cDNA cloning and 1.75 A˚ crystal structure determination of PPL2, an endochitinase and N-acetylglucosaminebinding hemagglutinin from Parkia platycephala seeds

Abstract

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 ± 15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed b(1–4) glycosidic bonds linking 2-acetoamido-2-deoxy-b-d-glucopyranose units in chitin. The full-length amino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 A˚ resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (ba)8 barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182.