The E358S mutant of Agrobacterium sp β-glucosidase is a greatly improved glycosynthase

被引:112
作者
Mayer, C
Zechel, DL
Reid, SP
Warren, RAJ
Withers, SG [1 ]
机构
[1] Univ British Columbia, Dept Chem, Prot Engn Network Ctr Excellence, Vancouver, BC V6T 1Z1, Canada
[2] Univ British Columbia, Dept Microbiol, Prot Engn Network Ctr Excellence, Vancouver, BC V6T 1Z1, Canada
关键词
enzymatic oligosaccharide synthesis; glycosyl transfer;
D O I
10.1016/S0014-5793(99)01751-2
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
Glycosynthases are nucleophile mutants of retaining glycosidases that catalyze the glycosylation of sugar accepters using glycosyl fluoride donors, thereby synthesizing oligosaccharides, The 'original' glycosynthase, derived from Agrobacterium sp, beta-glucosidase (Abg) by mutating the nucleophile glutamate to alanine (E358A), synthesizes oligosaccharides in yields exceeding 90% [Mackenzie, L.F., Wang, Q,, Warren, R.A.J. and Withers, S.G. (1998) J. Am. Chem. Soc. 120, 5583-5584], This mutant has now been re-cloned with a His(6)-tag into a pET-29b(+) vector, allowing gram scale production and single step chromatographic purification. A dramatic, 24-fold, improvement in synthetic rates has also been achieved by substituting the nucleophile with serine, resulting in improved product yields, reduced reaction times and an enhanced synthetic repertoire. Thus poor accepters for Abg E358A, such as PNP-GlcNAc, are successfully glycosylated by E358S, allowing the synthesis of PNP-beta-LacNAc. The increased glycosylation activity of Abg E358S likely originates from a stabilizing interaction between the Ser hydroxyl group and the departing anomeric fluorine of the alpha-glycosyl fluoride. (C) 2000 Federation of European Biochemical Societies.
引用
收藏
页码:40 / 44
页数:5
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