期刊
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
卷 1844, 期 4, 页码 793-802出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbapap.2014.02.013
关键词
Crystal structure; GH19 chitinase; Chitin oligosaccharide; NMR spectroscopy; Catalytic mechanism
资金
- MEXT (Ministry of Education, Culture, Sports, Science and Technology) [S1101035]
- Japan Society for the Promotion of Science [25-3639]
- Grants-in-Aid for Scientific Research [13J03639] Funding Source: KAKEN
Descriptions: The structure of a GH19 chitinase from the moss Bryum coronatum (BcChi-A) in complex with the substrate was examined by X-ray crystallography and NMR spectroscopy in solution. The X-ray crystal structure of the inactive mutant of BcChi-A (BcChi-A-E61A) liganded with chitin tetramer (GlcNAc)(4) revealed a clear electron density of the tetramer bound to subsites -2, -1, +1, and +2. Individual sugar residues were recognized by several amino acids at these subsites through a number of hydrogen bonds. This is the first crystal structure of GH19 chitinase liganded with oligosaccharide spanning the catalytic center. NMR titration experiments of chitin oligosaccharides into the BcChi-A-E61A solution showed that the binding mode observed in the crystal structure is similar to that in solution. The C-1 carbon of -1 GlcNAc, the O epsilon 1 atom of the catalytic base (Glu70), and the O gamma atom of Ser102 form a triangle surrounding the catalytic water, and the arrangement structurally validated the proposed catalytic mechanism of GH19 chitinases. The glycosidic linkage between -1 and +1 sugars was found to be twisted and under strain. This situation may contribute to the reduction of activation energy for hydrolysis. The complex structure revealed a more refined mechanism of the chitinase catalysis. (C) 2014 Elsevier B.V. All rights reserved.
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