Characterization and Structural Analysis of a Novel exo-Type Enzyme Acting on β-1,2-Glucooligosaccharides from Parabacteroides distasonis

Abstract

β-1,2-Glucan is a polysaccharide produced mainly by some Gram-negative bacteria as a symbiosis and infectious factor. We recently identified endo-β-1,2-glucanase from Chitinophaga pinensis ( CpSGL) as an enzyme comprising a new family. Here, we report the characteristics and crystal structure of a CpSGL homologue from Parabacteroides distasonis, an intestinal bacterium (BDI_3064 protein), which exhibits distinctive properties of known β-1,2-glucan-degrading enzymes. BDI_3064 hydrolyzed linear β-1,2-glucan and β-1,2-glucooligosaccharides with degrees of polymerization (DPs) of ≥4 to produce sophorose specifically but did not hydrolyze cyclic β-1,2-glucan. This result indicates that BDI_3064 is a new exo-type enzyme. BDI_3064 also produced sophorose from β-1,2-glucooligosaccharide analogues that have a modified reducing end, indicating that BDI_3064 acts on its substrates from the nonreducing end. The crystal structure showed that BDI_3064 possesses additional N-terminal domains 1 and 2, unlike CpSGL. Superimposition of BDI_3064 and CpSGL complexed with ligands showed that R93 in domain 1 overlapped subsite -3 in CpSGL. Docking analysis involving a β-1,2-glucooligosaccharide with DP4 showed that R93 completely blocks the nonreducing end of the docked β-1,2-glucooligosaccharide. This indicates that BDI_3064 employs a distinct mechanism of recognition at the nonreducing end of substrates to act as an exo-type enzyme. Thus, we propose 2-β-d-glucooligosaccharide sophorohydrolase (nonreducing end) as a systematic name for BDI_3064.

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

“…These results suggest that TfSGL is an endo-type enzyme but quite preferably releases Sop 2 from the reducing end of ␤-1,2-glucan. This is a different property from that of PdSGL, which releases Sop 2 from the nonreducing end of linear ␤-1,2-glucan (26).…”

“…Cyclic ␤-1,2-glucan with DP of 17-24 was kindly donated by Dr. M. Hisamatsu of Mie University (65). rSop n s with DP of 6 -11 were prepared by modifying Sop n s at the reducing end with NaBH 4 treatment as described by Shimizu et al (26). Approximately 10% Sop n solutions were added to 1 M NaBH 4 of 20 l, followed by incubation at room temperature for 5 min or more.…”

“…In 2017, Cpin6279 protein from Chitinophaga pinensis (CpSGL), a soil Gram-negative bacterium, was first identified as an SGL with a novel amino acid sequence, demonstrating a novel family, GH144 (25). Then, a sophorosyl hydrolase (nonreducing end), which releases sophorose from the nonreducing end of Sop n s (PdSGL), was found as an exo-type enzyme in Parabacteroides distasonis, an intestinal bacterium, among the GH144 homologs (26).…”

Effect of Long-Term Farming Practices on Agricultural Soil Microbiome Members Represented by Metagenomically Assembled Genomes (MAGs) and Their Predicted Plant-Beneficial Genes

“…Enzymes of GH144 prefer β-1,2-glucan as substrate. β-1,2-Glucan is a polysaccharide produced by Gram-negative bacteria with important roles in infection and symbiosis [98,99]. The metabolic profile of MAG_10 (Figure 6) also suggests its capability of metabolizing many different carbohydrates.…”

Characterization and structural analyses of a novel glycosyltransferase acting on the β-1,2-glucosidic linkages

“…This finding enables us to explore SGL homologs and SGL gene clusters. An SGL homolog possessing an unknown function region at the N terminus has been identified as a novel exolytic enzyme that releases Sop 2 from the nonreducing ends of Sop n s ( 30 ). A β-glucosidase preferably acting on longer Sop n s and β-1,2-glucan has also been found from an SGL gene cluster in Bacteroides thetaiotaomicron ( 31 ).…”