A glycosynthase derived from an inverting chitinase with an extended binding cleft

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.

Source: Journal of Biochemistry - July 27, 2016 Category: Biochemistry Authors: Ohnuma, T., Dozen, S., Honda, Y., Kitaoka, M., Fukamizo, T. Tags: Regular Papers Source Type: research