Enzymatic synthesis of complex glycosaminotrioses and study of their molecular recognition by hevein domains

Abstract

Hevein, a protein found in Hevea brasiliensis, has a CRD domain, which is known to bind chitin and GlcNAc-containing oligosaccharides. By using NMR and molecular modeling as major tools we have demonstrated that trisaccharides containing GalNAc and ManNAc residues are also recognized by hevein domains. Thus far unknown trisaccharides GlcNAcbeta(1-->4)GlcNAcbeta(1-->4)ManNAc (1) and GalNAcbeta(1-->4)GlcNAcbeta(1-->4)ManNAc (2) were synthesized with the use of beta-N-acetylhexosaminidase from Aspergillus oryzae. This method is based on the rather unique phenomenon that some fungal beta-N-acetylhexosaminidases cannot hydrolyze disaccharide GlcNAcbeta(1-->4)ManNAc (5) contrary to chitobiose GlcNAcbeta(1-->4)GlcNAc (4) that is cleaved and, therefore, cannot be used as an acceptor for further transglycosylation. Both trisaccharides 1 and 2 were prepared by transglycosylation from disaccharidic acceptor in good yields ranging from 35% to 40%. Our observations strongly indicate that the present nature of the modifications of chitotriose (GlcNAcbeta(1-->lcNAcbeta(1-->4)GlcNAc, 3) at either the non-reducing end (GalNAc instead of GlcNAc) or at the reducing end (ManNAc instead of GlcNAc) do not modify the mode of binding of the trisaccharide to hevein. The association constant values indicate that chitotriose (3) binding is better than that of 1 and 2, and that the binding of (with ManNAc at the reducing end) is favored with respect to that of 2 (with ManNAc at the reducing end with a non-reducing GalNAc moiety).

Highlights on Hevea brasiliensis (pro)hevein proteins

“…These hevein-carbohydrate interactions are enthalpically driven with estimated binding energies of approximately -2 to -5 kcal mol -1 [79,100]. The interaction of the carbohydrate-hevein complexes has been fully studied by NMR [81,83,101], molecular simulations [102][103][104] and isothermal titration calorimetry (ITC; [46,80,81,105]). Residues involved in binding include S19, W21, W23 and Y30, but also the loop region 13-16 [80,102].…”

Efficient and Regioselective Synthesis of β-GalNAc/GlcNAc-Lactose by a Bifunctional Transglycosylating β- N -Acetylhexosaminidase from Bifidobacterium bifidum

“…With the use of pNP-␤-GalNAc and pNP-␤-GlcNAc as donors, the ␤-Nacetylhexosaminidase from Trichoderma harzianum can transfer both GalNAc and GlcNAc residues to UDP-GlcNAc to form GalNAc␤1-4GlcNAc␣UDP and GlcNAcGlcNAc␣UDP (regioselectivity not identified) at 22% and 1.9% yields at 30°C for 8 and 14 h, respectively (25). In addition, when pNP-␤-GalNAc and pNP-␤-GlcNAc were used as donors, the ␤-N-acetylhexosaminidase from A. oryzae can transfer both GalNAc and GlcNAc residues to GlcNAc␤1-4ManNAc to form GalNAc␤1-4GlcNAc␤1-4Man-NAc and GlcNAc␤1-4GlcNAc␤1-4ManNAc at 41% and 36% yields at 37°C for 2 h and 50 min, respectively (24). In this work, BbhI transferred both GalNAc and GlcNAc residues from pNP-␤-GalNAc and pNP-␤-GlcNAc to lactose to form GalNAc␤1-3Gal␤1-4Glc and GlcNAc␤1-3Gal␤1-4Glc with 55.4% and 44.9% yields at 45°C for 4 h and at 55°C for 1.5 h, respectively.…”

“…Moreover, most ␤-N-acetylhexosaminidases with transglycosylation ability display low glycosyl transfer efficiency at 1% to 10% yield, as well as poor regioselectivity, resulting in isomer production with multiple glycosyl linkages that are difficult to isolate (33,35,36). Only two fungal ␤-N-acetylhexosaminidases can catalyze transfer of both GalNAc and GlcNAc residues for synthesis (24,25).…”

“…␤-N-Acetylhexosaminidases in the GH20 family have been successfully used to synthesize ␤-N-acetylhexosaminyl oligosaccharides, such as GlcNAc␤1-4GlcNAc␤1-4ManNAc, GalNAc␤1-4GlcNAc␣UDP, (GlcNAc␤1-4) 3 GlcNAc, and 6=-sulfodisaccharides (e.g., GlcNAc␤1-4Glc␣-OAll, GlcNAc␤1-3/1-6Gal␣-OAll, and GlcNAc␤1-4GlcNAc) (22)(23)(24)(25). However, the available enzyme sources for synthesis remain limited.…”

“…However, the available enzyme sources for synthesis remain limited. Most of the reported ␤-N-acetylhexosaminidases with transglycosylation ability are obtained from fungi, especially those belonging to the genera Aspergillus (22)(23)(24)(26)(27)(28)(29), Penicillium (28,30), and Talaromyces (31,32), and only three are obtained from the bacteria Nocardia orientalis and Serratia marcescens YS-1 (33)(34)(35). Moreover, most ␤-N-acetylhexosaminidases with transglycosylation ability display low glycosyl transfer efficiency at 1% to 10% yield, as well as poor regioselectivity, resulting in isomer production with multiple glycosyl linkages that are difficult to isolate (33,35,36).…”