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.
"Orf15 was assigned as the endolysin gene. Rather than sharing similarity with phage lysozymes, the orf15 product had a motif (aa 114–127) highly conserved in members of the GH19 chitinases family, [FHY]-G-R-G-[AP]-X-Q-[IL]-[ST]-[FHYW]-[HN]-[FY]-NY, that forms the substrate binding region  (Figure 4). Moreover, Glu50/Glu59 of ORF15 were similar to Glu68/Glu77 of Streptomyces coelicolor chitinase G experimentally identified as the active sites . "
[Show abstract][Hide abstract] ABSTRACT: Stenotrophomonas maltophilia is a ubiquitous Gram-negative bacterium previously named as Xanthomonas maltophilia. This organism is an important nosocomial pathogen associated with infections in immunocompromised patients. Clinical isolates of S. maltophilia are mostly resistant to multiple antibiotics and treatment of its infections is becoming problematic. Several virulent bacteriophages, but not temperate phage, of S. maltophilia have been characterized.
In this study, a temperate myophage of S. maltophilia (Smp131) was isolated and characterized. Sequence analysis showed that its genome is 33,525-bp long with 47 open reading frames (ORFs). Its similarity to P2-like phages and prophages in S. maltophilia and several Xanthomonas pathovars includes genomic organization, arrangement of several operons, and possession of a slippery sequence T7G for translational frameshifting in tail assembly genes. Smp131 encodes a tyrosine family integrase that shares low degrees of similarity with those of other phages and a lysin belonging to family 19 chitinase that is observed in plants and some bacteria, although not in phages. tRNA are the preferred sites for host integration of Smp131 and the related phages: tRNA-Thr for Smp131 and prophage of S. maltophilia K279a; tRNA-Lys for prophages of X. campestris pv. campestris ATCC33913, X. oryzae pv. oryzae strains MAFF311018, and KACC10331; and tRNA-Asn for prophage of X. oryzae pv. oryzae PXO99A and remnant of X. axonopodis pv. citri 306. Regions flanking the prophages are varied highly in nucleotide sequence and rich in transposase genes, suggesting that frequent insertion/excision had occurred.
Prevalence of closely related prophages in Stenotrophomonas and Xanthomonads may have contributed to the diversity of these closely related species owing to possible horizontal gene transfer mediated by the phages.
"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). "
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
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