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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Site-directed mutagenesis and a comparative characterisation of the kinetic parameters, pH dependency of activity and thermal stability of mutant and wild-type enzymes have been used in association with crystallographic analysis to delineate the functions of several active site residues in a novel glycoside hydrolase family 8 xylanase. Each of the residues investigated plays an essential role in this enzyme: E78 as the general acid, D281 as the general base and in orientating the nucleophilic water molecule, Y203 in maintaining the position of the nucleophilic water molecule and in structural integrity and D144 in sugar ring distortion and transition state stabilization. Interestingly, although crystal structure analyses and the pH-activity profiles clearly identify the functions of E78 and D281, substitution of these residues with their amide derivatives results in only a 250-fold and 700-fold reduction in their apparent k(cat) values, respectively. This, in addition to the observation that the proposed general base is not conserved in all glycoside hydrolase family 8 enzymes, indicates that the mechanistic architecture in this family of inverting enzymes is more complex than is conventionally believed and points to a diversity in the identity of the mechanistically important residues as well as in the arrangement of the intricate microenvironment of the active site among members of this family.
J Mol Biol 2005 Nov 25
PMID:Study of the active site residues of a glycoside hydrolase family 8 xylanase. 1624 70

Phytopathogenic fungi can degrade xylan, an abundant hemicellulose in plant cell walls, by the coordinate action of a group of extracellular enzymes. Among these, endo-beta-1,4-xylanases carry out the initial breakdown by cleaving internal bonds in the polymer backbone. We have isolated and characterized a gene, xyn11A, coding for an endo-beta-1,4-xylanase belonging to family 11 of glycosyl hydrolases. xyn11A was shown to be induced by xylan and repressed by glucose and to be expressed in planta. The disruption of xyn11A caused only a moderate decrease, about 30%, in the level of extracellular endo-beta-1-4-xylanase activity and in the growth rate, with beechwood xylan as the only carbon source. However, deletion of the gene had a more pronounced effect on virulence, delaying the appearance of secondary lesions and reducing the average lesion size by more than 70%. Reintroducing the wild-type gene into the mutant strains reversed this phenotype back to wild type.
Mol Plant Microbe Interact 2006 Jan
PMID:The endo-beta-1,4-xylanase xyn11A is required for virulence in Botrytis cinerea. 1640 50

We have screened 766 strains of fungi from the BIOTEC Culture Collection (BCC) for xylanases working in extreme pH and/or high temperature conditions, the so-called extreme xylanases. From a total number of 32 strains producing extreme xylanases, the strain BCC7928, identified by using the internal transcribed spacer (ITS) sequence of rRNA to be a Marasmius sp., was chosen for further characterization because of its high xylanolytic activity at temperature as high as 90 degrees C. The crude enzyme possessed high thermostability and pH stability. Purification of this xylanase was carried out using an anion exchanger followed by hydrophobic interaction chromatography, yielding the enzyme with >90% homogeneity. The molecular mass of the enzyme was approximately 40 kDa. The purified enzyme retained broad working pH range of 4-8 and optimal temperature of 90 degrees C. When using xylan from birchwood as substrate, it exhibits Km and Vmax values of 2.6 +/- 0.6 mg/ml and 428 +/- 26 U/mg, respectively. The enzyme rapidly hydrolysed xylans from birchwood, beechwood, and exhibited lower activity on xylan from wheatbran, or celluloses from carboxymethylcellulose and Avicel. The purified enzyme was highly stable at temperature ranges from 50 to 70 degrees C. It retained 84% of its maximal activity after incubation in standard buffer containing 1% xylan substrate at 70 degrees C for 3 h. This thermostable xylanase should therefore be useful for several industrial applications, such as agricultural, food and biofuel.
J Biochem Mol Biol 2006 Jan 31
PMID:Thermostable xylanase from Marasmius sp.: purification and characterization. 1647 80

Substituted xylan polymers constitute a major part of the hemicellulose fraction of plant cell walls, especially in monocotyledons. Endo-1,4-beta-xylanases (EC 3.2.1.8) are capable of hydrolyzing substituted xylan polymers into fragments of random size. Many herbivorous animals have evolved intimate relationships with endosymbionts to exploit their enzyme complexes for the degradation of xylan. Here, we report the first finding of a functional endo-1,4-beta-xylanase gene from an animal. The gene (Mi-xyl1) was found in the obligate plant-parasitic root-knot nematode Meloidogyne incognita, and encodes a protein that is classified as a member of glycosyl hydrolase family 5. The expression of Mi-xyl1 is localized in the subventral esophageal gland cells of the nematode. Previous studies have shown that M. incognita has the ability to degrade cellulose and pectic polysaccharides in plant cell walls independent of endosymbionts. Including our current data on Mi-xyl1, we show that the endogenous enzyme complex in root-knot nematode secretions targets essentially all major cell wall carbohydrates to facilitate a stealthy intercellular migration in the host plant.
Mol Plant Microbe Interact 2006 May
PMID:A symbiont-independent endo-1,4-beta-xylanase from the plant-parasitic nematode Meloidogyne incognita. 1667 39

The gene encoding an alkaline active xylanase of Bacillus halodurans S7, containing an open reading frame of 1188 nucleotides encoding 396 amino acids, was cloned and expressed in Escherchia coli. On the basis of sequence similarity, possible -10 and -35, ribosome binding, and transcription terminator regions were identified. Analysis of the deduced amino acid sequence revealed that the protein was a single domain enzyme belonging to family 10 and designated as xyn10A. The calculated molecular mass and isoelectric point (pI) of the mature peptide were 42.6 and 4.5 kDa, respectively. Xylanase activity expressed by the recombinant organism was detected in the cytoplasm, periplasm and the extracellular medium. In an 18-h old culture, about 39% of the xylanase was detected in the medium. The stability and activity profile of the recombinant xylanase was similar to the properties of the enzyme produced by the wild-type organism.
Mol Biotechnol 2006 Jun
PMID:Cloning, sequence analysis, and expression of a gene encoding an endoxylanase from Bacillus halodurans S7. 1675 2

Thermostability is an important property of industrially significant hydrolytic enzymes: understanding the structural basis for this attribute will underpin the future biotechnological exploitation of these biocatalysts. The Cellvibrio family 10 (GH10) xylanases display considerable sequence identity but exhibit significant differences in thermostability; thus, these enzymes represent excellent models to examine the structural basis for the variation in stability displayed by these glycoside hydrolases. Here, we have subjected the intracellular Cellvibrio mixtus xylanase CmXyn10B to forced protein evolution. Error-prone PCR and selection identified a double mutant, A334V/G348D, which confers an increase in thermostability. The mutant has a Tm 8 degrees C higher than the wild-type enzyme and, at 55 degrees C, the first-order rate constant for thermal inactivation of A334V/G348D is 4.1 x 10(-4) min(-1), compared to a value of 1.6 x 10(-1) min(-1) for the wild-type enzyme. The introduction of the N to C-terminal disulphide bridge into A334V/G348D, which increases the thermostability of wild-type CmXyn10B, conferred a further approximately 2 degrees C increase in the Tm of the double mutant. The crystal structure of A334V/G348D showed that the introduction of Val334 fills a cavity within the hydrophobic core of the xylanase, increasing the number of van der Waals interactions with the surrounding aromatic residues, while O(delta1) of Asp348 makes an additional hydrogen bond with the amide of Gly344 and O(delta2) interacts with the arabinofuranose side-chain of the xylose moiety at the -2 subsite. To investigate the importance of xylan decorations in productive substrate binding, the activity of wild-type CmXyn10B, the mutant A334V/G348D, and several other GH10 xylanases against xylotriose and xylotriose containing an arabinofuranose side-chain (AX3) was assessed. The enzymes were more active against AX3 than xylotriose, providing evidence that the arabinose side-chain makes a generic contribution to substrate recognition by GH10 xylanases.
J Mol Biol 2006 Jun 30
PMID:Probing the structural basis for the difference in thermostability displayed by family 10 xylanases. 1676 67

Endo-beta-1,4-xylanases (EC 3.2.1.8) are key enzymes in the degradation of xylan, the predominant hemicellulose in the cell walls of plants and the second most abundant polysaccharide on earth. A number of endoxylanases are produced by microbial phytopathogens responsible for severe crop losses. These enzymes are considered to play an important role in phytopathogenesis, as they provide essential means to the attacking organism to break through the plant cell wall. Plants have evolved numerous defense mechanisms to protect themselves against invading pathogens, amongst which are proteinaceous inhibitors of cell wall-degrading enzymes. These defense mechanisms are triggered when a pathogen-derived elicitor is recognized by the plant. In this review, the diverse aspects of endoxylanases in promoting virulence and in eliciting plant defense systems are highlighted. Furthermore, the role of the relatively recently discovered cereal endoxylanase inhibitor families TAXI (Triticum aestivum xylanase inhibitor) and XIP (xylanase inhibitor protein) in plant defense is discussed.
Mol Plant Microbe Interact 2006 Oct
PMID:Microbial endoxylanases: effective weapons to breach the plant cell-wall barrier or, rather, triggers of plant defense systems? 1702 71

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Shuffling techniques can be used on a collection of mutants of the same gene, or related families of genes can be shuffled to produce mutants encoding chimeric gene products. One difficulty with current shuffling procedures is the predominance of unshuffled ("parental") molecules in the pool of mutants. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled and reduces the regeneration of unshuffled parental genes. This procedure has the advantage of avoiding the use of endonucleases for gene fragmentation before shuffling and allows the use of random mutagenesis of selected segments of the gene as part of the procedure. We illustrate the use of the technique with a diverse family of beta-xylanase genes that possess widely different G and C contents.
Methods Mol Biol 2007
PMID:Degenerate oligonucleotide gene shuffling. 1704 Dec 66

Enzymes from cold-adapted species are significantly more active at low temperatures, even those close to zero Celsius, but the rationale of this adaptation is complex and relatively poorly understood. It is commonly stated that there is a relationship between the flexibility of an enzyme and its catalytic activity at low temperature. This paper gives the results of a study using molecular dynamics simulations performed for five pairs of enzymes, each pair comprising a cold-active enzyme plus its mesophilic or thermophilic counterpart. The enzyme pairs included alpha-amylase, citrate synthase, malate dehydrogenase, alkaline protease and xylanase. Numerous sites with elevated flexibility were observed in all enzymes; however, differences in flexibilities were not striking. Nevertheless, amino acid residues common in both enzymes of a pair (not present in insertions of a structure alignment) are generally more flexible in the cold-active enzymes. The further application of principle component analysis to the protein dynamics revealed that there are differences in the rate and/or extent of opening and closing of the active sites. The results indicate that protein dynamics play an important role in catalytic processes where structural rearrangements, such as those required for active site access by substrate, are involved. They also support the notion that cold adaptation may have evolved by selective changes in regions of enzyme structure rather than in global change to the whole protein.
J Mol Model 2007 Apr
PMID:Cold-active enzymes studied by comparative molecular dynamics simulation. 1723 16

Perception of general elicitors by plant cells initiates signal transduction cascades that are regulated by protein phosphorylation. The earliest signaling events occur within minutes and include ion fluxes across the plasma membrane, activation of MAPKs, and the formation of reactive oxygen species. The phosphorylation events that regulate these signaling cascades are largely unknown. Here we present a mass spectrometry-based quantitative phosphoproteomics approach that identified differentially phosphorylated sites in signaling and response proteins from Arabidopsis cells treated with either flg22 or xylanase. Our approach was sensitive enough to quantitate phosphorylation on low abundance signaling proteins such as calcium-dependent protein kinases and receptor-like kinase family members. With this approach we identified one or more differentially phosphorylated sites in 76 membrane-associated proteins including a number of defense-related proteins. Our data on phosphorylation indicate a high degree of complexity at the level of post-translational modification as exemplified by the complex modification patterns of respiratory burst oxidase protein D. Furthermore the data also suggest that protein translocation and vesicle traffic are important aspects of early signaling and defense in response to general elicitors. Our study presents the largest quantitative Arabidopsis phosphoproteomics data set to date and provides a new resource that can be used to gain novel insight into plant defense signal transduction and early defense response.
Mol Cell Proteomics 2007 Jul
PMID:Quantitative phosphoproteomics of early elicitor signaling in Arabidopsis. 1731 60


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