MOESM1 of Structural and functional characterization of a highly stable endo-β-1,4-xylanase from Fusarium oxysporum and its development as an efficient immobilized biocatalyst

Additional file 1. Additional Fig. 1. Schematic representation of Xyl2 topology. Additional Fig. 2. Docking of a xylose hexaoligosaccharide on Xyl2 (pH 5). Additional Fig. 3. Schematic representation of the rationale for random enzyme immobilization via the carrier or carrier-free approaches. Negative correlation between Xyl2 activity yield and functionalization degree in high and low agarose supports. Additional Table 1. Guiding values for binding capacities of commercial agarose beads employed for Xyl2 immobilization

Product inhibition patterns for TlGK.

<p>(<b>A</b>) Inhibition by Mg·AMP with Mg·ADP as variable substrate. Measurements were assayed at fixed Mg·AMP concentrations: 100 µM (□), 200 µM (▵), 600 µM (◊) and 1000 µM (▿). Control curve in absence of product (○) was also included in the graph. (<b>B</b>) Inhibition by Mg·AMP with D-glucose as variable substrate. Measurements were assayed at fixed concentrations of Mg·AMP: 435 µM (□), 858 µM (▵), 1738 µM (◊) and 3657 µM (▿). Control curve in the absence of product (○) was also included in the graph. (<b>C</b>) Product inhibition by glucose-6-P with Mg·ADP as variable substrate. Measurements were assayed at fixed glucose-6-P concentrations: 100 µM (□), 500 µM (▵), 1000 µM (◊) and 2000 µM (▿). Control without the product was included (○). (<b>D</b>) Product inhibition by D-glucose-6-P with D-glucose as variable substrate. Measurements were assayed at fixed glucose-6-P concentrations: 500 µM (□) and 2000 µM (▵). Control curve without the presence of product was included (○). Inset of all figures show the non-linear fit of the total data.</p

Kinetic parameters for ADP-dependent glucokinase from <i>T. litoralis</i>.

‡<p>Values obtained from fitting the data to the linear expression of bi-substrate ordered sequential model.</p

Interactions clusters between the small and large domain that mediate the transition from the open to the closed conformation.

<p>(A) Structure of the enzyme in the apo-form and (B) structure of the ternary TlGK·Mg·ADPβS·D-glucose complex. The small domain was colored in yellow (apo-enzyme) or blue (ternary complex) for clarity purposes. Side chains of residues are represented as sticks. The atoms involved in H-bonds are represented as spheres. The nucleotide (ADP), the cosolvent, glycerol (GOL) and water (W) are also shown. Cluster 1 achieves communication between the small and large domain through Glu188, Thr446 and Val447. Cluster 2 residues contribute to stabilize the ADP-induced conformational change. In the apo-enzyme, Arg202 of the large domain is over the active-site pocket and the side chain of Tyr354 is oriented toward the outside of the pocket. In contrast, in the TlGK·Mg·ADPβS·D-glucose ternary complex Arg202 and Tyr354 form a stacking interaction afforded by the rotation of their side-chains so as to allow a cation-π interaction. Cluster 3′s interaction network is rearranged upon ADP binding through the formation of H-bonds between the large and small domain that generates a net attraction. In cluster 4, Arg191 from the small domain H-bonds Ala441 and Ser442 from the small domain. The only net repulsive interaction is provided by cluster 5, involving Lys74 and Lys246; in the apo-enzyme Glu100 is H-bonded to Lys74 thus neutralizing its net charge, but in the ternary complex this stabilizing interaction is absent and Lys74 comes closer to Lys246 despite the associated unfavorable energy barrier.</p

TlGK radius of gyration (Rg) and distance between the small and large domains under different conditions.

<p>Rg^ξ radius of gyration calculated using probability density function graphs. Rg^¶ theoretical radius of gyration calculated with the software CRYSOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066687#pone.0066687-Svergun3" target="_blank">[48]</a>, using crystallographic data. D^† distance between the small and large domain calculate from crystal structures. For the apo-enzyme and the ternary ADPβS·D-glucose complex, we used the PDB structures solved in this work, whereas for the ADP complex the coordinates available from the PDB (1GC5) were used. N. A., not applicable.</p

Conservation of cluster residues involved in domain opening/closure across the ADP-dependent sugar kinases family.

<p>(<b>A</b>) Consensus phylogenetic tree for ADP-dependent sugar kinase family determined by Bayesian inference. Groups of enzymes from archaea are shown in color: GK from <i>Thermococcales</i> (blue), PFK from <i>Thermococcales</i> (pink), PFK from <i>Methanococcales</i> (purple) PFK from <i>Methanosarcinales</i> (dark green) and GK from <i>Methanosarcinales</i> (cyan). The group of enzymes from eukaryotic organisms that were used as an outgroup to establish the tree root is shown in gray. The posterior probability of some interesting groups is shown in its respective node. (<b>B</b>) Multiple sequence alignment of glucokinases from the <i>Thermococcales</i> group. Residues involved in clusters described in the texts are indicated by dots; cluster 1, red (Glu188-Thr446/Val447); cluster 2, yellow (Arg202-Tyr354); cluster 3, green (Arg117/Glu115-Gly386/Ser445) and brown (Lys382-Arg117). (<b>C</b>) Amino acids involved in clusters 1–3 are shown as spheres and colored as in B.</p

Comparison between SAXS envelope models and crystals structures.

<p>(<b>A</b>) Enzyme model obtained from the SAXS data in the absence of substrates; (<b>B</b>) in the presence of Mg·ADP and (<b>C</b>) in the presence of Mg·ADP and D-glucose. Every model was built using GASBOR with no symmetry constraints. (<b>D</b>), (<b>E</b>), (<b>F</b>) Surface representation of the enzyme’s structures in the absence of substrates; in the presence of ADP (PDB 1GC5) and in the presence of Mg·ADPβS and D-glucose, respectively.</p

Crystallographic data statistics and refinement.

<p>Crystallographic data statistics and refinement.</p

Structures of the <i>T. litoralis</i> glucokinase in the apo form and in the Mg·ADPβS·D-glucose ternary complex.

<p>(<b>A</b>) Ribbon representation of the enzyme in the absence of substrate. The large domain is shown in white, whereas the small domain is shown in yellow. (<b>B</b>) Ribbon representation of the enzyme in the presence of ADPβS and D-glucose. The large domain is shown in white and the small domain is shown in blue. (<b>C</b>) Structural alignment between the apo and holo (ADPβS·D-glucose) forms.</p

Scattering curves and pair distance distribution functions <i>P(r)</i>.

<p>(<b>A</b>) Scattering patterns for the different conditions explored: apoenzyme (□), enzyme-D-glucose (▪), enzyme-Mg·ADP (○) and in the presence of Mg·ADPβS and glucose (•). The curves were normalized to unity at their maximum value for comparison purposes. (<b>B</b>) P(r) graphs for each condition calculated by Fourier transformation using GNOM (28). The graphs were normalized to unity at their maximum value for comparison purposes.</p