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-β-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 1H-15N 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. Database The atomic coordinates and structural factors have been deposited in the Protein Data Bank, under the accession codes 4DWX (unliganded form) and 4DYG ((GlcNAc)4 complex). Chitinase, EC 184.108.40.206. Backbone assignment data were deposited in the Biological Magnetic Resonance Data Bank (http://www.bmrb.wisc.edu/bmrb/) with the code number 11467 Structured digital abstract RSC-c and RSC-c bind by x-ray crystallography (View interaction) Crystal structure of a 'loopful' GH19 chitinase from rye seeds in a complex with an N-acetylglucosamine tetramer, (GlcNAc)4, was solved, and revealed the binding mode of the tetramer to subsites +1, +2, +3, and +4. HPLC analysis of the reaction products and (GlcNAc)4 titration experiments using NMR spectroscopy confirmed the binding mode obtained by the crystallographic analysis.
!!!All Science Journal Classification (ASJC) codes