Polysaccharide peptides (or protein-bound polysaccharides, PSPs) are commonly found in mushrooms and plants and possess important nutritional properties and health benefits. The pathogenic bacterium Streptococcus zooepidemicus does not inherently produce PSPs but secretes the capsular polysaccharide hyaluronan. However, in a previous investigation of the catalytic mechanism of UDP-glucose dehydrogenase (UGDH), a PSP of peptide-bound hyaluronan was found to be produced by S. zooepidemicus through the in vivo expression of a mutant of the gene encoding UGDH. In the present study, this hyaluronan-derived PSP was structurally characterized by FT-IR, NMR, and high-performance liquid chromatography-mass spectrometry (HPLC-MS), and the data confirmed that the polysaccharide backbone, hyaluronan, is covalently bound to the side-chain peptides via an amide linkage. More importantly, the bacterial production of a PSP via this genetic modification method should inspire further research on the in vitro enzymatic synthesis of PSPs or even naturally occurring polysaccharide derivatives and may provide a theoretical foundation for investigating the in vivo synthetic mechanism of PSPs.

synthesis, Hyaluronan (HA), Polysaccharide peptide (PSP), Streptococcus zooepidemicus, UDP-Glucose dehydrogenase

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@H](O[C@@H]2[C@@H](C(=O)O)OC(O)[C@H](O)[C@H]2O)O[C@H](CO)[C@@H](O)[C@@H]1O

397.333 g/mol (C₁₄H₂₃NO₁₂)

Polysaccharide peptides (or protein-bound polysaccharides, PSPs) are commonly found in mushrooms and plants and possess important nutritional properties and health benefits. The pathogenic bacterium Streptococcus zooepidemicus does not inherently produce PSPs but secretes the capsular polysaccharide hyaluronan. However, in a previous investigation of the catalytic mechanism of UDP-glucose dehydrogenase (UGDH), a PSP of peptide-bound hyaluronan was found to be produced by S. zooepidemicus through the in vivo expression of a mutant of the gene encoding UGDH. In the present study, this hyaluronan-derived PSP was structurally characterized by FT-IR, NMR, and high-performance liquid chromatography-mass spectrometry (HPLC-MS), and the data confirmed that the polysaccharide backbone, hyaluronan, is covalently bound to the side-chain peptides via an amide linkage. More importantly, the bacterial production of a PSP via this genetic modification method should inspire further research on the in vitro enzymatic synthesis of PSPs or even naturally occurring polysaccharide derivatives and may provide a theoretical foundation for investigating the in vivo synthetic mechanism of PSPs.

synthesis, Hyaluronan (HA), Polysaccharide peptide (PSP), Streptococcus zooepidemicus, UDP-Glucose dehydrogenase

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

R1 = peptide
[1*]OC(=O)[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1NC(C)=O

396.325 g/mol (C₁₄H₂₂RNO₁₂, R = underdetermined substituent(s), not counted in mol weight)

Polysaccharide peptides (or protein-bound polysaccharides, PSPs) are commonly found in mushrooms and plants and possess important nutritional properties and health benefits. The pathogenic bacterium Streptococcus zooepidemicus does not inherently produce PSPs but secretes the capsular polysaccharide hyaluronan. However, in a previous investigation of the catalytic mechanism of UDP-glucose dehydrogenase (UGDH), a PSP of peptide-bound hyaluronan was found to be produced by S. zooepidemicus through the in vivo expression of a mutant of the gene encoding UGDH. In the present study, this hyaluronan-derived PSP was structurally characterized by FT-IR, NMR, and high-performance liquid chromatography-mass spectrometry (HPLC-MS), and the data confirmed that the polysaccharide backbone, hyaluronan, is covalently bound to the side-chain peptides via an amide linkage. More importantly, the bacterial production of a PSP via this genetic modification method should inspire further research on the in vitro enzymatic synthesis of PSPs or even naturally occurring polysaccharide derivatives and may provide a theoretical foundation for investigating the in vivo synthetic mechanism of PSPs.

synthesis, Hyaluronan (HA), Polysaccharide peptide (PSP), Streptococcus zooepidemicus, UDP-Glucose dehydrogenase

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

R1 = peptide
[*]O[C@@H]1[C@@H](C(=O)O)O[C@@H](O[C@H]2[C@H](O)[C@@H](CO)O[C@@H](O[C@@H]3[C@@H](C(=O)O[1*])O[C@@H](O[C@@H]4[C@@H](N)[C@H]([*])O[C@H](CO)[C@H]4O)[C@H](O)[C@H]3O)[C@@H]2NC(C)=O)[C@H](O)[C@H]1O

715.591 g/mol (C₂₆H₃₉R₃N₂O₂₁, R = next and previous repeats / underdetermined substituent(s), not counted in mol weight)

The glycosaminoglycan hyaluronan is involved in a diverse range of physiological and diseases processes and comprises repeated disaccharide units of N-acetyl-d-glucosamine (GlcNAc) and d-glucuronic acid (GlcA). A molecular description of the solution conformation of HA is required to account for this biology, which is best attained using nuclear magnetic resonance (NMR). NMR studies of the polymer, however, are frustrated by resonance overlap arising from the highly degenerate structure. In contrast, end-effects in oligosaccharides can produce some chemical shift dispersion, giving the possibility that their conformational properties can be measured and extrapolated to models of the polymer. We report the complete resolution and assignment of (1)H, (13)C and (15)N nuclei in hyaluronan oligosaccharides with seven different naturally occurring terminal rings. At 900MHz, all (1)H nuclei in the hexasaccharide GlcA-β-(1→3)-GlcNAc-β-(1→4)-GlcA-β-(1→3)-GlcNAc-β-(1→4)-GlcA-β-(1→3)-GlcNAc-OH were uniquely resolved and the two central rings were found to be a good model for the polymer environment. These assignments now allow resolved, unambiguous structural restraints to be acquired on this oligosaccharide and extrapolated to models for the solution conformation of the polymer.

coupling constant, resolution, hyaluronan, strong coupling, end-effect

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,2	Ac	?	25.232
4	bDGlcpN	103.380	58.212	76.811	72.582	78.662	63.410
	?DGlcpA


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,2	Ac	-	2.043
4	bDGlcpN	4.522	3.699	3.520	3.459	3.460	3.763-3.926
	?DGlcpA


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,2	Ac		25.232/2.043
4	bDGlcpN	103.380/4.522	58.212/3.699	76.811/3.520	72.582/3.459	78.662/3.460	63.410/3.763-3.926
	?DGlcpA

The spectrum also has 1 signal at unknown position (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@H](O[C@@H]2[C@@H](C(=O)O)OC(O)[C@H](O)[C@H]2O)O[C@H](CO)[C@@H](O)[C@@H]1O

397.333 g/mol (C₁₄H₂₃NO₁₂)