TRANSFER OF MAN(9)GLCNAC TO L-FUCOSE BY ENDO-BETA-N-ACETYLGLUCOSAMINIDASE FROM ARTHROBACTER-PROTOPHORMIAE

We have reported that transglycosylation activity of endo-beta-N-acety lglucosaminidase from Arthrobacter protophormiae (endo-A) can be enhan ced to near completion using GlcNAc as an acceptor in a medium contain ing 30% acetone (Fan J-Q, Takegawa K, Iwahara S, Kondo A, Kato I, Abey gunawardana C, Lee YC (1995) J Biol Chem 270: 17723-29). In this paper , we found that the endo-A can also transfer an oligosaccharide, Man(9 )GlcNAc, to L-Fuc using Man(9)GlcNAc(2)Asn as donor substrate in a med ium containing 35% acetone. The transglycosylation yield was greater t han 25% when 0.2 M L-Fuc was used as acceptor. The transglycosylation product was purified by high performance liquid chromatography on a gr aphitized carbon column and the presence of L-Fuc was confirmed by sug ar composition analysis and electrospray mass spectrometry. Sequential exo-glycosidase digestion of pyridyl-2-aminated transglycosylation pr oduct, Man(9)GlcNAc-L-Fuc-PA, revealed that a beta-anomeric configurat ion linkage was formed between GlcNAc and L-Fuc. The GlcNAc was found to be 1,2-linked to L-Fuc by two methods: i) collision-induced decompo sition on electrospray mass spectrometry after periodate oxidation, re duction and permethylation of Man(9)GlcNAc-L-Fuc; and ii) preparation of Man(9)GlcNAc-L-Fuc-PA, its periodate oxidation and reduction, follo wed by hydrolysis and HPLC analysis. Thus, the structure of the oligos accharide synthesized by endo-A transglycosylation was determined to b e Man(9)GlcNAc beta(1,2)-L-Fuc. Methyl beta-L-fucopyranoside, L-Gal ar e also accepters for the enzymic transglycosylation. However, transgly cosylation failed when methyl alpha-L-fucopyranoside, D-Fuc and D-Gal were used. These results indicate that the endo-A requires not only 3- OH and 4-OH to be equatorial but also a C-4(1)-conformation or equival ent conformation of the acceptor to perform transglycosylation.