Apo- and Cellopentaose-bound Structures of the Bacterial Cellulose Synthase Subunit BcsZ

Abstract

Cellulose, a very abundant extracellular polysaccharide, is synthesized in a finely tuned process that involves the activity of glycosyl-transferases and hydrolases. The cellulose microfibril consists of bundles of linear ␤-1,4-glucan chains that are synthesized inside the cell; however, the mechanism by which these polymers traverse the cell membrane is currently unknown. In Gram-negative bacteria, the cellulose synthase complex forms a trans-envelope complex consisting of at least four subunits. Although three of these subunits account for the synthesis and translocation of the polysaccharide, the fourth subunit, BcsZ, is a periplasmic protein with endo-␤-1,4-glucanase activity. BcsZ belongs to family eight of glycosyl-hydrolases, and its activity is required for optimal synthesis and membrane translocation of cellulose. In this study we report two crystal structures of BcsZ from Escherichia coli. One structure shows the wild-type enzyme in its apo form, and the second structure is for a catalytically inactive mutant of BcsZ in complex with the substrate cellopentaose. The structures demonstrate that BcsZ adopts an (␣/␣) 6 -barrel fold and that it binds four glucan moieties of cellopentaose via highly conserved residues exclusively on the nonreducing side of its catalytic center. Thus, the BcsZ-cellopentaose structure most likely represents a posthydrolysis state in which the newly formed nonreducing end has already left the substrate binding pocket while the enzyme remains attached to the truncated polysaccharide chain. We further show that BcsZ efficiently degrades ␤-1,4-glucans in in vitro cellulase assays with carboxymethyl-cellulose as substrate.Cellulose, one of the most abundant biopolymers in nature, is produced by most vascular plants and a large number of algae, but is also found in some bacteria and even tunicates (1, 2). The cellulose microfibril consists of linear chains of glucose molecules that are linked via ␤-1,4-glycosidic bonds (␤-1,4-glucan) and are bundled together to form cable-like structures outside the cell (3, 4). A key enzyme in the biosynthesis of cellulose is the membrane-embedded cellulose synthase, which catalyzes the polymerization of UDP-activated glucose molecules (5, 6) and, presumably, also the translocation of the growing polysaccharide across the cell membrane (7). The bacterial cellulose synthase (Bcs) 2 complex is predicted to form a trans-envelope secretion system comprising at least four subunits (6). Although the BcsA, -B, and -C subunits are involved in synthesizing and translocating the ␤-1,4-glucan across the inner and the outer membrane, the fourth subunit, BcsZ, is a periplasmic protein with endo-␤-1,4-glucanase activity (8).BcsZ belongs to family 8 of glycoside hydrolases (GH-8) that adopts an (␣/␣) 6 -barrel architecture, consisting of two rings of six parallel ␣-helices with opposing orientation in both rings (9). GH-8 enzymes hydrolyze glycosidic bonds with a pair of acidic residues in a reaction that inverts the anomeric configuration at the new reducing end...

Characterization of the Pseudomonas aeruginosa Glycoside Hydrolase PslG Reveals That Its Levels Are Critical for Psl Polysaccharide Biosynthesis and Biofilm Formation

“…This depressed activity may be a consequence of the incompatibility between the intrinsically compact structure of long polysaccharides and the requirement to bind in a thermodynamically disfavored extended conformation across an active site groove. The existence of polysaccharide aggregates has been previously suggested to result in low hydrolysis for the endo-acting GHs ExoK and ExsH from Rhizobium meliloti and BcsZ (16,66). In support of this hypothesis, BcsZ was more efficient at hydrolyzing in vitro synthesized cellulose in situ (67).…”

“…The deep active site groove of PslG, lined with conserved surface-exposed aromatic residues, is analogous to the active site groove of BcsZ, which facilitates binding and catalysis of cellulose (16), and a carbohydrate-binding groove in the C-terminal domain of PgaB, a putative GH involved in PNAG biosynthesis (63). These features are common to processive, endoacting enzymes (64,65), and polysaccharide binding is observed in the grooves of both BcsZ and PgaB.…”

“…PslG(31-442) exhibits low hydrolytic activity toward purified surface-associated Psl in solution but, comparable with BcsZ (16), exhibited no activity toward 4-nitrophenyl glycosides, which are substrates for exo-acting glycoside hydrolases. Low in vitro hydrolysis of carboxymethylcellulose by BcsZ could only be observed using Congo Red when the substrate was embedded in agar (16), and an ex vivo assay was required to demonstrate the glycoside hydrolase activity of L. monocytogenes PssZ (19). This depressed activity may be a consequence of the incompatibility between the intrinsically compact structure of long polysaccharides and the requirement to bind in a thermodynamically disfavored extended conformation across an active site groove.…”

Leucoagaricus gongylophorus Produces Diverse Enzymes for the Degradation of Recalcitrant Plant Polymers in Leaf-Cutter Ant Fungus Gardens

“…Previous metagenomic characterizations of bacterial communities in Atta and Acromyrmex gardens recovered mainly oligosaccharide-degrading enzymes and few CAZy families associated with the degradation of recalcitrant polysaccharides (17), suggesting that bacteria are not playing a prominent role in biomass degradation in fungus gardens. Moreover, our metaproteomic results revealed that although Ͼ81,000 spectra could be confidently mapped to diverse L. gongylophorus lignocellulases, only a single spectrum could be mapped to a bacterial GH8, a CAZyme family that in bacteria is often associated with cell wall modification rather than plant biomass degradation (49). Previous work identified a bacterial glucosidase produced in fungus gardens (17), however, and it remains a possibility that bacterial enzymes not present in the protein databases used in this study could be playing a role in biomass degradation.…”

Phylogenetic Distribution of Potential Cellulases in Bacteria

“…Based on our analysis, at least 75% of the cellulases from GH8 are involved in the cellulose production in Proteobacteria. These cellulases may be involved in a transglycosylation reaction but have hydrolytic activity on cellulose analogs (e.g., carboxymethyl cellulose [CMC]) (31,39,57) and are thus frequently identified as hydrolytic cellulases. Although we have been careful in identifying putative biosynthetic enzymes, it is possible that other genes may have been misclassified, as some of these enzymes have been located outside the BCS operon (31) and/or as part of other GH families (39,40).…”