Transfer of Man₉GlcNAc tol-fucose by endo-β-N-acetylglucosaminidase fromArthrobacter protophormiae

Abstract

We have reported that transglycosylation activity of endo-β-N-acetylglucosaminidase fromArthrobacter protophormiae (endo-A) can be enhanced to near completion using GlcNAc as an acceptor in a medium containing 30% acetone (Fan J-Q, Takegawa K, Iwahara S, Kondo A, Kato I, Abeygunawardana C, Lee YC (1995)J Biol Chem 270: 17723–29). In this paper, we found that the endo-A can also transfer an oligosaccharide, Man₉GlcNAc, tol-Fuc using Man₉GlcNAc₂Asn as donor substrate in a medium containing 35% acetone. The transglycosylation yield was greater than 25% when 0.2m l-Fuc was used as acceptor. The transglycosylation product was purified by high performance liquid chromatography on a graphitized carbon column and the presence ofl-Fuc was confirmed by sugar composition analysis and electrospray mass spectrometry. Sequential exo-glycosidase digestion of pyridyl-2-aminated transglycosylation product, Man₉GlcNAc-l-Fuc-PA, revealed that a β-anomeric configuration linkage was formed between GlcNAc andl-Fuc. The GlcNAc was found to be 1,2-linked tol-Fuc by two methods; i) collision-induced decomposition on electrospray mass spectrometry after periodate oxidation, reduction and permethylation of Man₉GlcNAc-l-Fuc; and ii) preparation of Man₉GlcNAc-l-Fuc-PA, its periodate oxidation and reduction, followed by hydrolysis and HPLC analysis. Thus, the structure of the oligosaccharide synthesized by endo-A transglycosylation was determined to be Man₉GlcNAcβ(1,2)-l-Fuc. Methyl β-l-fucopyranoside,l-Gal are also acceptors for the enzymic transglycosylation. However, transglycosylation failed when methyl α-l-fucopyranoside,d-Fuc andd-Gal were used. These results indicate that the endo-A requires not only 3-OH and 4-OH to be equatorial but also a⁴C₁-conformation or equivalent conformation of the acceptor to perform transglycosylation.