title: Modulation of the transglycosylation activity of plant family GH18 chitinase by removing or introducing a tryptophan side chain.
abstract: Transglycosylation (TG) activity of a family GH18 chitinase from the cycad, Cycas revoluta, (CrChiA) was modulated by removing or introducing a tryptophan side chain. The removal from subsite +3 through mutation of Trp168 to alanine suppressed TG activity, while introduction into subsite +1 through mutation of Gly77 to tryptophan (CrChiA-G77W) enhanced TG activity. The crystal structures of an inactive double mutant of CrChiA (CrChiA-G77W/E119Q) with one or two N-acetylglucosamine residues occupying subsites +1 or +1/+2, respectively, revealed that the Trp77 side chain was oriented toward +1 GlcNAc to be stacked with it face-to-face, but rotated away from subsite +1 in the absence of GlcNAc at the subsite. Aromatic residues in the aglycon-binding site are key determinants of TG activity of GH18 chitinases.
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Umemoto
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: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 Japan.
authors.forename: Takuo
authors.initials: T
authors.lastname: Osawa
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Numata
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan. Electronic address: fukamizo@nara.kindai.ac.jp.
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
mesh_terms: D020134:Catalytic Domain; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D029784:Cycas / Q000201:enzymology; D006031:Glycosylation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D014364:Tryptophan
chemical_list: D002686:Chitin; D014364:Tryptophan; D002688:Chitinases
keywords: Chitin oligosaccharide; Chitinase; Crystal structure; Cycas revoluta; Transglycosylation; Tryptophan residue