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Identification, characterization, and structural analyses of a fungal endo-β-1,2-glucanase reveal a new glycoside hydrolase family.

ABSTRACT: endo-β-1,2-Glucanase (SGL) is an enzyme that hydrolyzes β-1,2-glucans, which play important physiological roles in some bacteria as a cyclic form. To date, no eukaryotic SGL has been identified. We purified an SGL from Talaromyces funiculosus (TfSGL), a soil fungus, to homogeneity and then cloned the complementary DNA encoding the enzyme. TfSGL shows no significant sequence similarity to any known glycoside hydrolase (GH) families, but shows significant similarity to certain eukaryotic proteins with unknown functions. The recombinant TfSGL (TfSGLr) specifically hydrolyzed linear and cyclic β-1,2-glucans to sophorose (Glc-β-1,2-Glc) as a main product. TfSGLr hydrolyzed reducing-end-modified β-1,2-gluco-oligosaccharides to release a sophoroside with the modified moiety. These results indicate that TfSGL is an endo-type enzyme that preferably releases sophorose from the reducing end of substrates. Stereochemical analysis demonstrated that TfSGL is an inverting enzyme. The overall structure of TfSGLr includes an (α/α)₆ toroid fold. The substrate-binding mode was revealed by the structure of a Michaelis complex of an inactive TfSGLr mutant with a β-1,2-glucoheptasaccharide. Mutational analysis and action pattern analysis of β-1,2-gluco-oligosaccharide derivatives revealed an unprecedented catalytic mechanism for substrate hydrolysis. Glu-262 (general acid) indirectly protonates the anomeric oxygen at subsite -1 via the 3-hydroxy group of the Glc moiety at subsite +2, and Asp-446 (general base) activates the nucleophilic water via another water. TfSGLr is apparently different from a GH144 SGL in the reaction and substrate recognition mechanism based on structural comparison. Overall, we propose that TfSGL and closely-related enzymes can be classified into a new family, GH162.

SUBMITTER: Tanaka N

PROVIDER: S-EPMC6514616 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:As classified by the Carbohydrate-Active Enzymes (CAZy) database, enzymes in glycoside hydrolase (GH) family 10 (GH10) are all monospecific or bifunctional xylanases (except a tomatinase), and no endo-β-1,4-glucanase has been reported in the family. Here, we identified Arcticibacterium luteifluviistationis carboxymethyl cellulase (AlCMCase) as a GH10 endo-β-1,4-glucanase. AlCMCase originated from an Arctic marine bacterium, Arcticibacterium luteifluviistationis SM1504T It shows low identity (<35%) with other GH10 xylanases. The gene encoding AlCMCase was overexpressed in Escherichia coli Biochemical characterization showed that recombinant AlCMCase is a cold-adapted and salt-tolerant enzyme. AlCMCase hydrolyzes cello- and xylo-configured substrates via an endoaction mode. However, in comparison to its significant cellulase activity, the xylanase activity of AlCMCase is negligible. Correspondingly, AlCMCase has remarkable binding capacity for cello-oligosaccharides but no obvious binding capacity for xylo-oligosaccharides. AlCMCase and its homologs are grouped into a branch separate from other GH10 xylanases in a phylogenetic tree, and two homologs also displayed the same substrate specificity as AlCMCase. These results suggest that AlCMCase and its homologs form a novel subfamily of GH10 enzymes that have robust endo-β-1,4-glucanase activity. In addition, given the cold-adapted and salt-tolerant characters of AlCMCase, it may be a candidate biocatalyst under certain industrial conditions, such as low temperature or high salinity.IMPORTANCE Cellulase and xylanase have been widely used in the textile, pulp and paper, animal feed, and food-processing industries. Exploring novel cellulases and xylanases for biocatalysts continues to be a hot issue. Enzymes derived from the polar seas might have novel hydrolysis patterns, substrate specificities, or extremophilic properties that have great potential for both fundamental research and industrial applications. Here, we identified a novel cold-adapted and salt-tolerant endo-β-1,4-glucanase, AlCMCase, from an Arctic marine bacterium. It may be useful in certain industrial processes, such as under low temperature or high salinity. Moreover, AlCMCase is a bifunctional representative of glycoside hydrolase (GH) family 10 that preferentially hydrolyzes β-1,4-glucans. With its homologs, it represents a new subfamily in this family. Thus, this study sheds new light on the substrate specificity of GH10.

Project description:Endo-β-1,4-glucanase is a crucial glycoside hydrolase (GH) involved in the decomposition of cellulosic materials. In this study, to discover a novel cold-adapted β-1,4-D-glucan-degrading enzyme, the gene coding for an extracellular endo-β-1,4-glucanase (GluL) from Lichenicola cladoniae PAMC 26568, an Antarctic lichen (Cladonia borealis)-associated bacterium, was identified and recombinantly expressed in Escherichia coli BL21. The GluL gene (1044-bp) encoded a non-modular polypeptide consisting of a single catalytic GH8 domain, which shared the highest sequence identity of 55% with that of an uncharacterized protein from Gluconacetobacter takamatsuzukensis (WP_182950054). The recombinant endo-β-1,4-glucanase (rGluL: 38.0 kDa) most efficiently degraded sodium carboxymethylcellulose (CMC) at pH 4.0 and 45°C, and showed approximately 23% of its maximum degradation activity even at 3°C. The biocatalytic activity of rGluL was noticeably enhanced by >1.3-fold in the presence of 1 mM Mn2+ or NaCl at concentrations between 0.1 and 0.5 M, whereas the enzyme was considerably downregulated by 1 mM Hg2+ and Fe2+ together with 5 mM N-bromosuccinimide and 0.5% sodium dodecyl sulfate. rGluL is a true endo-β-1,4-glucanase, which could preferentially decompose D-cellooligosaccharides consisting of 3 to 6 D-glucose, CMC, and barley β-glucan, without other additional glycoside hydrolase activities. The specific activity (15.1 U mg-1) and k cat/K m value (6.35 mg-1 s-1mL) of rGluL toward barley β-glucan were approximately 1.8- and 2.2-fold higher, respectively, compared to its specific activity (8.3 U mg-1) and k cat/K m value (2.83 mg-1 s-1mL) toward CMC. The enzymatic hydrolysis of CMC, D-cellotetraose, and D-cellohexaose yielded primarily D-cellobiose, accompanied by D-glucose, D-cellotriose, and D-cellotetraose. However, the cleavage of D-cellopentaose by rGluL resulted in the production of only D-cellobiose and D-cellotriose. The findings of the present study imply that rGluL is a novel, acidic, and cold-adapted GH8 endo-β-1,4-glucanase with high specific activity, which can be exploited as a promising candidate in low-temperature processes including textile and food processes.

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Identification, characterization, and structural analyses of a fungal endo-β-1,2-glucanase reveal a new glycoside hydrolase family.

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