cDNA cloning and 1.75 Å crystal structure determination of PPL2, an endochitinase and N‐acetylglucosamine‐binding 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 Å 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.Abbreviations CTAB, cetyl triethylammonium bromide; GlcNac, N-acetyl-D-glucosamine; GSP, gene-specific forward primer; HPAEC-PAD, high-pH anion exchange chromatography with pulsed amperometric detection; PE, pyridylethylated; PPL1, Parkia platycephala lectin 1; PPL2, Parkia platycephala lectin 2; PTC, phenylisothiocyanate; PTH, phenylthiohydantoin.

ICChI, a glycosylated chitinase from the latex of Ipomoea carnea

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Crystal Structures of Bacillus cereus NCTU2 Chitinase Complexes with Chitooligomers Reveal Novel Substrate Binding for Catalysis

“…2) (8,10,13,(35)(36)(37)(38)(39)(40)(41)(42)(43)(44). The sequences share several highly conserved fragments among all chitinases in which three regions are important.…”

Structure, Catalysis, and Inhibition of OfChi-h, the Lepidoptera-exclusive Insect Chitinase

“…Several crystal structures of GH18 chitinases have been determined from archaea (20), bacteria (21)(22)(23)(24)(25)(26)(27)(28)(29), fungi (30 -34), plants (35)(36)(37)(38)(39)(40)(41), and mammals (42,43). These structures show that although all of the GH18 chitinases use the same catalytic mechanism, they have large discrepancies in the shape of the substrate binding cleft.…”