Biochemical and structural analyses of a bacterial endo-β-1,2-glucanase reveal a new glycoside hydrolase family

Abstract

β-1,2-Glucan is an extracellular cyclic or linear polysaccharide from Gram-negative bacteria, with important roles in infection and symbiosis. Despite β-1,2-glucan's importance in bacterial persistence and pathogenesis, only a few reports exist on enzymes acting on both cyclic and linear β-1,2-glucan. To this end, we purified an -β-1,2-glucanase to homogeneity from cell extracts of the environmental species, and an -β-1,2-glucanase candidate gene () was cloned from the related species The Cpin_6279 protein specifically hydrolyzed linear β-1,2-glucan with polymerization degrees of ≥5 and a cyclic counterpart, indicating that Cpin_6279 is an-β-1,2-glucananase. Stereochemical analysis demonstrated that the Cpin_6279-catalyzed reaction proceeds via an inverting mechanism. Cpin_6279 exhibited no significant sequence similarity with known glycoside hydrolases (GHs), and thus the enzyme defines a novel GH family, GH144. The crystal structures of the ligand-free and complex forms of Cpin_6279 with glucose (Glc) and sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) determined up to 1.7 Å revealed that it has a large cavity appropriate for polysaccharide degradation and adopts an (α/α)-fold slightly similar to that of GH family 15 and 8 enzymes. Mutational analysis indicated that some of the highly conserved acidic residues in the active site are important for catalysis, and the Cpin_6279 active-site architecture provided insights into the substrate recognition by the enzyme. The biochemical characterization and crystal structure of this novel GH may enable discovery of other β-1,2-glucanases and represent a critical advance toward elucidating structure-function relationships of GH enzymes.

Function and structure relationships of a β‐1,2‐glucooligosaccharide‐degrading β‐glucosidase

“…The gene cluster of the Bt BGL gene (BT_3566–BT_3569) is predicted to be a PUL, since BT_3568 and BT_3569 are annotated as an SusD‐like protein and an SusC‐like protein, respectively, in the Polysaccharide‐Utilization Loci DataBase . BT_3566 is a homolog of Cpin_6279, an endo ‐β‐1,2‐glucanase (GH144), which hydrolyzes β‐1,2‐glucans to produce Sop n s , this being consistent with the substrate specificity of Bt BGL. Based on the prediction of signal peptides in Gram‐negative bacteria , BT_3569 possesses a type‐I signal peptides like Bt BGL (BT_3567), suggesting periplasmic localization of the protein.…”

“…The primers for amplification of the btbgl gene were 5 0 -CTTCC TGCATATGGCAGCGCAGAAGTCGCC-3 0 (forward) and 5 0 -GTACGACTCGAGTTTTAGCGTGAATCGTGC-3 0 (reverse) (NdeI and XhoI sites are underlined). The primers were designed to exclude a signal peptide region (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and to fuse a His 6 -tag at the C-terminus. PCR was performed using KOD plus (Toyobo, Osaka, Japan) as a DNA polymerase as follows; 94°C for 2 min, and then 30 cycles of 94°C for 15 s, 50°C for 30 s, and 68°C for 2 min.…”

“…Sop n s and b-1,2-glucan (average DP 64) were prepared as previously reported by Abe et al and Nakajima et al [10,13], respectively. Laminarioligosaccharides (Lam n s), cellobiose (Cel 2 ), and gentiobiose (Gen 2 ) were purchased from Megazyme (Wicklow, Ireland), Wako Pure Chemical Industries (Osaka, Japan), and Tokyo Chemical Industries (Tokyo, Japan), respectively.…”

“…The identification of SOGP allowed the establishment of a practical preparation method for linear β‐1,2‐glucans . A bacterial endo ‐β‐1,2‐glucanase from Chitinophaga arvensicola has been induced using synthesized linear β‐1,2‐glucans as a sole carbon source and the gene encoding the enzyme has been successfully identified, leading to the foundation of a novel glycoside hydrolase (GH) family 144 . On the other hand, β‐1,2‐glucan‐inducible β‐glucosidase activity was reported along with the endo ‐β‐1,2‐glucanase activity in C. arvensicola , but the gene encoding the β‐glucosidase has not been identified.…”

Cyclic β-glucans at the bacteria-host cells interphase: One sugar ring to rule them all

“…CβG have a sugar ring-like structure with a varied degree of polymerisation (DP), with 17-25 glucose units although in some cases, as in Sinorhizobium meliloti, it can reach up to 40 glucoses (Figure 1c; Koizumi, Okada, Utamura, Hisamatsu, & Amenura, 1984). Second, the β-1,2-glycosidic bond is rare in nature; only a few β-1,2 glucanases have been described so far (Abe et al, 2017;Breedveld & Miller, 1994). CβG are stable biopolymers, and this is related to two unique structural characteristics.…”