The first crystal structures of a family 19 class IV chitinase: the enzyme from Norway spruce

Ubhayasekera, Wimal; Rawat, Reetika; Ho, Sharon Wing Tak; Wiweger, Małgorzata; Arnold, Sara von; Chye, Mee‐Len; Mowbray, Sherry L.

doi:10.1007/s11103-009-9523-9

Cited by 53 publications

(56 citation statements)

References 53 publications

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“…CHI4 has been demonstrated to have antifungal effects (Ubhayasekera et al, 2009) and has been indicated to mediate programmed cell death (PCD) during embryogenesis in Norway spruce (Wiweger et al, 2003). Mediation of PCD by CHI4 presumably occurs through action on endogenous arabinogalactan proteins or lipo-chitooligosaccharides (Wiweger et al, 2003), conceptually releasing oligosaccharides with signaling properties (Fossdal et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Using laser micro-dissection and qRT-PCR to analyze cell type-specific gene expression in Norway spruce phloem

Nagy¹,

Sikora

Krokene³

et al. 2014

PeerJ

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The tangentially oriented polyphenolic parenchyma (PP) and radially organized ray parenchyma in the phloem are central in the defense of conifer stems against insects and pathogens. Laser micro-dissection enables examination of cell-specific defense responses. To examine induced defense responses in Norway spruce stems inoculated with the necrotrophic blue-stain fungus Ceratocystis polonica, RNA extracted from laser micro-dissected phloem parenchyma and vascular cambium was analyzed using real-time RT-PCR (qRT-PCR) to profile transcript levels of selected resistance marker genes. The monitored transcripts included three pathogenesis-related proteins (class IV chitinase (CHI4), defensin (SPI1), peroxidase (PX3), two terpene synthesis related proteins (DXPS and LAS), one ethylene biosynthesis related protein (ACS), and a phenylalanine ammonia-lyase (PAL). Three days following inoculation, four genes (CHI4, PAL, PX3, SPI1) were differentially induced in individual cell and tissue types, both close to the inoculation site (5 mm above) and, to a lesser degree, further away (10 mm above). These resistance marker genes were all highly induced in ray parenchyma, supporting the important role of the rays in spruce defense propagation. CHI4 and PAL were also induced in PP cells and in conducting secondary phloem tissues. Our data suggests that different cell types in the secondary phloem of Norway spruce have overlapping but not fully redundant roles in active host defense. Furthermore, the study demonstrates the usefulness of laser micro-dissection coupled with qRT-PCR to characterize gene expression in different cell types of conifer bark.

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Section: Discussionmentioning

confidence: 99%

Using laser micro-dissection and qRT-PCR to analyze cell type-specific gene expression in Norway spruce phloem

Nagy¹,

Sikora

Krokene³

et al. 2014

PeerJ

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“…By contrast, the two lobes in the RSC-c•(GlcNAc) 4 complex are opened (green). Such opened conformations are frequently observed in unliganded forms of the GH19 chitinases [18]. Based on these conformational differences, Ubhayasekera et al [18] suggested that the binding of the substrate to the enzyme brings the two lobes close to each other in order to place the catalytic residues in optimal conformations for catalysis, forming a competent structure for the hydrolysis reaction.…”

Section: Activity and Mode Of Action Of W72a Rsc-cmentioning

confidence: 99%

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

et al. 2012

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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.

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“…On the other hand, a GH19 chitinase lacking the loops except Loop III (''loopless'' chitinases) was isolated from moss, Bryum coronatum [11]. Based on the amino acid sequence alignment, the structure of the moss enzyme is closely related to those of GH19 chitinases from Streptomyces griseus HUT6037 [12], Streptomyces coelicolor A3(2) [13], and Norway spruce [14]. Thus, the structural organization of the GH19 enzymes was intensively studied, and the structural data have been accumulated.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2013

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Edited by Richard Cogdell Keywords:Crystal structure GH19 chitinase Rye seed Chitin oligosaccharide Domain motion a b s t r a c t Crystallographic analysis of a mutated form of ''loopful'' GH19 chitinase from rye seeds a double mutant RSC-c, in which Glu67 and Trp72 are mutated to glutamine and alanine, respectively, (RSC-c-E67Q/W72A) in complex with chitin tetrasaccharide (GlcNAc) 4 revealed that the entire substrate-binding cleft was completely occupied with the sugar residues of two (GlcNAc) 4 molecules. One (GlcNAc) 4 molecule bound to subsites À4 to À1, while the other bound to subsites +1 to +4. Comparisons of the main chain conformation between liganded RSC-c-E67Q/W72A and unliganded wild type RSC-c suggested domain motion essential for catalysis. This is the first report on the complete subsite mapping of GH19 chitinase.

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The first crystal structures of a family 19 class IV chitinase: the enzyme from Norway spruce

Cited by 53 publications

References 53 publications

Using laser micro-dissection and qRT-PCR to analyze cell type-specific gene expression in Norway spruce phloem

Using laser micro-dissection and qRT-PCR to analyze cell type-specific gene expression in Norway spruce phloem

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

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

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