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Publication Abstract from PubMed

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 an aglycon binding site, subsites +1, +2, +3, and +4. This is the first crystallographic data showing the oligosaccharide-binding mode of a family GH-19 chitinase. From an 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. The 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, the 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 the 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 well consistent with that from the crystal structure. (c) 2012 The Authors Journal compilation (c) 2012 FEBS.

Crystal structure and chitin oligosaccharide-binding mode of a "loopful" family GH19 chitinase from rye, Secale cereal, seeds.,Ohnuma T, Numata T, Osawa T, Inanaga H, Okazaki Y, Shinya S, Kondo K, Fukuda T, Fukamizo T FEBS J. 2012 Jul 25. doi: 10.1111/j.1742-4658.2012.08723.x. PMID:22831795^[4]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.