title: Introduction of a tryptophan side chain into subsite +1 enhances transglycosylation activity of a GH-18 chitinase from Arabidopsis thaliana, AtChiC.
abstract: A tryptophan side chain was introduced into subsite +1 of family GH-18 (class V) chitinases from Nicotiana tabacum and Arabidopsis thaliana (NtChiV and AtChiC, respectively) by the mutation of a glycine residue to tryptophan (G74W-NtChiV and G75W-AtChiC). The specific activity toward glycol chitin of the two mutant enzymes was 70-71% of that of the wild type. Using chitin oligosaccharides, (GlcNAc)(n) (n = 4, 5 and 6), as the substrates, we found the transglycosylation reaction to be significantly enhanced in G74W-NtChiV and G75W-AtChiC when compared with the corresponding wild-type enzymes. The introduced tryptophan side chain might protect the oxazolinium ion intermediate from attack by a nucleophilic water molecule. The enhancement of transglycosylation activity was much more distinct in G75W-AtChiC than in G74W-NtChiV. Nuclear magnetic resonance titration experiments using the inactive double mutants, E115Q/G74W-NtChiV and E116Q/G75W-AtChiC revealed that the association constant of (GlcNAc)(5) was considerably larger for the latter. Amino acid substitutions at the acceptor binding site might have resulted in the larger association constant for G75W-AtChiC, giving rise to the higher transglycosylation activity of G75W-AtChiC.
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.forename: Takayuki
authors.initials: T
authors.lastname: Ohnuma
authors.forename: Mamiko
authors.initials: M
authors.lastname: Mizuhara
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Sato
authors.forename: Karen
authors.initials: K
authors.lastname: Skriver
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005998:Glycine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D002686:Chitin; D014364:Tryptophan; D002688:Chitinases; D005998:Glycine