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Query: UNIPROT:P06889 (Mol)
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Specific binding of interacting proteins generally depends on a limited set of amino acid residues located at the contact interface. We have applied a phage-display-based screening method to simultaneously evaluate the role of multiple residues of endo-beta-1,4-xylanase enzymes in conferring binding specificity towards two different endoxylanase inhibitors. Seven residues of the two beta-strand 'thumb' region of Trichoderma longibrachiatum endo-beta-1,4-xylanase XynII were targeted for randomization. The generated combinatorial library representing 62,208 site-directed variants was displayed on the surface of filamentous phage and selected against xylanase inhibitor protein (XIP) and Triticum aestivum xylanase inhibitor (TAXI). DNA sequence analysis of phagemid panning isolates provided information on the occurrence of particular amino acids at distinct positions. In particular, residues at positions 124 (Asn) and 131 (Thr) were found to be critical for specific inhibitor binding. These residue predictions derived from the combinatorial exploration of the thumb region and accompanying sequence analyses were experimentally confirmed by testing the inhibitor sensitivity of a limited set of recombinantly expressed XynII mutants. In addition, we successfully altered the inhibition susceptibility of the bacterial Bacillus subtilis endoxylanase XynA from XIP-insensitive to XIP-sensitive.
J Mol Recognit
PMID:Engineering molecular recognition of endoxylanase enzymes and their inhibitors through phage display. 1739 41

Fungal infection of plants involves degradation of the host cell wall through the action of lytic enzymes secreted by the pathogen. The role of these enzymes in virulence is difficult to determine due to their functional redundancy and, therefore, remains controversial. Here, we have studied XlnR, a zinc-finger transcription factor from the vascular wilt pathogen Fusarium oxysporum that is orthologous to the major transcriptional activator of xylanase genes in Aspergillus spp. Transcription of the xlnR gene was activated by inducing carbon sources such as oat spelt xylan (OSX) and repressed by glucose. Targeted knockout of xlnR in F. oxysporum resulted in lack of transcriptional activation of structural xylanase genes, both in culture and during infection of tomato plants, as well as in dramatically reduced extracellular xylanase activity. By contrast, overexpression of xlnR under the control of the Aspergillus nidulans gpdA promoter did not significantly increase xylanase activity, suggesting that XlnR is regulated not only at the transcriptional but also at the post-translational level. The deltaxlnR mutants were still fully virulent on tomato plants. Thus, XlnR, the major transcriptional activator of xylanase genes, is not an essential virulence determinant in F. oxysporum.
Mol Plant Microbe Interact 2007 Aug
PMID:Role of the transcriptional activator xlnR of Fusarium oxysporum in regulation of xylanase genes and virulence. 1772 1

Bacillus circulans xylanase (BcX) is a single-domain family 11 glycoside hydrolase. Using NMR-monitored titrations, we discovered that an inactive variant of this enzyme, E78Q-BcX, bound xylooligosaccharides not only within its pronounced active site (AS) cleft, but also at a distal surface region. Chemical shift perturbation mapping and affinity electrophoresis, combined with mutational studies, identified the xylan-specific secondary binding site (SBS) as a shallow groove lined by Asn, Ser, and Thr residues and with a Trp at one end. The AS and SBS bound short xylooligosaccharides with similar dissociation constants in the millimolar range. However, the on and off-rates to the SBS were at least tenfold faster than those of kon approximately 3x10(5) M(-1) s(-1) and koff approximately 1000 s(-1) measured for xylotetraose to the AS of E78Q-BcX. Consistent with their structural differences, this suggests that a conformational change in the enzyme and/or the substrate is required for association to and dissociation from the deep AS, but not the shallow SBS. In contrast to the independent binding of small xylooligosaccharides, high-affinity binding of soluble and insoluble xylan, as well as xylododecaose, occurred cooperatively to the two sites. This was evidenced by an approximately 100-fold increase in relative Kd values for these ligands upon mutation of the SBS. The SBS also enhances the activity of BcX towards soluble and insoluble xylan through a significant reduction in the Michaelis KM values for these polymeric substrates. This study provides an unexpected example of how a single domain family 11 xylanase overcomes the lack of a carbohydrate-binding module through the use of a secondary binding site to enhance substrate specificity and affinity.
J Mol Biol 2007 Oct 19
PMID:A secondary xylan-binding site enhances the catalytic activity of a single-domain family 11 glycoside hydrolase. 1782 16

Endo-beta1,4-xylanases (xylanases) hydrolyse the beta1,4 glycosidic bonds in the backbone of xylan. Although xylanases from glycoside hydrolase family 11 (GH11) have been extensively studied, several issues remain unresolved. Thus, the mechanism by which these enzymes hydrolyse decorated xylans is unclear and the structural basis for the variation in catalytic activity within this family is unknown. Furthermore, the mechanism for the differences in the inhibition of fungal GH11 enzymes by the wheat protein XIP-I remains opaque. To address these issues we report the crystal structure and biochemical properties of the Neocallimastix patriciarum xylanase NpXyn11A, which displays unusually high catalytic activity and is one of the few fungal GH11 proteins not inhibited by XIP-I. Although the structure of NpXyn11A could not be determined in complex with substrates, we have been able to investigate how GH11 enzymes hydrolyse decorated substrates by solving the crystal structure of a second GH11 xylanase, EnXyn11A (encoded by an environmental DNA sample), bound to ferulic acid-1,5-arabinofuranose-alpha1,3-xylotriose (FAX(3)). The crystal structure of the EnXyn11A-FAX(3) complex shows that solvent exposure of the backbone xylose O2 and O3 groups at subsites -3 and +2 allow accommodation of alpha1,2-linked 4-methyl-D-glucuronic acid and L-arabinofuranose side chains. Furthermore, the ferulated arabinofuranose side chain makes hydrogen bonds and hydrophobic interactions at the +2 subsite, indicating that the decoration may represent a specificity determinant at this aglycone subsite. The structure of NpXyn11A reveals potential -3 and +3 subsites that are kinetically significant. The extended substrate-binding cleft of NpXyn11A, compared to other GH11 xylanases, may explain why the Neocallimastix enzyme displays unusually high catalytic activity. Finally, the crystal structure of NpXyn11A shows that the resistance of the enzyme to XIP-I is not due solely to insertions in the loop connecting beta strands 11 and 12, as suggested previously, but is highly complex.
J Mol Biol 2008 Feb 01
PMID:Understanding the structural basis for substrate and inhibitor recognition in eukaryotic GH11 xylanases. 1807 55

The pH-dependence of the NMR chemical shift for titratable groups in proteins often deviate from a standard Henderson-Hasselbalch (HH) titration curve. A non-HH dependence of the chemical shift for a given residue can arise from a single-site, non-HH titrational event for that residue, or if the chemical shift of the group is influenced by additional titrational events occurring in other residues. We show that simultaneous fits of several non-HH NMR titration curves of interacting protein residues to a statistical mechanical model can be used to distinguish between these two cases. From fitting of non-HH titrations, we can extract electrostatic interaction energies between protein residues. Furthermore, by performing simultaneous fits of NMR titration curves and enzymatic pH-activity profiles, we can gain information on the identity and populations of the catalytically competent protonation states in enzymes. We apply the global fitting of titrational events (GloFTE) method to experimental data on five enzyme systems and on a single non-enzyme system, and show that the extracted electrostatic interaction energies and effective dielectric constants for a subset of these systems agree excellently with experimentally determined values as well as with theoretical calculations. In the case of reduced Escherichia coli thioredoxin we use GloFTE analysis to distinguish between two possible interpretations of the NMR titration curves of the active site residues. We also show that for the strongly coupled system of titratable groups in the active site of the Bacillus circulans xylanase (BCX) N35D mutant, GloFTE fits of a single titration curve and an enzymatic pH-activity profile can give a full description of the energetics of the titrational events in the enzyme's active site. Using only the X-ray crystallographic structure of the enzyme and the electrostatic interaction energies extracted from such a GloFTE fit, we can uniquely identify the three catalytic groups in this system. This raises the prospect of completely characterising active site titrational events from a single unassigned NMR titration curve and an enzymatic pH-activity profile.
J Mol Biol 2008 Feb 08
PMID:Determination of electrostatic interaction energies and protonation state populations in enzyme active sites. 1815 42

The PROPKA method for the prediction of the pK(a) values of ionizable residues in proteins is extended to include the effect of non-proteinaceous ligands on protein pK(a) values as well as predict the change in pK(a) values of ionizable groups on the ligand itself. This new version of PROPKA (PROPKA 2.0) is, as much as possible, developed by adapting the empirical rules underlying PROPKA 1.0 to ligand functional groups. Thus, the speed of PROPKA is retained, so that the pK(a) values of all ionizable groups are computed in a matter of seconds for most proteins. This adaptation is validated by comparing PROPKA 2.0 predictions to experimental data for 26 protein-ligand complexes including trypsin, thrombin, three pepsins, HIV-1 protease, chymotrypsin, xylanase, hydroxynitrile lyase, and dihydrofolate reductase. For trypsin and thrombin, large protonation state changes (|n| > 0.5) have been observed experimentally for 4 out of 14 ligand complexes. PROPKA 2.0 and Klebe's PEOE approach (Czodrowski P et al. J Mol Biol 2007;367:1347-1356) both identify three of the four large protonation state changes. The protonation state changes due to plasmepsin II, cathepsin D and endothiapepsin binding to pepstatin are predicted to within 0.4 proton units at pH 6.5 and 7.0, respectively. The PROPKA 2.0 results indicate that structural changes due to ligand binding contribute significantly to the proton uptake/release, as do residues far away from the binding site, primarily due to the change in the local environment of a particular residue and hence the change in the local hydrogen bonding network. Overall the results suggest that PROPKA 2.0 provides a good description of the protein-ligand interactions that have an important effect on the pK(a) values of titratable groups, thereby permitting fast and accurate determination of the protonation states of key residues and ligand functional groups within the binding or active site of a protein.
 ...
PMID:Very fast prediction and rationalization of pKa values for protein-ligand complexes. 1849 3

Protein phosphorylation is a key biological process that regulates reactions involved in plant-microbe interactions. The phosphorylated form of a protein often represents only a small fraction of the total population and can be problematic to analyze in a mass spectrometer. We demonstrate how a titanium dioxide (TiO(2)) resin can be employed for the enrichment of phosphoproteins, as well as a method to derivatize TiO(2)-purified phosphopeptides to facilitate determination of the exact site of phosphorylation. The use of these methods was exemplified by the identification of two plant proteins that were shown to be phosphorylated after the elicitation of Arabidopsis cells with Phytophthora infestans zoospores and xylanase. Both of the proteins that were identified, At5g54430.1 and At4g27320.1, were found to contain a universal stress protein domain with conserved residues for ATP binding.
Mol Plant Microbe Interact 2008 Oct
PMID:Enrichment of phosphoproteins and phosphopeptide derivatization identify universal stress proteins in elicitor-treated Arabidopsis. 1878 23

Efficient degradation of cellulose by the anaerobic thermophilic bacterium, Clostridium thermocellum, is carried out by the multi-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification. A C. thermocellum Coh module was thus fused to a carbohydrate-binding module, and the resultant fusion protein was applied directly onto beaded cellulose, thereby serving as a non-covalent "activation" procedure. A complementary Doc module was then fused to a model protein target: xylanase T-6 from Geobacillus stearothermophilus. However, the binding to the immobilized Coh was only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein were eluted from the affinity column. In order to improve protein elution, a series of truncated Docs were designed in which the calcium-coordinating function was impaired without appreciably affecting high-affinity binding to Coh. A shortened Doc of only 48 residues was sufficient to function as an effective affinity tag, and highly purified target protein was achieved directly from crude cell extracts in a single step with near-quantitative recovery of the target protein. Effective EDTA-mediated elution of the sequestered protein from the column was the key step of the procedure. The affinity column was reusable and maintained very high levels of capacity upon repeated rounds of loading and elution. Reusable Coh-Doc affinity columns thus provide an efficient and attractive approach for purifying proteins in high yield by modifying the calcium-binding loop of the Doc module.
J Mol Recognit
PMID:Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction. 1897 59

Xylan digestion by Corbicula japonica was investigated according to the hypothesis that C. japonica can breakdown xylan as well as cellulose. C. japonica showed relatively high xylanase activity compared to other bivalve species. Molecular cloning of a xylanase gene was performed in order to determine whether C. japonica possesses an endogenous xylanase, and resulted in the isolation of cDNA with an ORF of 2523 bp corresponding to 840 amino acids (CjXyn10A). CjXyn10A has a Glycoside Hydrolase Family 10 (GHF10) catalytic domain, N-terminal family 4 carbohydrate binding domain and novel C-terminal cysteine-rich domain. Phylogenetic analysis implies that this gene has common evolutional origin with other GHF10 genes of animal origin. Reverse transcription (RT)-PCR analysis and in situ hybridization revealed that CjXyn10A is likely to be expressed in the secretory cells in the digestive gland, suggesting that this enzyme is produced in the same site as previously reported endogenous cellulases of C. japonica. These findings suggest that CjXyn10A is an endogenous xylanase gene of this species. The occurrence of an endogenous xylanase gene in addition to cellulase genes in C. japonica strongly supports our hypothesis that this species can decompose plant-derived structural polysaccharides.
Comp Biochem Physiol B Biochem Mol Biol 2009 Sep
PMID:Putative endogenous xylanase from brackish-water clam Corbicula japonica. 1945 20

The Xyn2 gene, which encodes endo-beta-1,4-xylanase2, in Trichoderma reesei Rut C-30 was amplified by PCR from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to encode 190-amino-acid residues of a protein with a calculated molecular mass of 21 kDa. The cDNA was cloned into pET30alpha expression vector and subsequently expressed in Escherichia coli under the control of strong bacteriophage T7 transcription and translation signals. The enzyme activity assay verified the recombinant protein as xylanase. The isoelectric point and highest activity were 7.5 and 1,600 U/mg, respectively. Like with the T. reesei Xyn2, the highest activity of the recombinant Xyn2 was at 50 degrees C. However, the recombinant enzyme had an improved thermostability and more than 65% of its activity retained after 30 min incubation at 60 degrees C. In addition, the recombinant Xyn2 was active over the range of pH 3.5-7.5 with maximum activity at pH 5.0. Using birchwood xylan, the determined apparent K(m) and k(cat) values were 0.15 mg/ml and 119.7 s(-1), respectively. The enzyme was highly specific towards xylans and exhibited very low activity towards cellulosic substrates. Analysis of the products from birchwood xylan degradation confirmed that the enzyme was an endo-xylanase with xylobiose and xylose as the main degradation products. These properties should make the enzyme a suitable applicant in various industrial applications.
J Mol Microbiol Biotechnol 2009
PMID:Sequencing and expression of the xylanase gene 2 from Trichoderma reesei Rut C-30 and characterization of the recombinant enzyme and its activity on xylan. 1955 47


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