Kinetic constants determined for SACTE_2347 variants.

a<p>Pure β-1,4 d-mannan.</p>b<p>Acetylated glucomannan contain mannan (60%) and glucose (40%).</p>c<p>Locust bean gum is a natural galactomannan with composition of ∼3.5 mannose per galactose.</p>d<p>IL-pine has the following composition: 34% glucose; 9% xylose; 8% mannose; 4% arabinose, and 8% galactose.</p><p>SACTE_2347 did not hydrolyze cellulose, xylan and other polysaccharides described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094166#s4" target="_blank">Materials and Methods</a>, and likewise did not react with fluorogenic small molecule analogs.</p

Summary of crystal parameters, data collection, and refinement statistics.

<p>*R_merge = ∑_h ∑_i | I_i (h)−|/∑_h∑_i I_i(h), where I_i(h) is the intensity of an individual measurement of the reflection and is the mean intensity of the reflection.</p>§<p>R_cryst = ∑_h ||F_obs|−|F_calc||/∑_h |F_obs|, where F_obs and F_calc are the observed and calculated structure-factor amplitudes, respectively.</p>¶<p>R_free was calculated as R_cryst using 1.5% of randomly selected unique reflections that were omitted from the structure refinement.</p><p>Values in parentheses are for the highest resolution shell.</p

Insoluble substrate binding assays.

<p>SACTE_2347 variants were tested for binding to the insoluble substrates indicated using a pull-down format. A, SACTE_2347_FL. B, SACTE_2347_42kDa. C, SACTE_2347_34kDa. The presence of protein in the pellet fraction (P) indicates binding, as the control lane showed each variant was fully soluble in the conditions tested in the absence of an insoluble substrate. Black stars indicate the substrates for which binding was observed.</p

End products from exhaustive hydrolysis of locust bean gum by SACTE_2347 determined by HPLC.

<p>The three major products identified by comparison of elution times with purified commercial standards were ¹G,²G-M3 (<b>8</b>), ¹G-M2 (<b>5</b>), and M2 (<b>2</b>).</p

Schematic diagram of the binding subsites of SACTE_2347 correlated with reaction of purified oligomannosides and galactosyl-substituted oligomannosides.

<p>The active site schematic shows the positions of sugar binding subsites, the catalytic residues Glu178 and Glu272, and the position of loops L1 and L2. Mannosyl groups (grey circles) and galactosyl groups (black circles) of purified substrates studies are aligned in the −3 to +2 subsites under the schematic of the active site channel. Loop L1 blocks binding of a substituted mannosyl group in either the +1 of +2 subsites. The space between L1 and L2 allows placement of a substituted mannosyl group in the −1 subsite, while shortened L2 allows placement of a substituted mannosyl group into the −2 subsite. All reaction products can be rationalized to arise from hydrolysis of the glycosidic bond between the −1 and +1 subsites after accounting for steric interactions with L1 and L2.</p

Atomic resolution structure of SACTE_2347.

<p>A, 2Fo-Fc electron density map of eight conserved residues in the GH5 family, contoured at 1.3 σ. Hydrogen atoms were included in the refinement of the high-resolution data. B, Comparison of the active site channels of SACTE_2347 (green) and TfManA (blue). Residues that form the surface of the channel are highlighted in gray, and the positions of loops L1 and L2 are indicated. Positions of the catalytic residues (Glu178 and Glu273 in SACTE_2347_34kDa) are shown in red. Mannobiose observed in the −3 and −2 subsites of the TfManA structure is shown as ball and sticks.</p

Peptide sequences identified by mass spectrometry.

a<p>The full protein sequence of SACTE_2347, annotated with the positions of these peptides is found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094166#pone.0094166.s002" target="_blank">Figure S2</a>. The names of peptides are also used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094166#pone-0094166-g002" target="_blank">Figure 2</a>.</p>b<p>Observed <i>m/z</i>.</p

Phylogeny of GH5 subfamily 8 including SACTE_2347.

<p>The phylogenic tree was constructed from all sequences assigned to the GH5 subfamily 8. Names shown are GenBank accession codes. Black circles indicate enzymes that have been experimentally verified to exhibit β-mannanase activity; black stars indicate three enzymes whose structures have been determined besides SACTE_2347. These are: <i>Thermomonospora fusca</i>, AAZ54938.1, PDB 1BQC, 2MAN, 3MAN; <i>Bacillus</i> sp. N16-5, AAT06599.1, PDB 2WHJ, 2WJL, 3JUG; <i>Bacillus</i> sp. JAMB-602, BAD99527.1, PDB 1WKY.</p

Schematics of the domain structure in three variants of SACTE_2347.

<p>A, SACTE_2347_FL; B, SACTE_2347_42kDa; C, SACTE_2347_34kDa. Residues 1–41 correspond to the annotated twin arginine translocation peptide. Residue 51 corresponds to the experimentally determined N-terminus in the three variants. The GH5 domain spans residues 63–308, and boundaries of the Fn3 and CBM2 domains are as indicated. The location of C-terminal tryptic-digest polypeptides for each variant is shown (GH5, 34kDa-C, Fn3, 42kDa-C, CBM2; also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094166#pone-0094166-t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094166#pone.0094166.s001" target="_blank">Figure S1</a>).</p

PAGE analysis of SACTE_2347 preparations.

<p>A, Lane 1, <i>Streptomyces</i> sp. SirexAA-E secretome; lane 2, fraction from the ion exchange separation that showed the maximal mannanase activity; lane 3, recombinant SACTE_2347_FL purified from <i>E. coli</i> BL21(DE3); lane 4, recombinant SACTE_2347_42kDa; lane 4 recombinant SACTE_2347_34kDa. B, in-gel determination of mannan-degrading activity. Lane 1, native PAGE with Coomassie Blue staining of the fraction from A, lane 2 performed with mannan included in the gel; lane 2, Congo Red staining of the gel. Regions of the gel that contain polysaccharide bind the dye and appear orange; regions of the gel containing enzyme activity no longer contain polysaccharide and so have a grey-colored appearance.</p