Crystal structure and chitin oligosaccharide‐binding mode of a ‘loopful’ family GH19 chitinase from rye, Secale cereale, seeds

Abstract

The substrate-binding mode of a 26-kDa GH19 chitinase from rye, Secale cereale, seeds (RSC-c) was investigated by crystallography, sitedirected 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-bchitopentaoside. 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. DatabaseThe 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 3.2.1.14. Backbone assignment data were deposited in the Biological Magnetic Resonance Data Bank (http://www.bmrb.wisc.edu/bmrb) with the code number 11467Structured digital abstract l RSC-c and RSC-c bind by x-ray crystallography (View interaction) Abbreviations (GlcNAc) n , b-1,4-linked oligosaccharides of GlcNAc with a polymerization degree of n; GlcNAc, N-acetylglucosamine; pNP, p-nitrophenyl; RSC-c, a 'loopful' family GH19 chitinase from rye seeds; W72A RSC-c, RSC-c in which Trp 72 is mutated to alanine.

Complete subsite mapping of a “loopful” GH19 chitinase from rye seeds based on its crystal structure

“…The mode of (GlcNAc) 4 binding to subsites +1 to +4 was partly similar to that observed in the crystal structure reported for RSC-c liganded with (GlcNAc) 4 [18]; however, some interactions were rearranged in RSC-c-E67Q/W72A liganded with 2 Â (GlcNAc) 4 , as shown in Fig. 3.…”

“…We compared the main chain conformation of RSC-c-E67Q/ W72A liganded with 2 Â (GlcNAc) 4 to those of unliganded RSC-c (PDB code: 4DWX) and RSC-c liganded with (GlcNAc) 4 (PDB code: 4DYG), in which only one (GlcNAc) 4 molecule bound to subsites +1 to +4 [18]. As shown in Fig.…”

Comparative study of two GH19 chitinase‐like proteins from Hevea brasiliensis, one exhibiting a novel carbohydrate‐binding domain

“…Structural alignment of loop III, including 20 residues in each end, demonstrated that even though CatD1 was crystallized in the absence of ligands the loop III is in a closer conformation to the one present in the rye structure with bound ligands in the groove [40] (Fig. 5D) (rmsd 0.69 A), while the rmsd is 1.0 A when compared with the structure of the rye chitinase without ligand [39]. Loops IV and V showed similar displacements (Fig.…”

“…Recently, the crystal structure of a class IV chitinase from corn (Zea mays) that lacks the CBD provided evidence of the participation of residues Gln62, Arg177 and Glu165 in chitin hydrolysis and of Ser103 and Tyr106 in substrate binding; however, class IV chitinases have evolved to accommodate shorter substrates [42]. Compared with the chitinases of classes I, II and IV [39,[41][42][43], our CatDs exhibit all of the residues that have been implicated in catalysis, thereby allowing our mutants HbCLP1-A117E and HbCLP2-A147E to recover their chitinolytic activity.…”

“…5D). The closest structural models are from barley class II chitinase (PDB ID 1CNS) with an rmsd of 0.9 A, rice class I chitinase (PDB ID 2DKV, rmsd 1.0 A), mustard (Brassica juncea) class I chitinase (PDB ID 2Z38, rmsd 1.0 A), papaya class II chitinase (PDB ID 3CQL, rmsd 1.0 A) and rye class II chitinase (PDB ID 4DWX, rmsd 1.2 A; PDB ID 4DYG, rmsd 1.2 A; and PDB ID 4J0L, rmsd 0.9 A), which was crystallized alone and in complex with one and two molecules of N-acetyltetraose (GlcNAc) 4 , respectively [39,40].…”