title: A glycosynthase derived from an inverting chitinase with an extended binding cleft.
abstract: 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.
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.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204, Nakamachi, Nara 631-8505, Japan;
authors.forename: Satoshi
authors.initials: S
authors.lastname: Dozen
authors.affiliation: National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12, Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Honda
authors.affiliation: National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12, Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
authors.forename: Motomitsu
authors.initials: M
authors.lastname: Kitaoka
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204, Nakamachi, Nara 631-8505, Japan; fukamizo@nara.kindai.ac.jp.
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D002688:Chitinases / Q000737:chemistry*; D008958:Models, Molecular; D043367:Ralstonia / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D002688:Chitinases
keywords: chitin oligosaccharide; chitooligosyl fluoride; family GH19 chitinase; glycosynthase; site-directed mutagenesis