Table 1.
Data collection and refinement statistics.
Fig 1.
Schematic representation of genes coding for cellulose synthase subunits in a section of R. ornithinolytica strain S12 genome.
Bcs genes are arranged in operons, as illustrated. The genes coding for the endoglucanases AfmE1 and BcsZ are indicated by arrows. According to the classification of Bcs operons proposed by Römling & Galpering [1], AfmE1 and BcsZ integrate distinct operons of the subtypes Ib and IIa, respectively. NCBI genome accession: CP010557.1.
Fig 2.
Effect of pH and temperature on the catalytic activity of AfmE1.
(A) Determination of optimum pH. The hydrolytic activity was measured at different pHs at 40°C for 10 min. (B) Determination of optimum temperature. The hydrolytic activity was measured at temperatures ranging from 20 to 70°C. (C) Thermal stability assay. The enzyme was incubated at 45, 50 and 55°C for up to 4 h and residual activity was determined under the optimal reaction conditions. Error bars represent the standard deviation.
Table 2.
Biochemical characterization of AfmE1.
Fig 3.
Thin-layer chromatography analysis of degradation products derived from AfmE1-mediated hydrolysis of different cello-oligosaccharides. (A) cellobiose, cellotriose and cellotetraose; (B) cellopentaose and (C) cellohexaose. The first line of each panel corresponds to a mixture of the indicated standards. CZE electropherograms of the APTS-labeled products of cellopentaose (D) and cellohexaose (E) hydrolysis after 0, 2 and 4 h of incubation with AfmE1. CZE electropherograms of the APTS-labeled products from AfmE1-mediated hydrolysis of β-glucan (F) and CMC (G). The labeled cello-oligosaccharides are indicated, as inferred from a parallel run of a standard mixture. For all the analyses, control reactions were carried out in the absence of AfmE1 and run in parallel.
Fig 4.
(A) Circular dichroism spectrum of AfmE1 indicating that the recombinant protein was produced and purified in a folded conformation. CD (B) and DSC (C) thermal unfolding curves showed similar melting temperatures around 55°C. The second peak in the DSC curve corresponds to protein aggregation after denaturation. (D) AUC analysis of AfmE1 at different concentrations confirms that the protein is monomeric in solution with a molecular weight of approximately 39 kDa.
Fig 5.
The crystallographic structure of AfmE1.
(A) Cartoon representation of the AfmE1 (α/α)6 barrel fold with the inner and outer helices colored in light-blue and light-green, respectively. The β-motif is shown in pink and the region of the lacking helix-α11 in red. The residues critical for catalysis are depicted as sticks with carbon atoms in yellow. Superposition of AfmE1 structure with those of K. xylinus CMCax (PDB code 1WZZ) (B), E. coli BcsZ (PDB code 3QXQ) (C) and C. thermocellum CelA (PDB code 1CEM) (D). The presence (+) or absence (-) of the β-motif and helix-α11 is indicated and highlighted in blue, orange, pink and green in the structures of AfmE1, CMCax, BcsZ and CelA, respectively. The r.m.s.d. and sequence identity values for each structural alignment are indicated.
Fig 6.
AfmE1 substrate-binding cleft.
(A) Molecular surface of AfmE1 with its stacking residue Trp272 in subsite +3 represented as sticks with carbon atoms in green. The stacking residue Tyr369 in the corresponding region of CelA from C. thermocellum is similarly represented in magenta to evidence the differences in the subsite +3 configuration of these enzymes. The region containing the catalytic residues is highlighted in yellow. The substrate molecules, represented as deduced from the complex of CelA with cellopentaose (white) and cellotriose (blue) (PDB code 1KWF), as well as of BcsZ with cellopentaose (orange) (PDB code 3QXQ), are shown as sticks to indicate the position of the subsites. (B) AfmE1 substrate-binding cleft highlighting the catalytic (yellow) and the glucosyl-stacking residues (green) in its six subsites (dashed lines). The corresponding stacking residues of the proteins CMCax, BcsZ and CelA are shown in cyan, orange and magenta, respectively. The position of the glucosyl residues (blue) occupying the six subsites was predicted from the complex CelA-substrate [44].