A novel class of protein from wheat which inhibits xylanases. (1/428)

We have purified a novel class of protein that can inhibit the activity of endo-beta-1,4-xylanases. The inhibitor from wheat (Triticum aestivum, var. Soisson) is a glycosylated, monomeric, basic protein with a pI of 8.7-8.9, a molecular mass of 29 kDa and a unique N-terminal sequence of AGGKTGQVTVFWGRN. We have shown that the protein can inhibit the activity of two family-11 endo-beta-1, 4-xylanases, a recombinant enzyme from Aspergillus niger and an enzyme from Trichoderma viride. The inhibitory activity is heat and protease sensitive. The kinetics of the inhibition have been characterized with the A. niger enzyme using soluble wheat arabinoxylan as a substrate. The Km for soluble arabinoxylan in the absence of inhibitor is 20+/-2 mg/ml with a kcat of 103+/-6 s-1. The kinetics of the inhibition of this reaction are competitive, with a Ki value of 0.35 microM, showing that the inhibitor binds at or close to the active site of free xylanase. This report describes the first isolation of a xylanase inhibitor from any organism.  (+info)

Citric acid production from xylan and xylan hydrolysate by semi-solid culture of Aspergillus niger. (2/428)

Citric acid production from xylan and xylan hydrolysate was done by Aspergillus niger Yang no. 2 cultivated in a semi-solid culture using bagasse as a carrier. Yang no. 2 produced 72.4 g/l and 52.6 g/l of citric acid in 5 d from 140 g/l of xylose and arabinose, respectively. Yang no. 2 produced 51.6 g/l of citric acid in 3 d from a concentrated xylan hydrolysate prepared by cellulase treatment, containing 100 g/l of reducing sugars. Moreover, Yang no. 2 directly produced 39.6 g/l of citric acid maximally in 3 d from 140 g/l of xylan.  (+info)

Conserved sequence motifs in levansucrases and bifunctional beta-xylosidases and alpha-L-arabinases. (3/428)

Comparison of the amino acid sequences of two families of glycosyl hydrolases reveals that they are related in a region in the central part of the sequences. One of these families (GH family 68) includes levansucrases and the other one (glycosyl hydrolase family 43) includes bifunctional beta-xylosidases and alpha-L-arabinofuranosidases. The similarity of the primary structure of proteins from these families allows us to consider the invariant glutamate residue as a component of their active center. It is shown for the first time that glycosyl hydrolases recognizing different glycofuranoside residues can have a common sequence motif.  (+info)

Characterization of active-site aromatic residues in xylanase A from Streptomyces lividans. (4/428)

The role of four aromatic residues (W85, Y172, W266 and W274) in the structure-function relationship in xylanase A from Streptomyces lividans (XlnA) was investigated by site-directed mutagenesis where each residue was subjected to three substitutions (W85A/H/F; W266A/H/F; W274A/H/F and Y172A/F/S). These four amino acids are highly conserved among family 10 xylanases and structural data have implicated them in substrate binding at the active site. Far-UV circular dichroism spectroscopy was used to show that the overall structure of XlnA was not affected by any of these mutations. High-performance liquid chromatographic analysis of the hydrolysis products of birchwood xylan and xylopentaose showed that mutation of these aromatic residues did not alter the enzyme's mode of action. As expected, though, it did reduce the affinity of XlnA for birchwood xylan. A comparison of the kinetic parameters of different mutants at the same position demonstrated the importance of the aromatic nature of W85, Y172 and W274 in substrate binding. Replacement of these residues by a phenylalanine resulted in mutant proteins with a K(M) closer to that of the wild-type protein in comparison with the other mutations analyzed. The kinetic analysis of the mutant proteins at position W266 indicated that this amino acid is important for both substrate binding and efficient catalysis by XlnA. These studies also demonstrated the crucial role of these active site aromatic residues for the thermal stability of XlnA.  (+info)

Significant enhancement in the binding of p-nitrophenyl-beta-D-xylobioside by the E128H mutant F/10 xylanase from Streptomyces olivaceoviridis E-86. (5/428)

Mutagenesis studies were carried out to examine the effects of replacement of either the nucleophile Glu-236 or the acid/base Glu-128 residue of the F/10 xylanase by a His residue. To our surprise, the affinity for the p-nitrophenyl-beta-D-xylobioside substrate was increased by 10(3)-fold in the case of the mutant E128H enzyme compared with that of the wild-type F/10 xylanase. The catalytic activity of the mutant enzymes was low, despite the fact that the distance between the nucleophilic atom (an oxygen in the native xylanase and a nitrogen in the mutant) and the alpha-carbon was barely changed. Thus, the alteration of the acid/base functionality (Glu-128 to His mutation) provided a significantly favorable interaction within the E128H enzyme/substrate complex in the ground state, accompanying a reduction in the stabilization effect in the transition state.  (+info)

The glucuronic acid utilization gene cluster from Bacillus stearothermophilus T-6. (6/428)

A lambda-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting beta-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a beta-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of alpha-D-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (orf1, -2, -3, and -4) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-O-alpha-(4-O-methyl-alpha-D-glucuronosyl)-xylotriose] (MeGlcUAXyl3). The following two genes code for an intracellular alpha-glucuronidase (aguA) and a beta-xylosidase (xynB). Five more genes (kdgK, kdgA, uxaC, uxuA, and uxuB) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene, uxuR, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl3, the most likely molecular inducer.  (+info)

A unique eukaryotic beta-xylosidase gene from the phytopathogenic fungus Cochliobolus carbonum. (7/428)

The plant-pathogenic fungus Cochliobolus carbonum secretes one major beta-xylosidase (Xyp1) when grown on xylan or maize cell walls. cDNA and genomic DNA encoding Xyp1 were isolated using PCR primers based on peptide sequences from the purified protein. XYP1 contains three introns, has 5' and 3' untranslated regions of 74 and 145 bp, respectively, and is predicted to encode a protein of 328 amino acids (Mr 36700) with four N-glycosylation sites. Although it is secreted, Xyp1 has no predicted signal peptide. Furthermore, Xyp1 appears not to be processed at the N-terminus because one of the peptides isolated from the mature protein is located only six amino acids downstream of the translational start methionine. The primary sequence of Xyp1 is unrelated to any known eukaryotic beta-xylosidase but has 35% overall identity to two bacterial bifunctional beta-xylosidase/alpha-arabinosidases. Mutation of XYP1 by targeted gene replacement resulted in the loss of the major beta-xylosidase activity corresponding to the product of XYP1, but a significant amount of secreted beta-xylosidase activity (25% of wild-type) remained in the culture filtrates. The xyp1 mutant was still fully pathogenic on maize.  (+info)

Catalysis and specificity in enzymatic glycoside hydrolysis: a 2,5B conformation for the glycosyl-enzyme intermediate revealed by the structure of the Bacillus agaradhaerens family 11 xylanase. (8/428)

BACKGROUND: The enzymatic hydrolysis of glycosides involves the formation and subsequent breakdown of a covalent glycosyl-enzyme intermediate via oxocarbenium-ion-like transition states. The covalent intermediate may be trapped on-enzyme using 2-fluoro-substituted glycosides, which provide details of the intermediate conformation and noncovalent interactions between enzyme and oligosaccharide. Xylanases are important in industrial applications - in the pulp and paper industry, pretreating wood with xylanases decreases the amount of chlorine-containing chemicals used. Xylanases are structurally similar to cellulases but differ in their specificity for xylose-based, versus glucose-based, substrates. RESULTS: The structure of the family 11 xylanase, Xyl11, from Bacillus agaradhaerens has been solved using X-ray crystallography in both native and xylobiosyl-enzyme intermediate forms at 1.78 A and 2.0 A resolution, respectively. The covalent glycosyl-enzyme intermediate has been trapped using a 2-fluoro-2-deoxy substrate with a good leaving group. Unlike covalent intermediate structures for glycoside hydrolases from other families, the covalent glycosyl-enzyme intermediate in family 11 adopts an unusual 2,5B conformation. CONCLUSIONS: The 2,5B conformation found for the alpha-linked xylobiosyl-enzyme intermediate of Xyl11, unlike the 4C1 chair conformation observed for other systems, is consistent with the stereochemical constraints required of the oxocarbenium-ion-like transition state. Comparison of the Xyl11 covalent glycosyl-enzyme intermediate with the equivalent structure for the related family 12 endoglucanase, CelB, from Streptomyces lividans reveals the likely determinants for substrate specificity in this clan of glycoside hydrolases.  (+info)

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