A novel method for chemo-enzymatic synthesis of chitin oligosaccharide catalyzed by the mutant of inverting family GH19 chitinase using 4,6-dimethoxy-1,3,5-triazin-2-yl α-chitobioside as a glycosyl donor.

A novel method for the chemo-enzymatic synthesis of chitin oligosaccharide catalyzed by mutants of BcChi-A, an inverting family GH19 chitinase from Bryum coronatum, has been developed using 4,6-dimethoxy-1,3,5-triazin-2-yl α-chitobioside [DMT-α-(GlcNAc)2)] as a donor substrate. Based on the glycosynthase derived from BcChi-A, Glu70, which acts as a catalytic base, and Ser102, which fixes a nucleophilic water molecule, were changed to generate several single and double mutants of BcChi-A, which were employed in synthetic reactions. Among the double mutants tested, E70G/S102G, E70G/S102C and E70G/S102A were found to successfully synthesize chitotetraose [(GlcNAc)4] from DMT-α-(GlcNAc)2 and (GlcNAc)2; however, the single mutants, E70G, S102G, S102C and S102A, did not. Among the mutants, E70G/S102A showed the highest synthetic activity. This is the first report of a glycosynthase that employs a dimethoxytriazine-type glycoside as a donor substrate.

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.

A glycosynthase derived from an inverting GH19 chitinase from the moss Bryum coronatum.

BcChi-A, a GH19 chitinase from the moss Bryum coronatum, is an endo-acting enzyme that hydrolyses the glycosidic bonds of chitin, (GlcNAc)(n) [a ß-1,4-linked polysaccharide of GlcNAc (N-acetylglucosamine) with a polymerization degree of n], through an inverting mechanism. When the wild-type enzyme was incubated with α-(GlcNAc)2-F [α-(GlcNAc)(2) fluoride] in the absence or presence of (GlcNAc)(2), (GlcNAc)(2) and hydrogen fluoride were found to be produced through the Hehre resynthesis-hydrolysis mechanism. To convert BcChi-A into a glycosynthase, we employed the strategy reported by Honda et al. [(2006) J. Biol. Chem. 281, 1426-1431; (2008) Glycobiology 18, 325-330] of mutating Ser(102), which holds a nucleophilic water molecule, and Glu(70), which acts as a catalytic base, producing S102A, S102C, S102D, S102G, S102H, S102T, E70G and E70Q. In all of the mutated enzymes, except S102T, hydrolytic activity towards (GlcNAc)(6) was not detected under the conditions we used. Among the inactive BcChi-A mutants, S102A, S102C, S102G and E70G were found to successfully synthesize (GlcNAc)(4) as a major product from α-(GlcNAc)(2)-F in the presence of (GlcNAc)(2). The S102A mutant showed the greatest glycosynthase activity owing to its enhanced F(-) releasing activity and its suppressed hydrolytic activity. This is the first report on a glycosynthase that employs amino sugar fluoride as a donor substrate.