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Comparative study of two GH 19 chitinase‐like proteins from Hevea brasiliensis , one exhibiting a novel carbohydrate‐binding domain
Author(s) -
MartínezCaballero Siseth,
CanoSánchez Patricia,
MaresMejía Israel,
DíazSánchez Angel G.,
MacíasRubalcava Martha L.,
Hermoso Juan A.,
RodríguezRomero Adela
Publication year - 2014
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12962
Subject(s) - hevea brasiliensis , chitinase , chitin , biochemistry , chemistry , trimer , homology modeling , glycosyl , protein data bank (rcsb pdb) , stereochemistry , biology , enzyme , dimer , natural rubber , organic chemistry , chitosan
Plants express chitinase and chitinase‐like proteins ( CLP s) belonging to the glycosyl hydrolases of the GH 18 and GH 19 families, which exhibit varied functions. CLP s in the GH 18 family have been structurally and functionally characterized; however, there are no structures available for any member of the GH 19 family. In this study, two CLP s of the GH 19 family from the rubber tree Hevea brasiliensis (Hb CLP 1 and Hb CLP 2) were cloned, expressed and characterized. Hb CLP 1 was identical to the allergen Hev b 11.0101 previously described by others, while Hb CLP 2 was a novel isoform exhibiting an unusual half chitin‐binding domain before the catalytic domain. Sequence alignments showed that in the two proteins the catalytic residues Glu117 and Glu147 in HbCLP1 and HbCLP2, respectively, were mutated to Ala, accounting for the lack of activity. Nonetheless, both CLP s bound chitin and chitotriose (Glc NA c) 3 with high affinities, as evaluated with chitin‐affinity chromatography and tryptophan fluorescence experiments. The chitin‐binding domains also bound chitotriose with even higher affinities. The crystal structures of the Hb CLP 1‐isolated domains were determined at high resolution. The analysis of the crystallographic models and docking experiments using (Glc NA c) 6 oligosaccharides provides evidence of the residues involved in sugar binding. Endochitinase activity was restored in both proteins by mutating residues A117E (Hb CLP 1) and A147E (Hb CLP 2); the distance between the catalytic proton donor and the catalytic nucleophile in the in silico mutated residues was 9.5 Å, as occurs in inverting enzymes. Hb CLP 1 and Hb CLP 2 were highly thermostable and exhibited antifungal activity against Alternaria alternata , suggesting their participation in plant defense mechanisms. Database The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 4MST for the catalytic domain (CatD1) and 4MPI for the chitin‐binding domain (CBD1). Nucleotide sequence data are available in the GenBank database under the accession numbers KF648872 (chi‐L1) and KF648873 (chi‐L2).