Introduction of a tryptophan side chain into subsite +1 enhances transglycosylation activity of a GH-18 chitinase from Arabidopsis thaliana, AtChiC
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Introduction of a tryptophan side chain into subsite +1 enhances transglycosylation activity of a GH-18 chitinase from Arabidopsis thaliana, AtChiC. / Umemoto, Naoyuki; Ohnuma, Takayuki; Mizuhara, Mamiko; Sato, Hirokazu; Skriver, Karen; Fukamizo, Tamo.
I: Glycobiology, Bind 23, Nr. 1, 2013, s. 81-90.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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T1 - Introduction of a tryptophan side chain into subsite +1 enhances transglycosylation activity of a GH-18 chitinase from Arabidopsis thaliana, AtChiC
AU - Umemoto, Naoyuki
AU - Ohnuma, Takayuki
AU - Mizuhara, Mamiko
AU - Sato, Hirokazu
AU - Skriver, Karen
AU - Fukamizo, Tamo
PY - 2013
Y1 - 2013
N2 - 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.
AB - 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.
U2 - 10.1093/glycob/cws125
DO - 10.1093/glycob/cws125
M3 - Journal article
C2 - 22936594
VL - 23
SP - 81
EP - 90
JO - Glycobiology
JF - Glycobiology
SN - 0959-6658
IS - 1
ER -
ID: 44753447