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 β-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 Ser¹⁰², which holds a nucleophilic water molecule, and Glu⁷⁰, 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.