Role of the CBM11, Fn3, and CBM3 Domains in Enhancing the Multifunctional Enzymatic Activities of Glycoside Hydrolase Family 5 from Paenibacillus curdlanolyticus B-6
2
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
Glycoside hydrolase family 5 (GH5) from Paenibacillus curdlanolyticus B-6 (PcGH5) is a modular protein consisting of a GH5 catalytic domain and three non-catalytic domains (NCDs): a family 11 carbohydrate-binding module (CBM11), a fibronectin type 3 (Fn3), and a family 3 carbohydratebinding module (CBM3). PcGH5 exhibits unique properties compared with other GH5 members; it shows multifunctional endo-cellulase, endo-xylanase, endo-mannanase, and endo-1,3-1,4-β-glucanase activities. To understand the function of each NCD, recombinant full-length PcGH5 and truncated variants were analyzed. Compared with the GH5 catalytic domain alone, stepwise addition of CBM11, Fn3, and CBM3 enhanced substrate binding and improved enzymatic activities towards polysaccharides, as confirmed by Fourier transform infrared spectroscopy (FTIR). Each NCD contributed distinctly: CBM11 bound 1,3-1,4-β-glucan, xylan and mannan with limited branching; Fn3 bound only cellulose; and CBM3 significantly improved binding to 1,3-1,4-β-glucan and highly branched xylan and mannan. There was a higher percentage of surface-exposed aromatic amino acids in CBM11 and CBM3 of PcGH5, important for hydrophobic interactions with sugar rings, compared to other CBM11 and CBM3 members. Unlike other CBMs, sequence alignment and structural modelling revealed that PcGH5 CBM11 and CBM3 have extra and/or more surfaceexposed aromatic amino acids, which could interact with various oligosaccharide ligands such as hexose (cellotetraose and mannotetraose) and pentose (xylotetraose) via hydrophobic interactions, affecting enzyme activity. Mutagenesis confirmed Trp17 (CBM11) and Trp51 (CBM3) as key residues for insoluble substrates binding and enzymatic enhancement. Therefore, these aromatic amino acids are key factors improving substrates binding and enzymatic activities of PcGH5 towards different β-1,4 glycosidic polysaccharide substrates.
Abstract
Glycoside hydrolase family 5 (GH5) from Paenibacillus curdlanolyticus B-6 (PcGH5) is a modular protein consisting of a GH5 catalytic domain and three non-catalytic domains (NCDs): a family 11 carbohydrate-binding module (CBM11), a fibronectin type 3 (Fn3), and a family 3 carbohydratebinding module (CBM3). PcGH5 exhibits unique properties compared with other GH5 members; it shows multifunctional endo-cellulase, endo-xylanase, endo-mannanase, and endo-1,3-1,4-β-glucanase activities. To understand the function of each NCD, recombinant full-length PcGH5 and truncated variants were analyzed. Compared with the GH5 catalytic domain alone, stepwise addition of CBM11, Fn3, and CBM3 enhanced substrate binding and improved enzymatic activities towards polysaccharides, as confirmed by Fourier transform infrared spectroscopy (FTIR). Each NCD contributed distinctly: CBM11 bound 1,3-1,4-β-glucan, xylan and mannan with limited branching; Fn3 bound only cellulose; and CBM3 significantly improved binding to 1,3-1,4-β-glucan and highly branched xylan and mannan. There was a higher percentage of surface-exposed aromatic amino acids in CBM11 and CBM3 of PcGH5, important for hydrophobic interactions with sugar rings, compared to other CBM11 and CBM3 members. Unlike other CBMs, sequence alignment and structural modelling revealed that PcGH5 CBM11 and CBM3 have extra and/or more surfaceexposed aromatic amino acids, which could interact with various oligosaccharide ligands such as hexose (cellotetraose and mannotetraose) and pentose (xylotetraose) via hydrophobic interactions, affecting enzyme activity. Mutagenesis confirmed Trp17 (CBM11) and Trp51 (CBM3) as key residues for insoluble substrates binding and enzymatic enhancement. Therefore, these aromatic amino acids are key factors improving substrates binding and enzymatic activities of PcGH5 towards different β-1,4 glycosidic polysaccharide substrates.