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Query: UNIPROT:P06889 (Mol)
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A soybean chitinase which has an apparent molecular mass of 28 kDa by SDS-PAGE, and has chitinase specific activity of 133 units per mg protein at pH 5.2 and an apparent pI of 5.7, was purified from mature dry seeds. Based upon the selected part (the residue positions 10-17) of the determined N-terminal 38 amino acid sequence, a 23-mer degenerate oligonucleotide was synthesized and used for the PCR cloning of the chitinase cDNA. The resulting 1340 bp cDNA was comprised of a 5'-untranslated region of 39 bases, a coding region corresponding to a 25 amino acid signal sequence, followed by a mature 308 amino acid sequence (calculated molecular mass 34,269, calculated pI 4.7), and a 235 nucleotide 3'-terminal untranslated region including 24 bases of the poly(A) tail. By comparing the deduced primary sequence with those of plant chitinases known to date, this enzyme was more than 50% identical to every class III acidic chitinase, but has no significant similarity to other families of chitinases. The comparison also showed that the C-terminal region of this chitinase is markedly extended, by at least 31 residues. Northern blot analysis demonstrated that this mRNA species is remarkably transcribed from the early stage until the late middle stage of seed development, whilst it is hardly expressed in the leaves and the stems of soybean. Spatial and temporal expression of this single gene imply that this class III chitinase is mainly devoted to the seed defense, not only in development but also in dormancy of soybean seed. This is the first reported isolation and cDNA cloning of a class III acidic endochitinase from seeds. According to the chitinase nomenclature we propose that this enzyme would be classified into a new class of chitinase PR-8 family, together with a Sesbania homologue.
Plant Mol Biol 1998 Feb
PMID:A class III acidic endochitinase is specifically expressed in the developing seeds of soybean (Glycine max [L.] Merr.). 948 81

Chitin, an insoluble structural polysaccharide that occurs in the exoskeletal and gut linings of insects, is a metabolic target of selective pest control agents. One potential biopesticide is the insect molting enzyme, chitinase, which degrades chitin to low molecular weight, soluble and insoluble oligosaccharides. For several years, our laboratories have been characterizing this enzyme and its gene. Most recently, we have been developing chitinase for use as a biopesticide to control insect and also fungal pests. Chitinases have been isolated from the tobacco hornworm, Manduca sexta, and several other insect species, and some of their chemical, physical, and kinetic properties have been determined. Also, cDNA and genomic clones for the chitinase from the hornworm have been isolated and characterized. Transgenic plants that express hornworm chitinase constitutively have been generated and found to exhibit host plant resistance. A transformed entomopathogenic virus that produces the enzyme displayed enhanced insecticidal activity. Chitinase also potentiated the efficacy of the toxin from the microbial insecticide, Bacillus thuringiensis. Insect chitinase and its gene are now available for biopesticidal applications in integrated pest management programs. Current knowledge regarding the molecular biology and biopesticidal action of insect and several other types of chitinases is described in this mini-review.
Insect Biochem Mol Biol 1997 Nov
PMID:Insect chitinases: molecular biology and potential use as biopesticides. 950 15

A differentially displayed cDNA clone (MD17) was isolated from tobacco roots (nicotiana tabacum cv. Xanthi-nc) infected with the arbuscular mycorrhizal (AM) fungus Glomus intraradices. The isolated DNA fragment exhibited a reduced level of expression in response to AM establishment and 90% identity with the 3' noncoding sequence of two basic chitinases (EC 3.2.1.14) from N. tabacum. Northern (RNA) blots and Western blots (immunoblots), probed with tobacco basic chitinase gene-specific probe and polyclonal antibodies raised against the chitinase enzyme, yielded hybridization patterns similar to those of MD17. Moreover, the up-regulation of the 32-kDa basic chitinase gene expression in tobacco roots by (1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) was less effective in mycorrhizal roots than in nonmycorrhizal controls. Suppression of endogenous basic chitinase (32-kDa) expression was also observed in transgenic mycorrhizal plants that constitutively express the 34-kDa basic chitinase A isoform. When plants were grown with an increased phosphate supply, no suppression of the 32-kDa basic chitinase was obtained. These findings indicate that during the colonization and establishment of G. intraradices in tobacco roots, expression of the basic chitinase gene is down-regulated at the mRNA level.
Mol Plant Microbe Interact 1998 Jun
PMID:Suppression of tobacco basic chitinase gene expression in response to colonization by the arbuscular mycorrhizal fungus Glomus intraradices. 961 47

Among the four classes of chitinase, a class II chitinase had not yet been reported for rice. We have isolated and characterized a class II acidic chitinase, Rcht2, from rice (Oryza sativa L. cv. Cheongcheongbyeo). The protein consists of a single polypeptide chain of 261 amino acid residues and includes a putative signal sequence of 29 amino acids at its N-terminus. It has a calculated molecular mass of 27,642 Da and an isoelectric point of 5.56. The Rcht2 chitinase lacks the cysteine-rich and hinge domains in the N-terminal region of the protein, which is the criterion for its classification as a class II chitinase. Comparison of the genomic and the cDNA sequence revealed that the coding region of Rcht2 consist of three exons of 301, 112, and 370 bp separated by two introns of 89 and 984 bp. In suspension-cultured rice cells, the transcript level of Rcht2 was dramatically increased by treatment with both glycol chitin and fungal elicitor. The application of protein phosphatase 1 and 2A inhibitors, calyculin A and okadaic acid, effectively abolished the induction of Rcht2 in response to fungal elicitor. In contrast, the activation of Rcht2 transcript was not inhibited by both cycloheximide and protein kinase inhibitors. These results demonstrate that protein dephosphorylation events play a crucial role in the elicitor-mediated induction of Rcht2 in rice cells, while de novo protein synthesis is not required for induction.
Plant Mol Biol 1998 Jun
PMID:A new class II rice chitinase, Rcht2, whose induction by fungal elicitor is abolished by protein phosphatase 1 and 2A inhibitor. 961 19

cDNAs encoding chitinases were cloned and characterized from Bombyx mori and Hyphantria cunea, and their gene expression during the metamorphosis was also studied. The chitinase cDNA from B. mori encodes a protein of 565 amino acids with a calculated molecular mass of 63.4 kDa and the H. cunea chitinase cDNA encodes a protein of 553 amino acids with a calculated molecular mass of 62.0 kDa. Amino acid alignment of the two chitinases revealed 75% homology and 77-80% with M. sexta chitinase. The putative cleavage site of the signal peptide was between amino acid residues 20 and 21 for both chitinases. There were three potential N-glycosylation sites in the chitinase of B. mori at the amino acid residues 86-89, NFTS 304-307, NATG, 398-401, NYTV, whereas two potential N-glycosylation sites were present at the amino acid residues 86-89, NFTA and 304-307, NATG, in that of H. cunea. Southern blot analysis of total genomic DNA suggested that the B. mori genome has only one chitinase gene detectable by the cDNA probe and the H. cunea genome has one or two chitinase gene copies. Northern analysis indicated that gene expression was up-regulated during the molting process, larval-pupal transformation and pupal-adult transformation, when enzymatic degradation of cuticle was occurring.
Insect Biochem Mol Biol 1998 Mar
PMID:Molecular cloning of chitinase cDNAs from the silkworm, Bombyx mori and the fall webworm, Hyphantria cunea. 965 39

Degenerate primers were used to amplify by the polymerase chain reaction (PCR) DNA fragments from the chitinase genes of five insect species: Aedes aegypti, Anopheles freeborni, Anopheles gambiae, Anopheles stephensi and Drosophila melanogaster. As many as four different products were found for each species; each deduced protein sequence having greatest homology to chitinase sequences from other species of insects and the crustacean, Penaeus japonicus. The four PCR products of A. aegypti hybridize to two loci, with three of the products derived from either three tightly linked genes or a single gene with three catalytic domains. Southern blot hybridizations of the PCR products from the species of Anopheles suggest a similar arrangement.
Insect Mol Biol 1998 Aug
PMID:Chitinases are a multi-gene family in Aedes, Anopheles and Drosophila. 966 72

Based on first principles and molecular mechanics calculations, we conclude that the mechanism of hevamine (a family 18 chitinase) involves an oxazoline ion intermediate stabilized by the neighboring C2' acetamido group. In this intermediate, the acetamido carbonyl oxygen atom forms a covalent bond to C1' of N-acetyl-glucosamine and has a transferred positive charge from the pyranose ring onto the acetamido nitrogen atom, leading to an anchimeric stabilization of 38.1 kcal/mol when docked with hevamine. This double displacement mechanism involving an oxazoline intermediate distinguishes the family 18 chitinase (which have one acidic residue near the active site) from family 19 chitinase and from hen egg-white lysozyme, which have two acidic residues near the active site. The structural and electronic properties of the oxazoline intermediate are similar to the known chitinase inhibitor allosamidin, suggesting that allosamidins act as transition state analogs of an oxazoline intermediate. Structural and electronic features of the oxazoline ion likely to be important in the design of new chitinase inhibitors are discussed.
J Mol Biol 1998 Jul 31
PMID:Substrate assistance in the mechanism of family 18 chitinases: theoretical studies of potential intermediates and inhibitors. 967 59

A lysozyme (pI 5.5) was purified to homogeneity from heated acid extracts of Drosophila melanogaster larvae, using gel filtration in a Superose column and ion-exchange chromatography in a Mono Q column. The final yield was 67%. The purified lysozyme with Mr 13,700 (determined by SDS-polyacrylamide gel electrophoresis) decreases in activity and has its pH optimum displaced towards acidic values and Km increases as the ionic strength of the medium becomes higher. The lysozyme is resistant to a cathepsin D-like proteinase present in cyclorrhaphous Diptera and displays a chitinase activity which is 11-fold higher than that of chicken lysozyme. Microsequencing of an internal peptide of the purified lysozyme showed that this enzyme is the product of the previously sequenced Lys D gene. The results suggest that the product of the Lys P gene has pI 7.2, a pH optimum around 5 and is not a true digestive enzyme. The most remarkable sequence convergence of D. melanogaster lysozyme D and lysozymes from vertebrate foregut fermenters are serine 104 and a decrease in the number of basic amino acids, suggesting that these features are necessary for digestive function in an acid environment. Adaptive residues putatively conferring stability in an acid proteolytic environment differ between insects and vertebrates, probably because they depend on the overall three-dimensional structure of the lysozymes. A maximum likelihood phylogeny and inferences from insect lysozyme sequences showed that the recruitment of lysozymes as digestive enzymes is an ancestral condition of the flies (Diptera: Cyclorrhapha).
Insect Biochem Mol Biol
PMID:Molecular adaptation of Drosophila melanogaster lysozymes to a digestive function. 969 34

To understand the coordinated functions of the different classes of defense-related genes expressed in plant disease resistance, the expression patterns of pathogenesis related (PR) protein genes and genes involved in antioxidation and the production of secondary metabolites were examined. The expression patterns of the respective defense-related genes were monitored following TMV infection or salicylic acid treatment. Northern blot analyses showed that PR genes such as PR-1, beta-1,3-glucanase and chitinase were strongly induced in tobacco leaves upon TMV infection or salicylic acid treatment. 3-Hydroxy-3-methylglutaryl CoA reductase (HMGR) and phenylalanine ammonialyase (PAL), involved in isoprenoid and phenylpropanoid biosynthesis, respectively, were mildly induced at the late stage of normal hypersensitive response (HR) or after salicylic acid treatment when compared with the PR-gene expressions. However, in acute HR, they were strongly expressed at the early stage. Interestingly, the expression of the antioxidative genes, anionic peroxidase and ascorbate peroxidase, were inversely expressed following TMV infection and salicylic acid treatment. Differential expression of 3 groups of genes involved in plant defense responses are discussed in relation to different signal transduction pathways.
Mol Cells 1998 Aug 31
PMID:Coordinated expression of defense-related genes by TMV infection or salicylic acid treatment in tobacco. 974 24

To determine if the attached cells formed in Myosin II-deficient Saccharomyces cerevisiae result from deficient chitinase 1 (CTS1) expression, the activity of chitinase 1 was assayed. Secretion of this enzyme was not prevented by a MYO1 gene deficiency, and soluble and cell wall-associated Cts1p activity were increased approximately 5-fold and 20-fold, respectively, in these cells. The increase in soluble activity was correlated with an increase in enzyme levels. Likewise, intracellular chitinase activity was increased approximately 22-fold, and the chitin content of cell walls was elevated 2-fold. These data suggest that the origin of myo1-associated phenotypes is not due to deficient chitinase expression and may instead be due to a deregulation of cell wall metabolism in these cells.
Cell Mol Biol (Noisy-le-grand) 1998 Sep
PMID:Elevated expression of chitinase 1 and chitin synthesis in myosin II-deficient Saccharomyces cerevisiae. 976 95


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