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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↗

PubMed2005

Mutational analysis of target enzyme recognition of the beta-trefoil fold barley alpha-amylase/subtilisin inhibitor.

The barley alpha-amylase/subtilisin inhibitor (BASI) inhibits alpha-amylase 2 (AMY2) with subnanomolar affinity. The contribution of selected side chains of BASI to this high affinity is discerned in this study, and binding to other targets is investigated. Seven BASI residues along the AMY2-BASI interface and four residues in the putative protease-binding loop on the opposite side of the inhibitor were mutated. A total of 15 variants were compared with the wild type by monitoring the alpha-amylase and protease inhibitory activities using Blue Starch and azoalbumin, respectively, and the kinetics of binding to target enzymes by surface plasmon resonance. Generally, the mutations had little effect on k(on), whereas the k(off) values were increased up to 67-fold. The effects on the inhibitory activity, however, were far more pronounced, and the K(i) values of some mutants on the AMY2-binding side increased 2-3 orders of magnitude, whereas mutations on the other side of the inhibitor had virtually no effect. The mutants K140L, D150N, and E168T lost inhibitory activity, revealing the pivotal role of charge interactions for BASI activity on AMY2. A fully hydrated Ca(2+) at the AMY2-BASI interface mediates contacts to the catalytic residues of AMY2. Mutations involving residues contacting the solvent ligands of this Ca(2+) had weaker affinity for AMY2 and reduced sensitivity to the Ca(2+) modulation of the affinity. These results suggest that the Ca(2+) and its solvation sphere are integral components of the AMY2-BASI complex, thus illuminating a novel mode of inhibition and a novel role for calcium in relation to glycoside hydrolases.

Amino Acid Sequence↗

PubMed2001

Thiooligosaccharides as tools for structural biology.

Oligosaccharides in which at least one glycosidic oxygen atom is replaced with a sulfur atom can be routinely synthesized and act as competitive inhibitors of various glycoside hydrolases. Recent studies using both X-ray crystallography and other biophysical techniques provide structural insight into binding, recognition, and the catalytic mechanism of action of these enzymes.

Carbohydrate Conformation↗

PubMed2005

A novel alpha-glucosidase from the acidophilic archaeon Ferroplasma acidiphilum strain Y with high transglycosylation activity and an unusual catalytic nucleophile.

Ferroplasma acidiphilum strain Y (DSM 12658), a ferrous iron-oxidizing, acidophilic and mesophilic archaeon, was found to produce a membrane-bound alpha-glucosidase (alphaGluFa) showing no significant similarity to any of the known glycoside hydrolases classified in different families and having an unusual catalytic site consisting of a threonine and a histidine residue. The highest alpha-glucosidase activity was found at low pH, 2.4-3.5, and the substrate preference order was: sucrose>maltose>maltotriose >>maltotetraose>>malto-oligosaccharides from maltopentaose to maltoheptaose>>>soluble starch (kcat/K(m) was 293.0, 197.0, 18.8, 0.3 and 0.02 s(-1) x mM(-1) respectively). The enzyme was able to transfer glucosyl groups from maltose as donor, to produce exclusively maltotriose (up to 300 g/l). Chemical modification and electrospray ionization MS analysis of 5-fluoro-alpha-D-glucopyranosyl-enzyme derivatives, coupled with site-directed mutagenesis, strongly suggested that the putative catalytic nucleophile in this enzyme is Thr212. Iron was found to be essential for enzyme activity and integrity, and His390 was shown to be essential for iron binding. These results suggest that the metalloenzyme alphaGluFa is a new member of the glycosyl hydrolase family that uses a novel mechanism for sugar glycosylation and/or transglycosylation.

Archaea↗

PubMed2005

NAD+ and metal-ion dependent hydrolysis by family 4 glycosidases: structural insight into specificity for phospho-beta-D-glucosides.

The import of disaccharides by many bacteria is achieved through their simultaneous translocation and phosphorylation by the phosphoenolpyruvate-dependent phosphotransferase system (PEP-PTS). The imported phospho-disaccharides are, in some cases, subsequently hydrolyzed by members of the unusual glycoside hydrolase family GH4. The GH4 enzymes, occasionally found also in bacteria such as Thermotoga maritima that do not utilise a PEP-PTS system, require both NAD(+) and Mn(2+) for catalysis. A further curiosity of this family is that closely related enzymes may show specificity for either alpha-d- or beta-d-glycosides. Here, we present, for the first time, the three-dimensional structure (using single-wavelength anomalous dispersion methods, harnessing extensive non-crystallographic symmetry) of the 6-phospho-beta-glycosidase, BglT, from T.maritima in native and complexed (NAD(+) and Glc6P) forms. Comparison of the active-center structure with that of the 6-phospho-alpha-glucosidase GlvA from Bacillus subtilis reveals a striking degree of structural similarity that, in light of previous kinetic isotope effect data, allows the postulation of a common reaction mechanism for both alpha and beta-glycosidases. Given that the "chemistry" occurs primarily on the glycone sugar and features no nucleophilic attack on the intact disaccharide substrate, modulation of anomeric specificity for alpha and beta-linkages is accommodated through comparatively minor structural changes.

Bacterial Proteins↗

PubMed1999

Isolation and characterization of the gene encoding the starch debranching enzyme limit dextrinase from germinating barley.

The gene encoding the starch debranching enzyme limit dextrinase, LD, from barley (Hordeum vulgare), was isolated from a genomic phage library using a barley cDNA clone as probe. The gene encodes a protein of 904 amino acid residues with a calculated molecular mass of 98.6 kDa. This is in agreement with a value of 105 kDa estimated by SDS-PAGE. The coding sequence is interrupted by 26 introns varying in length from 93 bp to 825 bp. The 27 exons vary in length from 53 bp to 197 bp. Southern blot analysis shows that the limit dextrinase gene is present as a single copy in the barley genome. Gene expression is high during germination and the steady state transcription level reaches a maximum at day 5 of germination. The deduced amino acid sequence corresponds to the protein sequence of limit dextrinase purified from germinating malt, as determined by automated N-terminal sequencing of tryptic fragments coupled with matrix assisted laser desorption mass spectrometry. The sequenced peptide fragments cover 70% of the entire protein sequence, which shows 62% and 77% identity to that of starch debranching enzymes from spinach and rice and 37% identity to Klebsiella pullulanase. Sequence alignment supports the multidomain architecture and identifies both secondary structure elements of the catalytic (beta/alpha)8-barrel substrate, catalytic residues, and specificity associated motifs characteristic of members of the glycoside hydrolase family 13 which cleave alpha-1,6-glucosidic bonds. A remarkable distribution of the secondary structure elements to individual exons is observed.

Amino Acid Sequence↗

PubMed2002

Investigation of the microheterogeneity and aglycone specificity-conferring residues of black cherry prunasin hydrolases.

In black cherry (Prunus serotina Ehrh.) seed homogenates, (R)-amygdalin is degraded to HCN, benzaldehyde, and glucose by the sequential action of amygdalin hydrolase (AH), prunasin hydrolase (PH), and mandelonitrile lyase. Leaves are also highly cyanogenic because they possess (R)-prunasin, PH, and mandelonitrile lyase. Taking both enzymological and molecular approaches, we demonstrate here that black cherry PH is encoded by a putative multigene family of at least five members. Their respective cDNAs (designated Ph1, Ph2, Ph3, Ph4, and Ph5) predict isoforms that share 49% to 92% amino acid identity with members of glycoside hydrolase family 1, including their catalytic asparagine-glutamate-proline and isoleucine-threonine-glutamate-asparagine-glycine motifs. Furthermore, consistent with the vacuolar/protein body location and glycoprotein character of these hydrolases, their open reading frames predict N-terminal signal sequences and multiple potential N-glycosylation sites. Genomic sequences corresponding to the open reading frames of these PHs and of the previously isolated AH1 isoform are interrupted at identical positions by 12 introns. Earlier studies established that native AH and PH display strict specificities toward their respective glucosidic substrates. Such behavior was also shown by recombinant AH1, PH2, and PH4 proteins after expression in Pichia pastoris. Three amino acid moieties that may play a role in conferring such aglycone specificities were predicted by structural modeling and comparative sequence analysis and tested by introducing single and multiple mutations into isoform AH1 by site-directed mutagenesis. The double mutant AH ID (Y200I and G394D) hydrolyzed prunasin at approximately 150% of the rate of amygdalin hydrolysis, whereas the other mutations failed to engender PH activity.

Aldehyde-Lyases↗

PubMed2000

Biochemical analysis of recombinant fungal mutanases. A new family of alpha1,3-glucanases with novel carbohydrate-binding domains.

Nucleotide sequence analysis shows that Trichoderma harzianum and Penicillium purpurogenum alpha1,3-glucanases (mutanases) have homologous primary structures (53% amino acid sequence identity), and are composed of two distinct domains: a NH(2)-terminal catalytic domain and a putative COOH-terminal polysaccharide-binding domain separated by a O-glycosylated Pro-Ser-Thr-rich linker peptide. Each mutanase was expressed in Aspergillus oryzae host under the transcriptional control of a strong alpha-amylase gene promoter. The purified recombinant mutanases show a pH optimum in the range from pH 3.5 to 4.5 and a temperature optimum around 50-55 degrees C at pH 5.5. Also, they exhibit strong binding to insoluble mutan with K(D) around 0.11 and 0.13 microM at pH 7 for the P. purpurogenum and T. harzianum mutanases, respectively. Partial hydrolysis showed that the COOH-terminal domain of the T. harzianum mutanase binds to mutan. The catalytic domains and the binding domains were assigned to a new family of glycoside hydrolases and to a new family of carbohydrate-binding domains, respectively.

Amino Acid Sequence↗

PubMed2003

Two alpha-L-arabinofuranosidase genes in Arabidopsis thaliana are differentially expressed during vegetative growth and flower development.

Glycosyl hydrolases are important mediators of plant cell wall modification during plant development. These enzymes catalyse the hydrolytic release of specific sugars, such as L-arabinose, from the polysaccharide-rich cell wall matrix. The cloning and expression analysis of two genes, AtASD1 and AtASD2, encoding putative alpha-L-arabinofuranosidases in Arabidopsis thaliana are reported here. AtASD1 and AtASD2 identities were assigned on the basis of homology to plant and microbial family 51 glycoside hydrolases. Using RT-PCR, RNA gel blot analysis and reporter gene expression analysis, AtASD1 and AtASD2 were shown to have different developmental expression profiles. High levels of AtASD1 promoter activity are present in multiple tissues during vegetative and reproductive growth. AtASD1 expression is particularly intense in zones of cell proliferation, the vascular system, developing and regressing floral tissues, and floral abscission zones. By comparison, AtASD2 expression is limited to the vasculature of older root tissue and to some floral organs and floral abscission zones.

Amino Acid Sequence↗

PubMed2001

Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes.

The hydrolases and transferases that constitute the alpha-amylase family are multidomain proteins, but each has a catalytic domain in the form of a (beta/alpha)(8)-barrel, with the active site being at the C-terminal end of the barrel beta-strands. Although the enzymes are believed to share the same catalytic acids and a common mechanism of action, they have been assigned to three separate families - 13, 70 and 77 - in the classification scheme for glycoside hydrolases and transferases that is based on amino acid sequence similarities. Each enzyme has one glutamic acid and two aspartic acid residues necessary for activity, while most enzymes of the family also contain two histidine residues critical for transition state stabilisation. These five residues occur in four short sequences conserved throughout the family, and within such sequences some key amino acid residues are related to enzyme specificity. A table is given showing motifs distinctive for each specificity as extracted from 316 sequences, which should aid in identifying the enzyme from primary structure information. Where appropriate, existing problems with identification of some enzymes of the family are pointed out. For enzymes of known three-dimensional structure, action is discussed in terms of molecular architecture. The sequence-specificity and structure-specificity relationships described may provide useful pointers for rational protein engineering.

Amino Acid Sequence↗

PubMed2002

Cloning, sequencing and overexpression of a Sinorhizobium meliloti M5N1CS carboxymethyl-cellulase gene.

The EndS encoding sequence was isolated from Sinorhizobium meliloti M5N1CS DNA. Comparisons between the deduced amino acid sequence of the mature EndS (337 amino acids, molecular mass 36,418 Da, isoelectric point 4.92) and those of published beta-glycanases showed that this enzyme belongs to family 5 of the glycoside hydrolases. The protein was overproduced in Escherichia coli using a T7 expression system. When the purified overexpressed EndS protein was tested on cellulose-type components, the best substrate was CM-cellulose.

Base Sequence↗

PubMed2006

Induction of alpha-L-arabinofuranosidase activity by monomeric carbohydrates in Bifidobacterium longum and ubiquity of encoding genes.

Bifidobacterium longum can be isolated from human faeces, some strains being considered probiotics. B. longum NIZO B667 produces an exo-acting alpha-L-arabinofuranosidase, AbfB, previously purified by us, that releases L-arabinose from arabinan and arabinoxylan. This activity was subjected to two-seven-fold induction by L-arabinose, D-xylose, L-arabitol and xylitol and to repression by glucose. Maximum activity was obtained at 48 h incubation except for D-xylose that was at 24 h. High concentrations (200 mM) of L-arabitol also caused repression of the arabinofuranosidase. A unique band of activity showing the same migration pattern as the purified AbfB was found in zymograms of cell free extracts, indicating that the activity was likely due to this sole enzyme. The assessment of the influence of inducers and repressors on the activity of AbfB and on the expression of the abfB gene by real time PCR indicated that regulation was transcriptional. DNA amplifications using a pair of degenerated primers flanking an internal fragment within alpha-L-arabinofuranosidase genes of the family 51 of glycoside hydrolases evidenced that these enzymes are widespread in Bifidobacterium. The aminoacidic sequences of bifidobacteria included a fragment of four to six residues in the position 136-141 that was absent in other microorganisms.

Arabinose↗

PubMed2002

Purification, characterization, cloning, and expression of a novel xyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase.

A novel oligoxyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase (OXG-RCBH), with a molecular mass of 97 kDa and a pI of 6.1, was isolated from the fungus Geotrichum sp. M128. Analysis of substrate specificity using various xyloglucan oligosaccharide structures revealed that OXG-RCBH had exoglucanase activity. It recognized the reducing end of oligoxyloglucan and released two glucosyl residue segments from the main chain. The full-length cDNA encoding OXG-RCBH was cloned and sequenced, and it had a 2436-bp open reading frame encoding an 812amino acid protein. The deduced protein showed approximately 35% identity to members of glycoside hydrolase family 74. The cDNA encoding OXG-RCBH was then expressed in Escherichia coli. Although the recombinant protein was expressed as an inclusion body, renaturation was successful, and enzymatically active recombinant OXG-RCBH was obtained.

Amino Acid Sequence↗

PubMed2006

Crystallization and preliminary crystallographic analysis of the family GH78 alpha-L-rhamnosidase RhaB from Bacillus sp. GL1.

Alpha-L-rhamnosidases play important roles in the metabolism of plant cell walls, glycosides and bacterial biofilms. This enzyme is also used industrially for debittering citrus fruits by releasing rhamnose from the plant flavonoid naringin. Bacillus sp. GL1 alpha-L-rhamnosidase (RhaB) is a member of glycoside hydrolase (GH) family 78. Native and selenomethionine-derivative enzymes were crystallized at 293 K by hanging-drop vapour diffusion with polyethylene glycol 8000 as a precipitant. This is the first report of the crystallization of a family GH78 enzyme.

Bacillus↗

PubMed2006

Exo-oligosaccharides of Rhizobium sp. strain NGR234 are required for symbiosis with various legumes.

Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with beta-1,3, beta-1,4, beta-1,6, alpha-1,3, and alpha-1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGROmegaexoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGROmegaexoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)5(GlcA)2PvGal. When used as inoculants, both the exo-deficient mutants and NGROmegaexoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, approximately 50 microg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-beta-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes.

Bacterial Proteins↗

PubMed2005

The structure of a resuscitation-promoting factor domain from Mycobacterium tuberculosis shows homology to lysozymes.

Resuscitation-promoting factor (RPF) proteins reactivate stationary-phase cultures of (G+C)-rich Gram-positive bacteria including the causative agent of tuberculosis, Mycobacterium tuberculosis. We report the solution structure of the RPF domain from M. tuberculosis Rv1009 (RpfB) solved by heteronuclear multidimensional NMR. Structural homology with various glycoside hydrolases suggested that RpfB cleaved oligosaccharides. Biochemical studies indicate that a conserved active site glutamate is important for resuscitation activity. These data, as well as the presence of a clear binding pocket for a large molecule, indicate that oligosaccharide cleavage is probably the signal for revival from dormancy.

Bacterial Proteins↗

PubMed2004

Expression, purification, crystallization and preliminary X-ray analysis of alpha-L-arabinofuranosidase B from Aspergillus kawachii.

Alpha-L-Arabinofuranosidase (EC 3.2.1.55) is one of the hemicellulases that cleave the glycosidic bonds between L-arabinofuranoside side chains and various oligosaccharides. In this study, the first crystallization and preliminary X-ray analysis of alpha-L-arabinofuranosidase B from Aspergillus kawachii IFO4308 (AkAbfB), a family 54 glycoside hydrolase, is described. Recombinant AkAbfB was expressed in Escherichia coli and Pichia pastoris. The native crystals of recombinant AkAbfB produced by P. pastoris belong to the orthorhombic space group P2(1)2(1)2(1) (unit-cell parameters a = 39.5, b = 98.2, c = 144.0 A) and diffracted X-rays to a resolution of 1.82 A.

Aspergillus↗

PubMed2004

Chitobiose phosphorylase from Vibrio proteolyticus, a member of glycosyl transferase family 36, has a clan GH-L-like (alpha/alpha)(6) barrel fold.

Vibrio proteolyticus chitobiose phosphorylase (ChBP) belongs to glycosyl transferase family 36 (GT-36), and catalyzes the reversible phosphorolysis of chitobiose into alpha-GlcNAc-1-phosphate and GlcNAc with inversion of the anomeric configuration. As the first known structures of a GT-36 enzyme, we determined the crystal structure of ChBP in a ternary complex with GlcNAc and SO(4). It is also the first structures of an inverting phosphorolytic enzyme in a complex with a sugar and a sulfate ion, and reveals a pseudo-ternary complex structure of enzyme-sugar-phosphate. ChBP comprises a beta sandwich domain and an (alpha/alpha)(6) barrel domain, constituting a distinctive structure among GT families. Instead, it shows significant structural similarity with glycoside hydrolase (GH) enzymes, glucoamylases (GH-15), and maltose phosphorylase (GH-65) in clan GH-L. The structural similarity reported here, together with distant sequence similarities between ChBP and GHs, led to the reclassification of family GT-36 into a novel GH family, namely GH-94.

Amino Acid Sequence↗