Figure 1.
Phylogeny of GH5 subfamily 8 including SACTE_2347.
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: Thermomonospora fusca, AAZ54938.1, PDB 1BQC, 2MAN, 3MAN; Bacillus sp. N16-5, AAT06599.1, PDB 2WHJ, 2WJL, 3JUG; Bacillus sp. JAMB-602, BAD99527.1, PDB 1WKY.
Figure 2.
Schematics of the domain structure in three variants of SACTE_2347.
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 Table 2 and Figure S1).
Figure 3.
PAGE analysis of SACTE_2347 preparations.
A, Lane 1, Streptomyces 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 E. coli 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.
Table 1.
Peptide sequences identified by mass spectrometry.
Table 2.
Summary of crystal parameters, data collection, and refinement statistics.
Figure 4.
Atomic resolution structure of SACTE_2347.
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.
Table 3.
Kinetic constants determined for SACTE_2347 variants.
Figure 5.
Schematic diagram of the binding subsites of SACTE_2347 correlated with reaction of purified oligomannosides and galactosyl-substituted oligomannosides.
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.
Figure 6.
End products from exhaustive hydrolysis of locust bean gum by SACTE_2347 determined by HPLC.
The three major products identified by comparison of elution times with purified commercial standards were 1G,2G-M3 (8), 1G-M2 (5), and M2 (2).
Figure 7.
Insoluble substrate binding assays.
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.