title: A glycosynthase derived from an inverting GH19 chitinase from the moss Bryum coronatum.
abstract: 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 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.
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ohnuma
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Fukuda
authors.forename: Satoshi
authors.initials: S
authors.lastname: Dozen
authors.forename: Yuji
authors.initials: Y
authors.lastname: Honda
authors.forename: Motomitsu
authors.initials: M
authors.lastname: Kitaoka
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
mesh_terms: D044002:Bryophyta / Q000201:enzymology* / Q000235:genetics; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D009154:Mutation / Q000502:physiology; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary
chemical_list: D010940:Plant Proteins; D002688:Chitinases