Chitin, a β-1,4-linked polysaccharide of N
-acetylglucosamine (NAG), is hydrolyzed by chitinases (EC 220.127.116.11), which are widely distributed in living organisms and are responsible for self-defence, growth, morphogenesis, cuticle destabilization and stress tolerance (Kasprzewska, 2003
; Duo-Chuan, 2006
; Bhattacharya et al.
; Arakane & Muthukrishnan, 2010
; Donnelly & Barnes, 2004
). Several plant chitinase genes have been isolated and sequenced and the chitinases have been divided into at least five classes (classes I, II, III, IV and V) based on their deduced amino-acid sequences (Collinge et al.
; Melchers et al.
). Classes I, II and IV correspond to the GH-19 family and classes III and V to the GH-18 family, according to the CAZy database (Henrissat & Davies, 1997
The first crystal structure of these enzymes to be reported was that of the class II chitinase from barley seeds (Hart et al.
) and was followed by that of the class III chitinase from Hevea brasiliensis
(Terwisscha van Scheltinga et al.
). The class II enzyme is composed of two lobes, which are both rich in α-helical structures, with the substrate-binding cleft between the lobes. In contrast, the class III enzyme exhibits a typical (α/β)8
-barrel fold and the substrate binds to the top of the barrel. Although the class III and class V chitinases belong to the GH-18 family, the molecular masses of the class V chitinases (about 40 kDa) are higher than those of the class III chitinases (about 30 kDa) and their amino-acid sequence similarity is low. In addition, the physiological role of the class V chitinases appears to differ from that of the class III chitinases (Melchers et al.
; Takenaka et al.
). Subsequent to these structural reports, the crystal structures of family GH-19 chitinases (classes I, II and IV) from several plants, including jack bean, mustard greens, papaya, Norway spruce and rice, have been reported (Hahn et al.
; Ubhayasekera et al.
; Huet et al.
; Kezuka et al.
). As expected from their amino-acid sequences, the three-dimensional structures of their catalytic domains resemble that of the barley class II chitinase. The structure of class II chitinase complexed with NAG monomers has also been reported and the two NAGs were found to separately bind to subsites −2 and +1, respectively, in the complex structure (Huet et al.
). However, no crystal structure of a class V chitinase has been reported to date.
A class V chitinase (NtChiV) was first isolated by Melchers et al.
) from tobacco leaves inoculated with tobacco mosaic virus. Expression of the chitinase gene (NtChiV
) is up-regulated by ultraviolet irradiation and wounding in addition to viral attack. The recombinant NtChiV protein exhibited antifungal activity towards Trichoderma viride
. The enzyme appears to be responsible for tolerance not only to biotic stress but also to abiotic stress. Interestingly, the amino-acid sequence of NtChiV is highly homologous to that of a bacterial chitinase, Serratia marcescens
chitinase B (Brurberg, 1995
). Since the Serratia
enzyme has been intensively studied with respect to its structure and function (van Aalten et al.
; Synstad et al.
), the crystal structure of NtChiV would provide important information on the function of a plant class V chitinase by comparison with the Serratia
In this study, we produced recombinant NtChiV protein using an Escherichia coli expression system and purified it to homogeneity. The purified NtChiV was employed for crystallization and X-ray diffraction experiments.