Chitinase III in pomegranate seeds (Punica granatum Linn.): a high‐capacity calcium‐binding protein in amyloplasts

Abstract

). † These authors contributed equally to this work. SUMMARYChitinases are a class of ubiquitous proteins that are widely distributed in plants. Defense is the major natural role for chitinases, primarily against fungal pathogens. Little is known regarding their non-defensive roles in seeds. In this study, a new class III chitinase from pomegranate seeds (pomegranate seed chitinase, PSC) was isolated and purified to homogeneity. The native state of PSC is a monomer with a molecular weight of approximately 30 kDa. This chitinase naturally binds calcium ions with high capacity and low affinity, suggesting that PSC is a calcium storage protein. Consistent with this idea, its amino acid sequence (inferred from cDNA) is rich in acidic amino acid residues, especially Asp, similar to reported calcium storage proteins. The presence of calcium considerably improves the stability of the protein but has little effect on its enzymatic activity. Transmission electron microscopy analyses indicate that, similar to phytoferritin, this enzyme is widely distributed in the stroma of amyloplasts of the embryonic cells, suggesting that amyloplasts in seeds could serve as an alternative plastid for calcium storage. Indeed, the transmission electron microscopy results showed that, within the embryonic cells, calcium ions are mainly distributed in the stroma of the amyloplasts, consistent with a role for PSC in calcium storage. Thus, the plant appears to have evolved a new plastid for calcium storage in seeds. During seed germination, the content of this enzyme decreases with time, suggesting that it is involved in the germination process.

High-capacity calcium-binding chitinase III from pomegranate seeds (Punica granatumLinn.) is located in amyloplasts

“…8,16 This result is in good agreement with its isoelectric point (4.6). Furthermore, MALDI-TOF-MS and X-ray diffraction (XRD) analyses showed that these acidic amino acid residues in PSC are responsible for the binding of 10 calcium ions 14 with a similar way to reported Ca-storage proteins.…”

“…cells of pomegranate seeds. 14 On the other hand, PSC binds calcium ions with high capacity and low affinity, suggesting that PSC could be a calcium storage protein in pomegranate seeds. If this were the case, we would expect that the amyloplasts of the embryonic cells are rich in calcium ions.…”

“…10 We have purified the chitinase from the pomegranate seeds (PSC) which has been classified into the chitinase III according to the structural properties and amino acid sequences. [11][12][13][14] Differently, this enzyme is mainly distributed in the amyloplasts of the pomegranate seeds, and naturally binds calcium ions with a stoichiometry of approximately ten moles Ca 2+ per mole of protein, 14 suggesting that PSC may be the Ca-storage protein. The content of this enzyme decreases with time during seed germination.…”

Crystal structure of class III chitinase from pomegranate provides the insight into its metal storage capacity

“…3(a)), suggesting that the PSC is active class III chitinase. In fact, we demonstrated the chitin hydrolytic activity of the PSC in our previous report, 11) the k cat /K m value of PSC is much lower than that of another active class III chitinase hevamine. 24,25) Activity center and conserved non-proline cis-peptide The active site (Asp125 and Glu127) of PSC is well superimposable with that of hevamine, whereas the glutamate is substituted by glutamine in concanavalin B and both of the two residues are absent in the sequence of chi-lectin.…”

Purification and Biochemical Characterization of a Pathogenesis Related Endochitinase Arachis hypogaea

“…To date, different types of chitinases have been purified and characterized from different plants like sugar beet (El Sayed et al, 2000), soya bean (Gijzen et al, 2001), pineapple (Taira et al, 2005), pomegranate (Yang et al, 2011), and persimmon (Zhang et al, 2013).…”