title: Transfer of Man9GlcNAc to L-fucose by endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae.
abstract: We have reported that transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter 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, to L-Fuc using Man9GlcNAc2Asn as donor substrate in a medium containing 35% acetone. The transglycosylation yield was greater than 25% when 0.2 M L-Fuc was used as acceptor. The transglycosylation produce was purified by high performance liquid chromatography on a graphitized carbon column and the presence of L-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 beta-anomeric configuration 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 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 Man9GlcNAc beta (1,2)-L-Fuc. Methyl-beta-L-fucopyranoside, L-Gal are also acceptors for the enzymic transglycosylation. However, transglycosylation 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 4C1-conformation or equivalent conformation of the acceptor to perform transglycosylation.
authors.affiliation: Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: J Q
authors.initials: JQ
authors.lastname: Fan
authors.forename: L H
authors.initials: LH
authors.lastname: Huynh
authors.forename: B B
authors.initials: BB
authors.lastname: Reinhold
authors.forename: V N
authors.initials: VN
authors.lastname: Reinhold
authors.forename: K
authors.initials: K
authors.lastname: Takegawa
authors.forename: S
authors.initials: S
authors.lastname: Iwahara
authors.forename: A
authors.initials: A
authors.lastname: Kondo
authors.forename: I
authors.initials: I
authors.lastname: Kato
authors.forename: Y C
authors.initials: YC
authors.lastname: Lee
mesh_terms: D000118:Acetylglucosaminidase / Q000737:chemistry* / Q000378:metabolism; D001173:Arthrobacter / Q000201:enzymology*; D005643:Fucose / Q000737:chemistry*; D006031:Glycosylation; D008351:Mannans / Q000737:chemistry*; D013058:Mass Spectrometry; D013379:Substrate Specificity
chemical_list: D008351:Mannans; D005643:Fucose; C057328:mannosyl(9)-N-acetylglucosamine; D000118:Acetylglucosaminidase