Molecular characterization of four chitinase cDNAs obtained fromCladosporium fulvum-infected tomato

Complementary DNA clones encoding acidic and basic isoforms of tomato chitinases were isolated fromCladosporium fulvum-infected leaves. The clones were sequenced and found to encode the 30 kDa basic intracellular and the 26 and 27 kDa acidic extracellular tomato chitinases previously purified (M.H.A.J. Joostenet al., in preparation). A fourth truncated cDNA which appears to encode an extracellular chitinase with 82% amino acid similarity to the 30 kDa intracellular chitinase was also isolated. Characterization of the clones revealed that the 30 kDa basic intracellular protein is a class I chitinase and that the 26 and 27 kDa acidic extracellular proteins which have 85% peptide sequence similarity are class II chitinases. The characterized cDNA clones represent four from a family of at least six tomato chitinases. Southern blot analysis indicated that, with the exception of the 30 kDa basic intracellular chitinase, the tomato chitinases are encoded by one or two genes. Northern blot analysis showed that the mRNA encoding the 26 kDa acidic extracellular chitinase is induced more rapidly during an incompatibleC. fulvum-tomato interaction than during a compatible interaction. This difference in timing of mRNA induction was not observed for the 30 kDa basic intracellular chitinase.     

Purification, characterization, and molecular cloning of a chitinase from the seeds of Benincasa hispida

A chitinase was purified from the seeds of Benincasa hispida, a medicinal plant also called white gourd, and a member of the Cucurbitaceae family. Purification was done by using a procedure consisting of only two fractionation steps: an acid denaturation step followed by ion-exchange chromatography. The sequence of the N-terminal forty amino acid residues was analyzed and the sequence indicated that the enzyme is a class III chitinase. The enzyme, which is a basic chitinase, is one of at least five chitinases detected in the seed extract of B. hispida. Like other class III chitinases, this enzyme also has lysozyme activity. A genomic clone of the gene encoding the enzyme was isolated and sequenced. The gene has the potential to encode a protein of 301 amino acid residues. The deduced amino acid sequence of the protein, as expected from the N-terminal amino acid sequence, shares high degrees of similarity with other class III chitinases.     

Molecular cloning of a genomic DNA encoding yam class IV chitinase

Genomic DNA for a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves and sequenced. The deduced amino acid sequence shows 50 to 59% identity to class IV chitinases from other plants. The yam chitinase, however, has an additional sequence of 8 amino acids (a C-terminal extension) following the cysteine that was reported as the last amino acid for other class IV chitinases; this extension is perhaps involved in subcellular localization. A homology model based on the structure of a class II chitinase from barley was used as an aid to interpreting the available data. The analysis suggests that the class IV enzyme recognizes an even shorter segment of the substrate than class I or II enzymes. This observation might help to explain why class IV enzymes are better suited to attack against pathogen cell walls.     

A tobacco gene encoding a novel basic class II chitinase: a putative ancestor of basic class I and acidic class II chitinase genes

Various chitinases have been identified in plants and categorized into several groups based on the analysis of their sequences and domains. We have isolated a tobacco gene that encodes a predicted polypeptide consisting of a 20-amino acid N-terminal signal peptide, followed by a 245-amino acid chitinolytic domain. Although the predicted mature protein is basic and shows greater sequence identity to basic class I chitinases (75%) than to acidic class II chitinases (67%), it lacks the N-terminal cysteine-rich domain and the C-terminal vacuolar targeting signal that is diagnostic for class I chitinases. Therefore, this gene appears to encode a novel, basic, class II chitinase, which we have designated NtChia2;B1. Accumulation of Chia2;B1 mRNA was induced in leaves in association with the local-lesion response to tobacco mosaic virus (TMV) infection, and in response to treatment with salicylic acid, but was only slightly induced by treatment with ethephon. Little or no Chia2;B1 mRNA was detected in roots, flowers, and cell-suspension cultures, in which class I chitinase mRNAs accumulate to high concentrations. Sequence comparisons of Chia2;B1 with known tobacco class I and class II chitinase genes suggest that Chia2;B1 might encode an ancestral prototype of the present-day class I and class II isoforms. Possible mechanisms for chitinase gene evolution are discussed.     

A tobacco gene encoding a novel basic class II chitinase: a putative ancestor of basic class I and acidic class II chitinase genes

Various chitinases have been identified in plants and categorized into several groups based on the analysis of their sequences and domains. We have isolated a tobacco gene that encodes a predicted polypeptide consisting of a 20-amino acid N-terminal signal peptide, followed by a 245-amino acid chitinolytic domain. Although the predicted mature protein is basic and shows greater sequence identity to basic class I chitinases (75%) than to acidic class II chitinases (67%), it lacks the N-terminal cysteine-rich domain and the C-terminal vacuolar targeting signal that is diagnostic for class I chitinases. Therefore, this gene appears to encode a novel, basic, class II chitinase, which we have designated NtChia2;B1. Accumulation of Chia2;B1 mRNA was induced in leaves in association with the local-lesion response to tobacco mosaic virus (TMV) infection, and in response to treatment with salicylic acid, but was only slightly induced by treatment with ethephon. Little or no Chia2;B1 mRNA was detected in roots, flowers, and cell-suspension cultures, in which class I chitinase mRNAs accumulate to high concentrations. Sequence comparisons of Chia2;B1 with known tobacco class I and class II chitinase genes suggest that Chia2;B1 might encode an ancestral prototype of the present-day class I and class II isoforms. Possible mechanisms for chitinase gene evolution are discussed. Received: 25 May 1998 / Accepted: 29 June 1998     

High-capacity calcium-binding chitinase III from pomegranate seeds (Punica granatum Linn.) is located in amyloplasts

We have recently identified a new class III chitinase from pomegranate seeds (PSC). Interestingly, this new chitinase naturally binds calcium ions with high capacity and low affinity, suggesting that PSC is a Ca-storage protein. Analysis of the amino acid sequence showed that this enzyme is rich in acidic amino acid residues, especially Asp, which are responsible for calcium binding. Different from other known chitinases, PSC is located in the stroma of amyloplasts in pomegranate seeds. Transmission electron microscopy (TEM) analysis indicated that the embryonic cells of pomegranate seeds are rich in calcium ions, most of which are distributed in the stroma and the starch granule of the amyloplasts, consistent with the above idea that PSC is involved in calcium storage, a newly non-defensive function.     

The primary structure of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex

The primary structure of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex, has been determined predominantly with conventional non-automatic methods. The positions of three disulfide bridges have been determined. The sequence has about 60% identity with that of a chitinase from cucumber and 95% with the N-terminal sequence of the lysozyme/chitinase of Parthenocissus quinquefolia. The half-cystine residues in hevein and cucumber chitinase are located at identical positions. Hevamine is a basic protein from the lutoids (vacuoles) of rubber latex and may have a role in plugging the latex vessels and cessation of latex flow. The differences in cellular location, charge properties and sequence between hevamine and cucumber chitinase are similar to those between class I and class II chitinases from tobacco and other plant species.     

Acidic and basic class III chitinase mRNA accumulation in response to TMV infection of tobacco

Complementary DNA clones encoding acidic and basic isoforms of the class III chitinase were isolated from Nicotiana tabacum. The clones share ca. 65% identity, are equally homologous to the class III chitinases from cucumber and Arabidopsis, and are members of small gene families in tobacco. An acidic class III chitinase was purified from the intercellular fluid of tobacco leaves infected with tobacco mosaic virus (TMV). Partial amino acid sequencing of the protein confirmed that it was encoded by one of the cDNA clones. The mRNAs of the class III chitinases are coordinately expressed in response to TMV infection, both in infected and uninfected tissue. The acidic and basic class III chitinases constitute previously undescribed pathogenesis-related proteins in tobacco.     

Isolation and sequence of a novel amphibian pancreatic chitinase

An approximately 60-kDa protein with chitinase activity was purified from the pancreas of the toad Bufo japonicus. Its specific activity was 4.5 times higher than that of a commercial bacterial chitinase in fragmenting crab shell chitin, and its optimal pH was approximately 6.0. A cDNA clone encoding a protein consisting of 488 amino acid residues, including part of the peptide sequence determined from the isolated protein, was obtained from a toad pancreas cDNA library. The deduced amino acid sequence indicated that the protein contained regions with high homology to those present in chitinases from different species, with the amino acid residues for the chitinase activity and the chitin-binding ability being completely conserved. We designate the protein as toad pancreatic chitinase (tPCase). Northern blot analysis revealed the mRNA of this enzyme to be expressed exclusively in the pancreas. Toad PCase is the first amphibian chitinase to be identified as well as the first pancreatic chitinase identified in a vertebrate.     

Amino acid sequence of chitinase from Streptomyces erythraeus

The amino acid sequence of chitinase from Streptomyces erythraeus was determined by the conventional method. The amino acid sequences of tryptic peptides of the reduced and S-carboxymethylated protein were determined. The tryptic peptides were aligned by overlapping the amino acid sequences of chymotryptic peptides, lysyl endopeptidase peptides and cyanogen bromide fragments. S. erythraeus chitinase consists of 290 amino acid residues with the molecular weight of 30,400 and has two disulfide bridges at Cys(45)-Cys(89) and Cys(265)-Cys(272). The enzyme has no significant homology with other chitinases, lysozymes, and other proteins.     

Molecular cloning and expression of the gene encoding family 19 chitinase from Streptomyces sp. J-13-3

The gene encoding chitinase from Streptomyces sp. (strain J-13-3) was cloned and its nucleotide structure was analyzed. The chitinase consisted of 298 amino acids containing a signal peptides (29 amino acids) and a mature protein (269 amino acids), and had calculated molecular mass of 31,081 Da. The calculated molecular mass (28,229 Da) of the mature protein was almost same as that of the native chitinase determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometer. Comparison of the encoded amino acid sequences with those of other chitinases showed that J-13-3 chitinase was a member of the glycosyl-hydrolase family 19 chitinases and the mature protein had a chitin binding domain (65 amino acids) containing AKWWTQ motif and a catalytic domain (204 amino acids). The J-13-3 strain had a single chitinase gene. The chitinase (298 amino acids) with C-terminal His tag was overexpressed in Escherichia coli BL21(DE3) cells. The recombinant chitinase purified from the cell extract had identical N-terminal amino acid sequence of the mature protein in spite of confirmation of the nucleotide sequence, suggesting that the signal peptide sequence is successfully cut off at the predicted site by signal peptidase from E. coli and will be a useful genetic tool in protein engineering for production of soluble recombinant protein. The optimum temperature and pH ranges of the purified chitinase were at 35-40 degrees C and 5.5-6.0, respectively. The purified chitinase hydrolyzed colloidal chitin and trimer to hexamer of N-acetylglucosamine and also inhibited the hyphal extension of Tricoderma reesei.     

Expression of a novel chitinase by the fungal endophyte in Poa ampla

Many wild and cultivated cool-season grass species are naturally infected with fungal endophytes of the genera Neotyphodium and Epichlo?. These associations generally are considered mutualistic with the plants benefiting from reduced herbivory and the fungi benefiting from nutrients supplied by the plants. The fungi secrete proteins that might have a role in the interspecies symbiosis. In the interaction between Poa ampla Merr. and the endophyte Neotyphodium sp., a fungal chitinase was detected in the apoplastic protein fraction. The chitinase was also the major protein secreted in culture. Sequence analysis of the chitinase revealed it has a low level of amino acid sequence identity to other fungal chitinases and one of the conserved active site residues is altered. DNA gel-blot analysis indicated the chitinase was encoded by a single gene. Expression of similar chitinases also was detected in endophyte-infected tall fescue (Festuca arundinacea Schreb.), perennial ryegrass (Lolium perenne L.) and Chewings fescue (Festuca rubra L. subsp. fallax [Thuill] Nyman). This is the first report of an endophyte chitinase expressed in the infected host grass. As a secreted hydrolytic enzyme, the chitinase might have roles in the nutrition, growth or defense of the endophyte.     

Molecular Cloning of a Genomic DNA Encoding Yam Class IV Chitinase

Genomic DNA for a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves and sequenced. The deduced amino acid sequence shows 50 to 59% identity to class IV chitinases from other plants. The yam chitinase, however, has an additional sequence of 8 amino acids (a C-terminal extension) following the cysteine that was reported as the last amino acid for other class IV chitinases; this extension is perhaps involved in subcellular localization. A homology model based on the structure of a class II chitinase from barley was used as an aid to interpreting the available data. The analysis suggests that the class IV enzyme recognizes an even shorter segment of the substrate than class I or II enzymes. This observation might help to explain why class IV enzymes are better suited to attack against pathogen cell walls.     

Cloning of a cluster of chitinase genes from Aeromonas sp. No. 10S-24

A gene encoding chitinases from Aeromonas sp. No. 10S-24 was cloned into Escherichia coli DH5α using pUC19, and its nucleotides were sequenced. The chitinase gene was clustered in ORFs (open reading frame) 1 to 4, in a 8-kb fragment of DNA. ORF-1 consisted of 1608 bp encoding 535 amino acid residues, and ORF-2 consisted of 1425 bp encoding 474 amino acid residues. ORF-3 was 1617 bp long and encodes a protein consisting of 538 amino acids. ORF-4 encodes 287 amino acids of the N-terminal region. The amino acid sequences of ORF-1 and ORF-3 share sequence homology with chitinase D from Bacillus circulans, and chitinase A and B from Streptomyces lividans. The amino acid sequence of ORF-2 shared sequence homology with chitinase II from Aeromonas sp. No. 10S-24, and chitinase from Saccharopolyspora erythraea. A region of the sequence starting from Ala-28 of the amino acid sequence of ORF-3 coincided with the N-terminal amino acid sequence of chitinase III from Aeromonas sp. No. 10S-24.     

Amino acid sequence of the N-terminal domain of yam (Dioscorea japonica) aerial tuber acidic chitinase. Evidence for the presence of a wheat germ agglutinin domain in matured acidic chitinase from unstressed tuber

The amino acid sequence of the N-terminal domain of acidic chitinase from unstressed aerial tuber was determined and proved the presence of an N-terminal domain in acidic chitinase. The amino acid sequence was determined on a pyroglutamylaminopeptidase-treated N-terminal fragment of V8 protease and on chymotryptic peptides of this fragment. The sequence determined revealed 8 residues deletion and 2 residues insertion as compared with the N-terminal domain of tobacco basic chitinase. The N-terminal domain determined showed a homology of 40% and 52% with the N-terminal domain of tobacco basic chitinase and wheat germ agglutinin, respectively.Abbreviations DABITC,4-N,N dimethylaminoazobenzene 4-isothiocyanate - PITC phenylisothiocyanate - Cm carboxymethyl - WGA wheat germ agglutinin - TFA trifluoroacetic acid - PGAP pyroglutamylaminopeptidase     

Molecular cloning and sequence analysis of the chitinase gene from Bacillus thuringiensis serovar alesti

Endogenous chitinase plays a positive role in the pathogenicity of Bacillus thuringiensis to insect pests. The chitinase gene was cloned from B. thuringiensis serovar alesti strain HD-16, and the deduced 676 amino acid sequence showed a high degree of similarity with other Bacillus chitinases. Additionally, the deduced amino acid sequence showed that the protein contained an amino terminus signal peptide and consisted of a catalytic domain, a fibronectin type III domain and a chitin-binding domain. All three domains showed conserved sequences when compared to other bacterial chitinase or cellulase sequences.     

Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class III plant chitinases.

The gene (chiD) encoding the precursor of chitinase D was found to be located immediately upstream of the chiA gene, encoding chitinase A1, which is a key enzyme in the chitinase system of Bacillus circulans WL-12. Sequencing analysis revealed that the deduced polypeptide encoded by the chiD gene was 488 amino acids long and the distance between the coding regions of the chiA and chiD genes was 103 bp. Remarkable similarity was observed between the N-terminal one-third of chitinase D and the C-terminal one-third of chitinase A1. The N-terminal 47-amino-acid segment (named ND) of chitinase D showed a 61.7% amino acid match with the C-terminal segment (CA) of chitinase A1. The following 95-amino-acid segment (R-D) of chitinase D showed 62.8 and 60.6% amino acid matches, respectively, to the previously reported type III-like repeating units R-1 and R-2 in chitinase A1, which were shown to be homologous to the fibronectin type III sequence. A 73-amino-acid segment (residues 247 to 319) located in the putative activity domain of chitinase D was found to show considerable sequence similarity not only to other bacterial chitinases and class III higher-plant chitinases but also to Streptomyces plicatus endo-beta-N-acetylglucosaminidase H and the Kluyveromyces lactis killer toxin alpha subunit. The evolutionary and functional meanings of these similarities are discussed.     

Cloning and characterization of a small family 19 chitinase from moss (Bryum coronatum)

Chitinase-A (BcChi-A) was purified from a moss, Bryum coronatum, by several steps of column chromatography. The purified BcChi-A was found to be a molecular mass of 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 3.5. A cDNA encoding BcChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1012 nucleotides and encoded an open reading frame of 228 amino acid residues. The predicted mature BcChi-A consists of 205 amino acid residues and has a molecular weight of 22,654. Sequence analysis indicated that BcChi-A is glycoside hydrolase family-19 (GH19) chitinase lacking loops I, II, IV and V, and a C-terminal loop, which are present in the catalytic domain of plant class I and II chitinases. BcChi-A is a compact chitinase that has the fewest loop regions of the GH19 chitinases. Enzymatic experiments using chitooligosaccharides showed that BcChi-A has higher activity toward shorter substrates than class II enzymes. This characteristic is likely due to the loss of the loop regions that are located at the end of the substrate-binding cleft and would be involved in substrate binding of class II enzymes. This is the first report of a chitinase from mosses, nonvascular plants.