AfmE1 substrate-binding cleft.

<p>(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 <i>C</i>. <i>thermocellum</i> 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176550#pone.0176550.ref044" target="_blank">44</a>].</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Effect of pH and temperature on the catalytic activity of AfmE1.

<p>(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.</p

Biophysical characterization.

<p>(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.</p

Structure and function of a novel GH8 endoglucanase from the bacterial cellulose synthase complex of <i>Raoultella ornithinolytica</i>

<div><p>Cellulose synthesis in bacteria is a complex process involving the concerted action of several enzymes whose genes are often organized in operons. This process influences many fundamental physiological aspects such as bacteria and host interaction, biofilm formation, among others. Although it might sound contradictory, the participation of cellulose-degrading enzymes is critical to this process. The presence of endoglucanases from family 8 of glycosyl hydrolases (GH8) in bacterial cellulose synthase (Bcs) complex has been described in different bacteria, including the model organism <i>Komagataeibacter xylinus</i>; however, their role in this process is not completely understood. In this study, we describe the biochemical characterization and three-dimensional structure of a novel GH8 member from <i>Raoultella ornithinolytica</i>, named AfmE1, which was previously identified by our group from the metagenomic analysis of the giant snail <i>Achatina fulica</i>. Our results demonstrated that AfmE1 is an endo-β-1,4-glucanase, with maximum activity in acidic to neutral pH over a wide temperature range. This enzyme cleaves cello-oligosaccharides with a degree of polymerization ≥ 5 and presents six glucosyl-binding subsites. The structural comparison of AfmE1 with other GH8 endoglucanases showed significant structural dissimilarities in the catalytic cleft, particularly in the subsite +3, which correlate with different functional mechanisms, such as the recognition of substrate molecules having different arrangements and crystallinities. Together, these findings provide new insights into molecular and structural features of evolutionarily conserved endoglucanases from the bacterial cellulose biosynthetic machinery.</p></div

AfmE1 mode of action.

<p>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.</p

Biochemical characterization of AfmE1.

<p>Biochemical characterization of AfmE1.</p