Mutants of Mucor hiemalis endo-β-N-acetylglucosaminidase show enhanced transglycosylation and glycosynthase-like activities

被引:185
作者
Umekawa, Midori [3 ]
Huang, Wei [1 ,2 ]
Li, Bing [1 ,2 ]
Fujita, Kiyotaka [4 ]
Ashida, Hisashi [3 ]
Wang, Lai-Xi [1 ,2 ]
Yamamoto, Kenji [3 ]
机构
[1] Univ Maryland, Sch Med, Inst Human Virol, Baltimore, MD 21201 USA
[2] Univ Maryland, Sch Med, Dept Biochem & Mol Biol, Baltimore, MD 21201 USA
[3] Kyoto Univ, Div Integrated Life Sci, Grad Sch Biostudies, Sakyo Ku, Kyoto 6068502, Japan
[4] Kagoshima Univ, Dept Biochem Sci & Technol, Fac Agr, Kagoshima 8900065, Japan
关键词
D O I
10.1074/jbc.M707137200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Endo-beta-N-acetylglucosaminidase from Mucor hiemalis(Endo-M), a family 85 glycoside hydrolase, acts on the beta 1,4 linkage of N,N'-diacetylchitobiose moiety in the N-linked glycans of glycoproteins and catalyzes not only the hydrolysis reaction but also the transglycosylation reaction that transfers the releasing sugar chain to an acceptor other than water to form a new glycosidic linkage. The transglycosylation activity of Endo-M holds a great promise for the chemo-enzymatic synthesis and glyco-engineering of glycoproteins, but the inherent hydrolytic activity for product hydrolysis and low transglycosylation have hampered its broad applications. This paper describes the site-directed mutagenesis on residues in the putative catalytic region of Endo-M to generate mutants with superior transglycosylation activity. Two interesting mutants were discovered. The Y217F mutant was found to possess much enhanced transglycosylation activity and yet much diminished hydrolytic activity in comparison with the wild-type Endo-M. Kinetic analyses revealed that the K-m value of Y217F for an acceptor substrate 4-methylumbel-liferyl-beta-D-N-acetylglucosaminide was only one-tenth of that of the wild-type, implicating a much higher affinity of Y217F for the acceptor substrate than the wild-type. The other mutant, N175A, acts like a glycosynthase. It was found that mutation at Asn175"knocked out" the hydrolytic activity, but the mutant was able to take the highly active sugar oxazolines ( the transition state mimics) as donor substrates for transglycosylation. This is the first glycosynthase derived from endo-beta-N-acetylglucosaminidases that proceed via a substrate-assisted mechanism. Our findings provide further insights on the substrate-assisted mechanism of GH85. The usefulness of the novel glycosynthase was exemplified by the efficient synthesis of a human immunodeficiency deficiency virus, type 1 (HIV-1) glycopeptide with potent anti-HIV activity.
引用
收藏
页码:4469 / 4479
页数:11
相关论文
共 38 条
[11]   A novel fluorescence-based assay for the transglycosylation activity of endo-β-N-acetylglucosaminidases [J].
Hauser, S ;
Song, HJ ;
Li, HG ;
Wang, LX .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2005, 328 (02) :580-585
[12]   Identification of an endo-β-N-acetylglucosaminidase gene in Caenorhabditis elegans and its expression in Escherichia coli [J].
Kato, T ;
Fujita, K ;
Takeuchi, M ;
Kobayashi, K ;
Natsuka, S ;
Ikura, K ;
Kumagai, H ;
Yamamoto, Y .
GLYCOBIOLOGY, 2002, 12 (10) :581-587
[13]  
Li B, 2005, J AM CHEM SOC, V127, P9692, DOI 10.1021/ja051715a
[14]   A highly efficient chemoenzymatic approach toward glycoprotein synthesis [J].
Li, Bing ;
Song, Haijing ;
Hauser, Steven ;
Wang, Lai-Xi .
ORGANIC LETTERS, 2006, 8 (14) :3081-3084
[15]   Chemoenzymatic synthesis of HIV-1V3 glycopeptides carrying two N-glycans and effects of glycosylation on the peptide domain [J].
Li, HG ;
Li, B ;
Song, HJ ;
Breydo, L ;
Baskakov, IV ;
Wang, LX .
JOURNAL OF ORGANIC CHEMISTRY, 2005, 70 (24) :9990-9996
[16]   Chemoenzymatic synthesis of CD52 glycoproteins carrying native N-glycans [J].
Li, HG ;
Singh, S ;
Zeng, Y ;
Song, HJ ;
Wang, LX .
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 2005, 15 (04) :895-898
[17]   Synthesis of a glycopeptide containing oligosaccharides: Chemoenzymatic synthesis of eel calcitonin analogues having natural N-linked oligosaccharides [J].
Mizuno, M ;
Haneda, K ;
Iguchi, R ;
Muramoto, I ;
Kawakami, T ;
Aimoto, S ;
Yamamoto, K ;
Inazu, T .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1999, 121 (02) :284-290
[18]   Oligosaccharide synthesis by glycosynthases [J].
Perugino, G ;
Trincone, A ;
Rossi, M ;
Moracci, M .
TRENDS IN BIOTECHNOLOGY, 2004, 22 (01) :31-37
[19]   CRYSTAL-STRUCTURE OF ENDO-BETA-N-ACETYLGLUCOSAMINIDASE-H AT 1.9 ANGSTROM RESOLUTION - ACTIVE-SITE GEOMETRY AND SUBSTRATE RECOGNITION [J].
RAO, VH ;
GUAN, CD ;
VANROEY, P .
STRUCTURE, 1995, 3 (05) :449-457
[20]   Synthesis of N-glycan oxazolines: donors for endohexosaminidase catalysed glycosylation [J].
Rising, Thomas W. D. F. ;
Claridge, Timothy D. W. ;
Davies, Nicola ;
Gamblin, David P. ;
Moir, James W. B. ;
Fairbanks, Antony J. .
CARBOHYDRATE RESEARCH, 2006, 341 (10) :1574-1596