Presence of N-L-lactyl-D-perosamine residue in the sheath-forming polysaccharide of Thiothrix fructosivorans

Yuta Kawasaki, Keiko Kondo, Rie Narizuka, Tomoyuki Endo, Masato Katahira, Izuru Kawamura, Michio Sato, Minoru Takeda

Abstract

Thiothrix fructosivorans forms a microtube (sheath) that encloses a line of cells. This sheath is an assemblage of [→4)-GlcN-(1→4)-Glc-(1→]n with side chains of Rha4N-(1→3)-Fuc(1→ at position 3 of Glc. The sheath-forming polysaccharide (SFP) may have some substitutions but this is not yet confirmed. To investigate the possible substitutions, the sheath was prepared by mild treatments. Solid-state NMR analysis suggested the presence of N-substitution. The sheath was hydrolyzed with concentrated HCl at 0°C, followed by derivatization with 4-aminobenzoic acid ethyl ester (ABEE). The presence of N-lactyl-Rha4N-Fuc-ABEE was suggested by NMR spectroscopy. Lactic acid was determined to be the l-isomer by chiral HPLC analysis. To estimate the N-lactylation degree, the sheath was N-acetylated. N-Acetyl-Rha4N-Fuc-ABEE and N-lactyl-Rha4N-Fuc-ABEE were then collectively recovered, and their abundance ratio was determined to be 1:4 by NMR analysis. When hydrolysis was performed at 40°C, GlcNAc-ABEE was obtained. For estimation of the N-acetylation degree, the sheath was N-acetylated with deuterated acetic anhydride and then N-acetyl-GlcN-ABEE was prepared. The content of deuterated N-acetyl-GlcN-ABEE was determined to be 50% based on the relative intensity of the acetyl proton signal in the 1D-1H NMR spectrum. It was concluded that Rha4N is mostly N-L-lactylated and GlcN is substoichiometrically N-acetylated.

Original languageEnglish
Pages (from-to)772-779
Number of pages8
JournalInternational Journal of Biological Macromolecules
Volume82
DOIs
StatePublished - 2016 Jan 1

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Aminobenzoates
Polysaccharides
Esters
Benzocaine
Anhydrides
Acetylation
Acetic Acid
Protons
Lactic Acid
Hydrolysis
Magnetic Resonance Spectroscopy
High Pressure Liquid Chromatography

Keywords

  • N-Lactylation
  • Perosamine
  • Polysaccharide
  • Sheath
  • Thiothrix fructosivorans

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Cite this

Presence of N-L-lactyl-D-perosamine residue in the sheath-forming polysaccharide of Thiothrix fructosivorans. / Kawasaki, Yuta; Kondo, Keiko; Narizuka, Rie; Endo, Tomoyuki; Katahira, Masato; Kawamura, Izuru; Sato, Michio; Takeda, Minoru.

In: International Journal of Biological Macromolecules, Vol. 82, 01.01.2016, p. 772-779.

Research output: Contribution to journalArticle

Kawasaki, Yuta; Kondo, Keiko; Narizuka, Rie; Endo, Tomoyuki; Katahira, Masato; Kawamura, Izuru; Sato, Michio; Takeda, Minoru / Presence of N-L-lactyl-D-perosamine residue in the sheath-forming polysaccharide of Thiothrix fructosivorans.

In: International Journal of Biological Macromolecules, Vol. 82, 01.01.2016, p. 772-779.

Research output: Contribution to journalArticle

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T1 - Presence of N-L-lactyl-D-perosamine residue in the sheath-forming polysaccharide of Thiothrix fructosivorans

AU - Kawasaki,Yuta

AU - Kondo,Keiko

AU - Narizuka,Rie

AU - Endo,Tomoyuki

AU - Katahira,Masato

AU - Kawamura,Izuru

AU - Sato,Michio

AU - Takeda,Minoru

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N2 - Thiothrix fructosivorans forms a microtube (sheath) that encloses a line of cells. This sheath is an assemblage of [→4)-GlcN-(1→4)-Glc-(1→]n with side chains of Rha4N-(1→3)-Fuc(1→ at position 3 of Glc. The sheath-forming polysaccharide (SFP) may have some substitutions but this is not yet confirmed. To investigate the possible substitutions, the sheath was prepared by mild treatments. Solid-state NMR analysis suggested the presence of N-substitution. The sheath was hydrolyzed with concentrated HCl at 0°C, followed by derivatization with 4-aminobenzoic acid ethyl ester (ABEE). The presence of N-lactyl-Rha4N-Fuc-ABEE was suggested by NMR spectroscopy. Lactic acid was determined to be the l-isomer by chiral HPLC analysis. To estimate the N-lactylation degree, the sheath was N-acetylated. N-Acetyl-Rha4N-Fuc-ABEE and N-lactyl-Rha4N-Fuc-ABEE were then collectively recovered, and their abundance ratio was determined to be 1:4 by NMR analysis. When hydrolysis was performed at 40°C, GlcNAc-ABEE was obtained. For estimation of the N-acetylation degree, the sheath was N-acetylated with deuterated acetic anhydride and then N-acetyl-GlcN-ABEE was prepared. The content of deuterated N-acetyl-GlcN-ABEE was determined to be 50% based on the relative intensity of the acetyl proton signal in the 1D-1H NMR spectrum. It was concluded that Rha4N is mostly N-L-lactylated and GlcN is substoichiometrically N-acetylated.

AB - Thiothrix fructosivorans forms a microtube (sheath) that encloses a line of cells. This sheath is an assemblage of [→4)-GlcN-(1→4)-Glc-(1→]n with side chains of Rha4N-(1→3)-Fuc(1→ at position 3 of Glc. The sheath-forming polysaccharide (SFP) may have some substitutions but this is not yet confirmed. To investigate the possible substitutions, the sheath was prepared by mild treatments. Solid-state NMR analysis suggested the presence of N-substitution. The sheath was hydrolyzed with concentrated HCl at 0°C, followed by derivatization with 4-aminobenzoic acid ethyl ester (ABEE). The presence of N-lactyl-Rha4N-Fuc-ABEE was suggested by NMR spectroscopy. Lactic acid was determined to be the l-isomer by chiral HPLC analysis. To estimate the N-lactylation degree, the sheath was N-acetylated. N-Acetyl-Rha4N-Fuc-ABEE and N-lactyl-Rha4N-Fuc-ABEE were then collectively recovered, and their abundance ratio was determined to be 1:4 by NMR analysis. When hydrolysis was performed at 40°C, GlcNAc-ABEE was obtained. For estimation of the N-acetylation degree, the sheath was N-acetylated with deuterated acetic anhydride and then N-acetyl-GlcN-ABEE was prepared. The content of deuterated N-acetyl-GlcN-ABEE was determined to be 50% based on the relative intensity of the acetyl proton signal in the 1D-1H NMR spectrum. It was concluded that Rha4N is mostly N-L-lactylated and GlcN is substoichiometrically N-acetylated.

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KW - Thiothrix fructosivorans

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