glucosidase/metabolismoRESUMO
BACKGROUND: Xyloglucan (XyG) is a ubiquitous and fundamental polysaccharide of plant cell walls. Due to its structural complexity, XyG requires a combination of backbone-cleaving and sidechain-debranching enzymes for complete deconstruction into its component monosaccharides. The soil saprophyte Cellvibrio japonicus has emerged as a genetically tractable model system to study biomass saccharification, in part due to its innate capacity to utilize a wide range of plant polysaccharides for growth. Whereas the downstream debranching enzymes of the xyloglucan utilization system of C. japonicus have been functionally characterized, the requisite backbone-cleaving endo-xyloglucanases were unresolved. RESULTS: Combined bioinformatic and transcriptomic analyses implicated three glycoside hydrolase family 5 subfamily 4 (GH5_4) members, with distinct modular organization, as potential keystone endo-xyloglucanases in C. japonicus. Detailed biochemical and enzymatic characterization of the GH5_4 modules of all three recombinant proteins confirmed particularly high specificities for the XyG polysaccharide versus a panel of other cell wall glycans, including mixed-linkage beta-glucan and cellulose. Moreover, product analysis demonstrated that all three enzymes generated XyG oligosaccharides required for subsequent saccharification by known exo-glycosidases. Crystallographic analysis of GH5D, which was the only GH5_4 member specifically and highly upregulated during growth on XyG, in free, product-complex, and active-site affinity-labelled forms revealed the molecular basis for the exquisite XyG specificity among these GH5_4 enzymes. Strikingly, exhaustive reverse-genetic analysis of all three GH5_4 members and a previously biochemically characterized GH74 member failed to reveal a growth defect, thereby indicating functional compensation in vivo, both among members of this cohort and by other, yet unidentified, xyloglucanases in C. japonicus. Our systems-based analysis indicates distinct substrate-sensing (GH74, GH5E, GH5F) and attack-mounting (GH5D) functions for the endo-xyloglucanases characterized here. CONCLUSIONS: Through a multi-faceted, molecular systems-based approach, this study provides a new insight into the saccharification pathway of xyloglucan utilization system of C. japonicus. The detailed structural-functional characterization of three distinct GH5_4 endo-xyloglucanases will inform future bioinformatic predictions across species, and provides new CAZymes with defined specificity that may be harnessed in industrial and other biotechnological applications.
RESUMO
Activity-based protein profiling has emerged as a powerful tool for visualizing glycosidases in complex biological samples. Several configurational cyclophellitol isomers have been shown to display high selectivity as probes for glycosidases processing substrates featuring the same configuration. Here, a set of deoxygenated cyclophellitols are presented which enable inter-class profiling of ß-glucosidases and ß-galactosidases.
RESUMO
The essential biological roles played by glycosidases, coupled to the diverse therapeutic benefits of pharmacologically targeting these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivalents of epoxides in organic synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here we present the synthesis, conformational analysis, and application of novel 1,6-cyclophellitol cyclosulfates. We show that 1,6-epi-cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose 4C1 chair conformation matches that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (ß-cyclosulfate) reacts more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions.
RESUMO
The conformational analysis of glycosidases affords a route to their specific inhibition through transition-state mimicry. Inspired by the rapid reaction rates of cyclophellitol and cyclophellitol aziridine-both covalent retaining ß-glucosidase inhibitors-we postulated that the corresponding carba "cyclopropyl" analogue would be a potent retaining ß-glucosidase inhibitor for those enzymes reacting through the 4H3 transition-state conformation. Ab initio metadynamics simulations of the conformational free energy landscape for the cyclopropyl inhibitors show a strong bias for the 4H3 conformation, and carba-cyclophellitol, with an N-(4-azidobutyl)carboxamide moiety, proved to be a potent inhibitor (Ki = 8.2 nM) of the Thermotoga maritima TmGH1 ß-glucosidase. 3-D structural analysis and comparison with unreacted epoxides show that this compound indeed binds in the 4H3 conformation, suggesting that conformational strain induced through a cyclopropyl unit may add to the armory of tight-binding inhibitor designs.
Assuntos
Cicloexanóis/farmacologia , Inibidores de Glicosídeo Hidrolases/farmacologia , alfa-Glucosidases/metabolismo , Cicloexanóis/química , Inibidores de Glicosídeo Hidrolases/química , Modelos Moleculares , Estrutura Molecular , Teoria Quântica , Thermotoga maritima/enzimologiaRESUMO
O-GlcNAc hydrolase (OGA) removes O-linked N-acetylglucosamine (O-GlcNAc) from a myriad of nucleocytoplasmic proteins. Through co-expression and assembly of OGA fragments, we determined the three-dimensional structure of human OGA, revealing an unusual helix-exchanged dimer that lays a structural foundation for an improved understanding of substrate recognition and regulation of OGA. Structures of OGA in complex with a series of inhibitors define a precise blueprint for the design of inhibitors that have clinical value.
Assuntos
Modelos Moleculares , beta-N-Acetil-Hexosaminidases/química , Acetilglucosamina/metabolismo , Sítios de Ligação , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Ligantes , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
The industrial conversion of cellulosic plant biomass into useful products such as biofuels is a major societal goal. These technologies harness diverse plant degrading enzymes, classical exo- and endo-acting cellulases and, increasingly, cellulose-active lytic polysaccharide monooxygenases, to deconstruct the recalcitrant ß-D-linked polysaccharide. A major drawback with this process is that the exo-acting cellobiohydrolases suffer from severe inhibition from their cellobiose product. ß-D-Glucosidases are therefore important for liberating glucose from cellobiose and thereby relieving limiting product inhibition. Here, the three-dimensional structures of two industrially important family GH3 ß-D-glucosidases from Aspergillus fumigatus and A. oryzae, solved by molecular replacement and refined at 1.95â Å resolution, are reported. Both enzymes, which share 78% sequence identity, display a three-domain structure with the catalytic domain at the interface, as originally shown for barley ß-D-glucan exohydrolase, the first three-dimensional structure solved from glycoside hydrolase family GH3. Both enzymes show extensive N-glycosylation, with only a few external sites being truncated to a single GlcNAc molecule. Those glycans N-linked to the core of the structure are identified purely as high-mannose trees, and establish multiple hydrogen bonds between their sugar components and adjacent protein side chains. The extensive glycans pose special problems for crystallographic refinement, and new techniques and protocols were developed especially for this work. These protocols ensured that all of the D-pyranosides in the glycosylation trees were modelled in the preferred minimum-energy (4)C1 chair conformation and should be of general application to refinements of other crystal structures containing O- or N-glycosylation. The Aspergillus GH3 structures, in light of other recent three-dimensional structures, provide insight into fungal ß-D-glucosidases and provide a platform on which to inform and inspire new generations of variant enzymes for industrial application.
Assuntos
Aspergillus/enzimologia , Proteínas Fúngicas/química , beta-Glucosidase/química , Sequência de Aminoácidos , Configuração de Carboidratos , Sequência de Carboidratos , Domínio Catalítico , Celulose/química , Cristalografia por Raios X , Glicoproteínas/química , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
The enzyme-catalysed degradation of starch is central to many industrial processes, including sugar manufacture and first-generation biofuels. Classical biotechnological platforms involve steam explosion of starch followed by the action of endo-acting glycoside hydrolases termed α-amylases and then exo-acting α-glucosidases (glucoamylases) to yield glucose, which is subsequently processed. A key enzymatic player in this pipeline is the `Termamyl' class of bacterial α-amylases and designed/evolved variants thereof. Here, the three-dimensional structure of one such Termamyl α-amylase variant based upon the parent Geobacillus stearothermophilus α-amylase is presented. The structure has been solved at 1.9â Å resolution, revealing the classical three-domain fold stabilized by Ca2+ and a Ca2+-Na+-Ca2+ triad. As expected, the structure is similar to the G. stearothermophilus α-amylase but with main-chain deviations of up to 3â Å in some regions, reflecting both the mutations and differing crystal-packing environments.
Assuntos
Proteínas de Bactérias/química , Geobacillus stearothermophilus/enzimologia , alfa-Amilases/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Secundária de Proteína , Homologia Estrutural de ProteínaRESUMO
ß-N-acetylhexosaminidases (HEX) are glycosidases that catalyze the glycosidic linkage hydrolysis of gluco- and galacto-configured N-acetyl-ß-d-hexosaminides. These enzymes are important in human physiology and are candidates for the biocatalytic production of carbohydrates and glycomimetics. In this study, the three-dimensional structure of the wild-type and catalytically impaired E302Q HEX variant from the soil bacterium Streptomyces coelicolor A3(2) (ScHEX) were solved in ligand-free forms and in the presence of 6-acetamido-6-deoxy-castanospermine (6-Ac-Cas). The E302Q variant was also trapped as an intermediate with oxazoline bound to the active center. Crystallographic evidence highlights structural variations in the loop 3 environment, suggesting conformational heterogeneity for important active-site residues of this GH20 family member. The enzyme was investigated for its ß-N-acetylhexosaminidase activity toward chitooligomers and pNP-acetyl gluco- and galacto-configured N-acetyl hexosaminides. Kinetic analyses confirm the ß(1-4) glycosidic linkage substrate preference, and HPLC profiles support an exoglycosidase mechanism, where the enzyme cleaves sugars from the nonreducing end of substrates. ScHEX possesses significant activity toward chitooligosaccharides of varying degrees of polymerization, and the final hydrolytic reaction yielded pure GlcNAc without any byproduct, promising high applicability for the enzymatic production of this highly valued chemical. Thermostability and activation assays further suggest efficient conditions applicable to the enzymatic production of GlcNAc from chitooligomers.
Assuntos
Streptomyces coelicolor/enzimologia , beta-N-Acetil-Hexosaminidases/química , beta-N-Acetil-Hexosaminidases/metabolismo , Catálise , Domínio Catalítico/genética , Cristalografia por Raios X , Ativação Enzimática/genética , Variação Genética , Humanos , Streptomyces coelicolor/genética , Streptomyces lividans/enzimologia , Relação Estrutura-Atividade , beta-N-Acetil-Hexosaminidases/genéticaRESUMO
The recently discovered futalosine pathway is a promising target for the development of new antibiotics. The enzymes involved in this pathway are crucial for the biosynthesis of the essential prokaryotic respiratory compound menaquinone, and as the pathway is limited to few bacterial species such as the gastric pathogen Helicobacter pylori it is a potential target for specific antibiotics. In this report, the crystal structure of an H. pylori methylthioadenosine nucleosidase (MTAN; an enzyme with broad specificity and activity towards 6-amino-6-deoxyfutalosine), which is involved in the second step of menaquinone biosynthesis, has been elucidated at a resolution of 1.76â Å and refined with R factors of Rwork = 17% and Rfree = 21%. Activity studies on the wild type and active-site mutants show that the hydrolysis of 6-amino-6-deoxyfutalosine follows a mechanism similar to that of Escherichia coli MTAN. Further evidence for this mode of action is supplied by the crystal structures of active-site mutants. Through the use of reaction intermediates, the structures give additional evidence for the previously proposed nucleosidase mechanism. These structures and the confirmed reaction mechanism will provide a structural basis for the design of new inhibitors targeting the futalosine pathway.
Assuntos
Helicobacter pylori/enzimologia , Nucleosídeos/metabolismo , Purina-Núcleosídeo Fosforilase/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Helicobacter pylori/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Purina-Núcleosídeo Fosforilase/metabolismo , Transdução de SinaisRESUMO
The enzymatic degradation of plant polysaccharides is emerging as one of the key environmental goals of the early 21st century, impacting on many processes in the textile and detergent industries as well as biomass conversion to biofuels. One of the well known problems with the use of nonstarch (nonfood)-based substrates such as the plant cell wall is that the cellulose fibers are embedded in a network of diverse polysaccharides, including xyloglucan, that renders access difficult. There is therefore increasing interest in the "accessory enzymes," including xyloglucanases, that may aid biomass degradation through removal of "hemicellulose" polysaccharides. Here, we report the biochemical characterization of the endo-ß-1,4-(xylo)glucan hydrolase from Paenibacillus polymyxa with polymeric, oligomeric, and defined chromogenic aryl-oligosaccharide substrates. The enzyme displays an unusual specificity on defined xyloglucan oligosaccharides, cleaving the XXXG-XXXG repeat into XXX and GXXXG. Kinetic analysis on defined oligosaccharides and on aryl-glycosides suggests that both the -4 and +1 subsites show discrimination against xylose-appended glucosides. The three-dimensional structures of PpXG44 have been solved both in apo-form and as a series of ligand complexes that map the -3 to -1 and +1 to +5 subsites of the extended ligand binding cleft. Complex structures are consistent with partial intolerance of xylosides in the -4' subsites. The atypical specificity of PpXG44 may thus find use in industrial processes involving xyloglucan degradation, such as biomass conversion, or in the emerging exciting applications of defined xyloglucans in food, pharmaceuticals, and cellulose fiber modification.