Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Apr 27;12(4):e0176550.
doi: 10.1371/journal.pone.0176550. eCollection 2017.

Structure and Function of a Novel GH8 Endoglucanase From the Bacterial Cellulose Synthase Complex of Raoultella Ornithinolytica

Free PMC article

Structure and Function of a Novel GH8 Endoglucanase From the Bacterial Cellulose Synthase Complex of Raoultella Ornithinolytica

Sandra Mara Naressi Scapin et al. PLoS One. .
Free PMC article


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 Komagataeibacter xylinus; 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 Raoultella ornithinolytica, named AfmE1, which was previously identified by our group from the metagenomic analysis of the giant snail Achatina fulica. 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Fig 1
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
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.
Fig 3
Fig 3. AfmE1 mode of action.
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
Fig 4. Biophysical characterization.
(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
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
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].

Similar articles

See all similar articles

Cited by 3 articles


    1. Romling U, Galperin MY. Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions. Trends Microbiol. 2015;23(9):545–57. doi: 10.1016/j.tim.2015.05.005 - DOI - PMC - PubMed
    1. Kawano S, Tajima K, Kono H, Erata T, Munekata M, Takai M. Effects of endogenous endo-β-1,4-glucanase on cellulose biosynthesis in Acetobacter xylinum ATCC23769. J Biosci Bioeng. 2002;94(3):275–81. - PubMed
    1. Tonouchi N, Tahara N, Tsuchida T, Yoshinaga F, Beppu T, Horinouchi S. Addition of a small amount of an endoglucanase enhances cellulose production by Acetobacter xylinum. Biosci, Biotechnol, and Biochem. 1995;59(5):805–8.
    1. Koo HM, Song SH, Pyun YR, Kim YS. Evidence that a β-1,4-endoglucanase secreted by Acetobacter xylinum plays an essential role for the formation of cellulose fiber. Biosci Biotechnol Biochem. 1998;62(11):2257–9. doi: 10.1271/bbb.62.2257 - DOI - PubMed
    1. Nakai T, Sugano Y, Shoda M, Sakakibara H, Oiwa K, Tuzi S, et al. Formation of highly twisted ribbons in a carboxymethylcellulase gene-disrupted strain of a cellulose-producing bacterium. J Bacteriol. 2013;195(5):958–64. doi: 10.1128/JB.01473-12 - DOI - PMC - PubMed

Grant support

This work was funded by the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2015/26982-0) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).