Fig 1.
A set of 5 genes proposed to be involved in O-acetylated cellulose exopolysaccharide biosynthesis was identified in Clostridioides difficile 630 and appears conserved in other bacteria of class Clostridia. These genes are not homologous to the described Gram-negative cellulose synthase components, but instead comprise a mosaic of distinct evolutionary lineages that encode apparent functional equivalents, including equivalents to the glycosyltransferase BcsA (orange) and the glycosyl hydrolase BcsZ (red), in addition to a putative MBOAT (green) and an O-acetyltransferase (blue) that may O-acetylate cellulose exopolysaccharides, as in P. fluorescens SBW25. The accession codes (GenBank) for genes are listed below each gene.
Fig 2.
The ccsZABHI gene cluster shares predicted structural and functional similarity, but not homology, to the Gram-negative O-acetylated cellulose synthase.
Proteins are coloured as in Fig 1. (A) The model of the P. fluorescens SBW25 cellulose synthase based on Spiers et al. [11] that contains putative O-acetyltransferases resembling those of the alginate biosynthesis pathway in P. aeruginosa. (B) Our proposed model of the cellulose synthase from selected Clostridia. The synthases we identified (CcsA) contain the hallmark GT-2 and PilZ domains of Gram-negative cellulose synthases, in addition to CcsB, a membrane-bound extracellular protein of unknown function. O-Acetylation of cellulose in this pathway is likely carried out by CcsH and CcsI, which resemble PatA1 and PatB1 from B. cereus, as well as WssH and WssI from P. fluorescens SBW25, and cleavage is carried out by the GH-5 enzyme CcsZ reported here.
Table 1.
Diffraction data processing, refinement statistics and model validation.
Fig 3.
The overall structure of CcsZ.
(A) The (α/β)8 fold of CcsZ is shown as a cartoon representation from a front (left) and top (right) view. The catalytic resides Glu-164 and Glu-281 are shown in orange. (B) Topology cartoon of the CcsZ fold, coloured as in panel A. (C) The substrate-binding cleft of CcsZ (yellow box) has an overall strong negative charge, an unusual feature for GH-5 enzymes.
Fig 4.
Substrate accommodation by CcsZ and TmCel5A.
(A) The cellotetraose-bound structure of TmCel5A. The cellotetraose molecule is bound at subsites adjacent to the active site residues Glu-136 and Glu-253, guided by ring-stacking with Trp-210. (B) The equivalent substrate-binding site of CcsZ. The active site residues are present as Glu-164 and Glu-281. The equivalent to the -2 subsite, shaped by W210 in TmCel5A, is obstructed by the orientation of Tyr-237 in CcsZ, preventing substrate accommodation in the same fashion.
Fig 5.
The product-bound structure of CcsZ.
(A) A glucose molecule is bound at the apparent -2 subsite adjacent the catalytic architecture and confined by the orientation of Y237. (B) The bound glucose molecule makes polar contacts with W314 via the C1 hydroxyl and N48 via the C2 hydroxyl and H123 via the C6 hydroxyl. (C) The polder omit map electron density (blue mesh) for the glucose ligand contoured at 3 σ.
Fig 6.
(A) CMC hydrolysis by CcsZ and the T. reesei cellulase cocktail positive control is evident by the zone of clearing in the Congo Red stained CMC plate-based zymogram. (B) CMC hydrolysis by CcsZ. The reducing sugar concentration of a CMC solution (8.3 g/L) was increased by treatment with CcsZ over 4 h at 37°C in a CcsZ concentration-dependent manner.
Fig 7.
pH and substrate specificity of CcsZ.
(A) The pH profile of CcsZ displays a clear preference for acidic conditions with a pH optimum of 4.5. Activity was calculated using the DNS method with solubilized CMC substrate. Buffer solutions used (50 mM) are listed above data points. (B) Substrate utilization profile of CcsZ using common GH-5 substrates. CcsZ exhibited five-fold greater activity on mixed-linkage β-glucan as compared to CMC when assayed using the DNS method. CcsZ activity on arabinoxylan, lichenin, xylan and β(1,3)-linked curdlan was not significantly different from an enzyme-free control under our assay conditions. Assays were performed with at least n ≥ 3 replicates for all groups. ns = not significant and asterisks denote significant activity (t-tests, t(2) = 23.33 and p = 0.0018 for CMC, t(2) = 21.30 and p = 0.0022 for β-glucan). All other groups were not significantly different from 0 (p > 0.05).
Fig 8.
Representative time-resolved mass spectra of the CcsZ products.
CcsZ is capable of mixed exo- and endo-glucanase activity on substrates at least 4 saccharide units in length, since all possible glucanase products were observed with complete digestion of the starting cellopentaose material after 90 min incubation with CcsZ.
Fig 9.
Endocellulase activity was assessed using a bimodified cellopentaose substrate that contains terminal p-nitrophenyl and 3-ketobutylidene groups. CcsZ and the positive control (a cellulase cocktail from Trichoderma reesei) demonstrated endocellulase activity in the coupled enzyme assay with an ancillary exo-β-glucosidase. CcsZ did not demonstrate exo-activity, since degradation of the endo-released products and concomitant release of p-nitrophenolate was not observed in the absence of the supplied exo-β-glucosidase. Data are the results of at least 4 replicates. ns = not significant (ANOVA, F (3, 12) = 3770, q = 1.920, p = 0.1834).