4DYG: Crystal Structure Of A Family Gh-19 Chitinase From Rye Seeds In Complex With (glcnac)4

Citation:
Abstract
The substrate-binding mode of a 26-kDa GH19 chitinase from rye, Secale cereale, seeds (RSC-c) was investigated by crystallography, site-directed mutagenesis and NMR spectroscopy. The crystal structure of RSC-c in a complex with an N-acetylglucosamine tetramer, (GlcNAc)(4) , was successfully solved, and revealed the binding mode of the tetramer to be an aglycon-binding site, subsites +1, +2, +3, and +4. These are the first crystallographic data showing the oligosaccharide-binding mode of a family GH19 chitinase. From HPLC analysis of the enzymatic reaction products, mutation of Trp72 to alanine was found to affect the product distribution obtained from the substrate, p-nitrophenyl penta-N-acetyl-beta-chitopentaoside. Mutational experiments confirmed the crystallographic finding that the Trp72 side chain interacts with the +4 moiety of the bound substrate. To further confirm the crystallographic data, binding experiments were also conducted in solution using NMR spectroscopy. Several signals in the (1) H-(15) N HSQC spectrum of the stable isotope-labeled RSC-c were affected upon addition of (GlcNAc)(4) . Signal assignments revealed that most signals responsive to the addition of (GlcNAc)(4) are derived from amino acids located at the surface of the aglycon-binding site. The binding mode deduced from NMR binding experiments in solution was consistent with that from the crystal structure.
PDB ID: 4DYGDownload
MMDB ID: 101992
PDB Deposition Date: 2012/2/29
Updated in MMDB: 2012/08
Experimental Method:
x-ray diffraction
Resolution: 1.7  Å
Source Organism:
Similar Structures:
Biological Unit for 4DYG: monomeric; determined by author and by software (PISA)
Molecular Components in 4DYG
Label Count Molecule
Protein (1 molecule)
1
Basic Endochitinase C
Molecule annotation
Chemicals (11 molecules)
1
4
2
1
3
2
4
4
* Click molecule labels to explore molecular sequence information.

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