Chitin, a natural homopolymer composed of β(1–4)-linked N
, is a major component of the exoskeleton of fungi (comprising up to 30% of fungal cell walls) and invertebrates. It is easily obtained from marine invertebrates, insects and algae (Patil et al.
The complete enzymatic hydrolysis of chitin to free N
-acetylglucosamine residues is performed by a chitinolic system and is known to be a continuous reaction. Different organisms produce a wide variety of hydrolytic enzymes that exhibit different substrate specificities. Some of them are called chitinases, which are enzymes that catalyze the hydrolysis of chitin. These proteins are a large and diverse group of enzymes that differ in their molecular structure, substrate specificity and catalytic mechanism (Kasprzewska, 2003
). Specificity for chitin oligosaccharide is not a feature that is exclusive to the chitinases. Proteins named ‘chitin-binding lectins’ or ‘hevein-like lectins’ also possess affinity for N
-acetylglucosamine residues, but cannot catalyze the hydrolysis of chitin (Van Damme et al.
Several chitinases have been found in plants (angiosperms and gymnosperms) and are present in diverse tissues. Most are expressed by stress factors such as infection. Plants use chitinases as a defence against pathogenic fungi, but the enzymes may also perform other functions (Peumans et al.
). Some chitinases have industrial and agricultural applications, such as in the biocontrol of pathogenic fungi and insects, as a target for biopesticides and in the production of chitooligosaccharides (Kasprzewska, 2003
; Patil et al.
Plant lectins with chitinase activity are poorly described in the literature. The acidic chitinase from Brassica juncea
shows a structure that is distinct from those observed for chitinases studied previously. This difference is characterized by the presence of two chitin-binding sites (Zhao & Chye, 1999
), which permit this protein to agglutinate cells and may provide an advantage over other chitinases in antimicrobial and antifungal activity (Chye et al.
Many carbohydrate-binding proteins have been reported, in particular those purified from plants (Moreno et al.
; Gadelha et al.
). The majority are from the Leguminosae family and comprise lectins and chitinases from diverse sources. Legume lectins have been well studied as a model of carbohydrate recognition. In the subfamily Mimosoideae, however, apart from Parkia plathycephala
2 (PPL-2), only the seed lectins from P. speciosa
(Suvachittanont & Peutpaiboon, 1992
), P. javanica
(Utarabhand & Akkayanont, 1995
), P. platycephala
(Cavada et al.
) and P. discolor
(Cavada et al.
) have been isolated and characterized in detail. Moreover, crystal structures are only available for P. platycephala
lectin (PPL-1) in native form (PDB code 1zgr
) and in complex with 5-bromo-4-chloro-3-indolyl-α-d
-mannose (PDB code 1zgs
Mass-spectrometric analysis indicates that the PPL-2 monomer is not glycosylated and contains six cysteine residues that are involved in three disulfide bonds; PPL-2 gives a main mass peak at 29 407. Functional analysis reveals that PPL-2 recognizes carbohydrates on red blood cells and agglutinates trypsin-treated rabbit erythrocytes (128 haemagglutinating units per millilitre). In addition, PPL-2 can hydrolyze β(1–4)-glycosidic linkages between 2-acetoamido-2-deoxy-β-d
-glucopyranoses present in chitin. The exact mechanism of glycoside hydrolysis has been described by Cavada et al.
) and this mechanism reveals an endochitinase activity to be associated with PPL-2 from the elution times found for the GlcNAc, (GlcNAc)2
standards. Hence, PPL-2 is the first and is a remarkable chimerolectin from the Mimosoideae, with the dual property of hydrolyzing chitin and binding sugar moieties on red blood cells (Cavada et al.
In order to establish the crystal structure of this new member of the chitin-binding proteins, this work reports the crystallization and preliminary X-ray diffraction analysis of a hevamine-like protein from P. platycephala
seeds, named PPL-2, that has the ability to agglutinate cells and shows inhibitory effects in the growth of bacterial colonies and nematode-egg eclosion (Castellón, 2004
; Cavada et al.