Publication Date:
2016-07-30
Description:
We created a glycosynthase from a GH19 chitinase from rye seeds (RSC-c), that has a long-extended binding cleft consisting of eight subsites; -4, -3, -2, -1, +1, +2, +3 and +4. When wild-type RSC-c was incubated with α-(GlcNAc) 3 -F [α-(GlcNAc) 3 fluoride], (GlcNAc) 3 and hydrogen fluoride were produced through the Hehre resynthesis–hydrolysis mechanism. Glu89, which acts as a catalytic base, and Ser120, which fixes a nucleophilic water molecule, were mutated to produce two single mutants, E89G and S120A, and a double mutant, E89G/S120A. E89G only produced a small amount of (GlcNAc) 7 from α-(GlcNAc) 3 -F in the presence of (GlcNAc) 4 . S120A, with the highest F – -releasing activity, produced a larger amount of (GlcNAc) 7 , a fraction of which was decomposed by its own residual hydrolytic activity. However, the double mutant E89G/S120A, of which the hydrolytic activity was completely abolished while its F – -releasing activity was only moderately affected, produced the largest amount of (GlcNAc) 7 from α-(GlcNAc) 3 -F and (GlcNAc) 4 without decomposition. We concluded that E89G/S120A was an efficient glycosynthase, that enabled the addition of a three-sugar unit.
Print ISSN:
0021-924X
Electronic ISSN:
1756-2651
Topics:
Biology
,
Chemistry and Pharmacology
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