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The E358S mutant of Agrobacterium sp. β‐glucosidase is a greatly improved glycosynthase
Author(s) -
Mayer Christoph,
Zechel David L.,
Reid Stephen P.,
Warren R.Antony J.,
Withers Stephen G.
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)01751-2
Subject(s) - chemistry , anomer , glycosylation , nucleophile , stereochemistry , glycosyl , mutant , glycosyl donor , glycoside hydrolase , hydrolysis , biochemistry , catalysis , gene
Glycosynthases are nucleophile mutants of retaining glycosidases that catalyze the glycosylation of sugar acceptors using glycosyl fluoride donors, thereby synthesizing oligosaccharides. The ‘original’ glycosynthase, derived from Agrobacterium sp. β‐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 acceptors for Abg E358A, such as PNP‐GlcNAc, are successfully glycosylated by E358S, allowing the synthesis of PNP‐β‐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 α‐glycosyl fluoride.