Structural determination of the sheath-forming polysaccharide of Sphaerotilus montanus using thiopeptidoglycan lyase which recognizes the 1,4 linkage between α-d-GalN and β-d-GlcA
| Autor: | Ryoji Usami, Ichiro Suzuki, Yuta Kawasaki, Minoru Takeda, Izuru Kawamura, Masato Katahira, Tadashi Nittami, Daisuke Kashiwabara, Daisuke Kan, Keiko Kondo, Michio Sato |
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| Rok vydání: | 2021 |
| Předmět: |
Disulfide Linkage
Glycoconjugate Stereochemistry Peptide Sphaerotilus 02 engineering and technology Polysaccharide Biochemistry 03 medical and health sciences Polysaccharides Structural Biology Molecular Biology Sphaerotilus montanus Polysaccharide-Lyases 030304 developmental biology chemistry.chemical_classification 0303 health sciences integumentary system biology Sphaerotilus natans General Medicine Sheath 021001 nanoscience & nanotechnology biology.organism_classification Lyase Enzyme chemistry Thiopeptidoglycan lyase 0210 nano-technology Paenibacillus Cysteine |
| Zdroj: | International Journal of Biological Macromolecules. 183:992-1001 |
| ISSN: | 0141-8130 |
| DOI: | 10.1016/j.ijbiomac.2021.05.001 |
| Popis: | Sphaerotilus natans is a filamentous sheath-forming bacterium commonly found in activated sludge. Its sheath is assembled from a thiolic glycoconjugate called thiopeptidoglycan. S. montanus ATCC-BAA-2725 is a sheath-forming member of stream biofilms, and its sheath is morphologically similar to that of S. natans. However, it exhibits heat susceptibility, which distinguishes it from the S. natans sheath. In this study, chemical composition and solid-state NMR analyses suggest that the S. montanus sheath is free of cysteine, indicating that disulfide linkage is not mandatory for sheath formation. The S. montanus sheath was successfully solubilized by N-acetylation, allowing solution-state NMR analysis to determine the sugar sequence. The sheath was susceptible to thiopeptidoglycan lyase prepared from the thiopeptidoglycan-assimilating bacterium, Paenibacillus koleovorans. The reducing ends of the enzymatic digests were labeled with 4-aminobenzoic acid ethyl ester, followed by HPLC. Two derivatives were detected, and their structures were determined. We found that the sheath has no peptides and is assembled as follows: [→4)-β- d -GlcA-(1→4)-β- d -Glc-(1→3)-β- d -GalNAc-(1→4)-α- d -GalNAc-(1→4)-α- d -GalN-(1→]n (β- d -Glc and α- d -GalNAc are stoichiometrically and substoichiometrically 3-O-acetylated, respectively). Thiopeptidoglycan lyase was thus confirmed to cleave the 1,4 linkage between α- d -GalN and β- d -GlcA, regardless of the peptide moiety. Furthermore, vital fluorescent staining of the sheath demonstrated that elongation takes place at the tips, as with the S. natans sheath. |
| Databáze: | OpenAIRE |
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