X-ray structure of papaya chitinase reveals the substrate binding mode of glycosyl hydrolase family 19 chitinases
ABSTRACT The crystal structure of a chitinase from Carica papaya has been solved by the molecular replacement method and is reported to a resolution of 1.5 A. This enzyme belongs to family 19 of the glycosyl hydrolases. Crystals have been obtained in the presence of N-acetyl- d-glucosamine (GlcNAc) in the crystallization solution and two well-defined GlcNAc molecules have been identified in the catalytic cleft of the enzyme, at subsites -2 and +1. These GlcNAc moieties bind to the protein via an extensive network of interactions which also involves many hydrogen bonds mediated by water molecules, underlying their role in the catalytic mechanism. A complex of the enzyme with a tetra-GlcNAc molecule has been elaborated, using the experimental interactions observed for the bound GlcNAc saccharides. This model allows to define four major substrate interacting regions in the enzyme, comprising residues located around the catalytic Glu67 (His66 and Thr69), the short segment E89-R90 containing the second catalytic residue Glu89, the region 120-124 (residues Ser120, Trp121, Tyr123, and Asn124), and the alpha-helical segment 198-202 (residues Ile198, Asn199, Gly201, and Leu202). Water molecules from the crystal structure were introduced during the modeling procedure, allowing to pinpoint several additional residues involved in ligand binding that were not previously reported in studies of poly-GlcNAc/family 19 chitinase complexes. This work underlines the role played by water-mediated hydrogen bonding in substrate binding as well as in the catalytic mechanism of the GH family 19 chitinases. Finally, a new sequence motif for family 19 chitinases has been identified between residues Tyr111 and Tyr125.
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- "The sequential motifs capable of uniquely identifying the GH19 family chitinase are searched in the major clusters (C1, C2, and C3) identified through phylogenetic analysis. A motif, M1 (Y[YF]GRGPIQ[LI][ST][WY]N[YF]NYG[AP][AC] GRA) is highly conserved across the GH19 family chitinases and it has already been reported (Huet et al. 2008). The second identified motif M2 (DA[ITV]CK[RK] [ES][LAI]A[AT]F[LF]A[NQH][VF][SA][HQ]E[TS]GG[LH] x[YA][VI]VExN) is also conserved across the GH19 family chitinases and a part of the M2 motif corresponds to the proposed active site region identified in plant chitinases (Tang et al. 2004). "
ABSTRACT: The discovery of GH (Glycoside Hydrolase) 19 chitinases in Streptomyces sp. raises the possibility of the presence of these proteins in other bacterial species, since they were initially thought to be confined to higher plants. The present study mainly concentrates on the phylogenetic distribution and homology conservation in GH19 family chitinases. Extensive database searches are performed to identify the presence of GH19 family chitinases in the three major super kingdoms of life. Multiple sequence alignment of all the identified GH19 chitinase family members resulted in the identification of globally conserved residues. We further identified conserved sequence motifs across the major sub groups within the family. Estimation of evolutionary distance between the various bacterial and plant chitinases are carried out to better understand the pattern of evolution. Our study also supports the horizontal gene transfer theory, which states that GH19 chitinase genes are transferred from higher plants to bacteria. Further, the present study sheds light on the phylogenetic distribution and identifies unique sequence signatures that define GH19 chitinase family of proteins. The identified motifs could be used as markers to delineate uncharacterized GH19 family chitinases. The estimation of evolutionary distance between chitinase identified in plants and bacteria shows that the flowering plants are more related to chitinase in actinobacteria than that of identified in purple bacteria. We propose a model to elucidate the natural history of GH19 family chitinases.Journal of Molecular Evolution 05/2010; 70(5):466-78. DOI:10.1007/s00239-010-9345-z · 1.86 Impact Factor
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ABSTRACT: The cDNA, genomic DNA, and promoter sequence of FaChit1, a class I chitinase gene from Festuca arundinacea, were isolated and characterized in the present work. The deduced amino acid sequence of FaChit1 contains the chitin binding, catalytic, and proline and glycine-rich domains characteristic for most class I chitinases, but no C-terminal extension region. FaChit1 is induced effectively by fungal elicitors, dehydration, and ethylene, but only slightly by mechanical wounding. To identify potential stress-related cis-acting elements, 5′ sequences 935, 651, and 233bp upstream of the FaChit1 start codon were fused to the GUS reporter gene and analyzed in transgenic tobacco. The results indicated that the 935bp fragment closely mirrored endogenous gene expression and that the 651bp fragment was sufficient to direct reporter the gene expression in response to fungal elicitors, ethylene, dehydration, or mechanical wounding due to both known and presently uncharacterized cis-acting elements.Plant Molecular Biology Reporter 09/2009; 27(3):305-314. DOI:10.1007/s11105-008-0086-8 · 2.37 Impact Factor
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ABSTRACT: We have studied the degradation of soluble heteropolymeric chitosans with a bacterial family 19 chitinase, ChiG from Streptomyces coelicolor A3(2), to obtain insight into the mode of action of ChiG, to determine subsite preferences for acetylated and deacetylated sugar units, and to evaluate the potential of ChiG for production of chito-oligosaccharides. Degradation of chitosans with varying degrees of acetylation was followed using NMR for the identity (acetylated/deacetylated) of new reducing and nonreducing ends as well as their nearest neighbors and using gel filtration to analyze the size distribution of the oligomeric products. Degradation of a 64% acetylated chitosan yielded a continuum of oligomers, showing that ChiG operates according to a nonprocessive, endo mode of action. The kinetics of the degradation showed an initial rapid phase dominated by cleavage of three consecutive acetylated units (A; occupying subsites -2, -1, and +1), and a slower kinetic phase reflecting the cleavage of the glycosidic linkage between a deacetylated unit (D, occupying subsite -1) and an A (occupying subsite +1). Characterization of isolated oligomer fractions obtained at the end of the initial rapid phase and at the end of the slower kinetic phase confirmed the preference for A binding in subsites -2, -1, and +1 and showed that oligomers with a deacetylated reducing end appeared only during the second kinetic phase. After maximum conversion of the chitosan, the dimers AD/AA and the trimer AAD were the dominating products. Degradation of chitosans with varying degrees of acetylation to maximum degree of scission produced a wide variety of oligomer mixtures, differing in chain length and composition of acetylated/deacetylated units. These results provide insight into the properties of bacterial family 19 chitinases and show how these enzymes may be used to convert chitosans to several types of chito-oligosaccharide mixtures.Biomacromolecules 03/2009; 10(4):892-9. DOI:10.1021/bm801418p · 5.75 Impact Factor