Chitinase accumulates systemically in wounded poplar trees

Young leaves of poplar ( Populus spp.) trees accumulate novel messenger RNAs shortly after the mature leaves have been mechanically wounded. These systemically wound‐induced ( win ) mRNAs are thought to encode proteins involved in plant defense. In the present paper, transgenic tobacco plants that ectopically expressed a win6 cDNA contained a novel chitinase activity that was not present in normal tobacco. This demonstrated Win6 was a chitinase. Win6 and a related protein Win8 accumulated in wounded poplars. Win6 and Win8 had low isoelectric points (ca 4) as predicted from their nucleotide sequence. The wound‐inducible increase in Win6 and Win8 was correlated with an increase in chitinase (EC 3.2.1.14) activity in poplar leaf extracts. We conclude that mechanical wounding induces chitinase in poplar trees, and speculate that the induced chitinase activity could act to increase the tolerance of poplars to opportunistic wound pathogens.     

Characterization of salicylic acid-induced genes in Chinese cabbage

Salicylic acid is a messenger molecule in the activation of defense responses in plants. In this study, we isolated four cDNA clones representing salicylic acid-induced genes in Chinese cabbage (Brassica rapa subsp. pekinensis) by subtractive hybridization. Of the four clones, the BC5-2 clone encodes a putative glucosyltransferase protein. The BC5-3 clone is highly similar to an Arabidopsis gene encoding a putative metal-binding farnesylated protein. The BC6-1 clone is a chitinase gene with similarities to a rapeseed class IV chitinase. Class IV chitinases have deletions in the chitin-binding and catalytic domains and the BC6-1 chitinase has an additional deletion in the catalytic domain. The BCP8-1 clone is most homologous to an Arabidopsis gene that contains a tandem array of two thiJ-like sequences. These four cabbage genes were barely expressed in healthy leaves, but were strongly induced by salicylic acid and benzothiadiazole. Expression of the three genes represented by the BC5-2, BC5-3 and BCP8-1 clones were also induced by Pseudomonas syringae pv. tomato, a nonhost pathogen that elicits a hypersensitive response in Chinese cabbage. None of these four genes, however, was strongly induced by methyl jasmonate or by ethylene.     

High-multiplicity of chitinase genes in Streptomyces coelicolor A3(2).

Six different genes for chitinase from ordered cosmids of the chromosome of Streptomyces coelicolor A3(2) were identified by hybridization, using the chitinase genes from other Streptomyces spp. as probes, and cloned. The genes were sequenced and analyzed. The genes, together with an additional chitinase gene obtained from the data bank, can be classified into either family 18 or family 19 of the glycosyl hydrolase classification. The five chitinases that fall into family 18 show diversity in their multiple domain structures as well as in the amino acid sequences of their catalytic domains. The remaining two chitinases are members of family 19 chitinases, since their C-terminus shares more than 70% identity with the catalytic domain of ChiC of Streptomyces griseus, the sole gene for family 19 chitinase so far found in an organism other than higher plants.     

A novel type of family 19 chitinase from Aeromonas sp. No.10S-24. Cloning, sequence, expression, and the enzymatic properties.

A family 19 chitinase gene from Aeromonas sp. No.10S-24 was cloned, sequenced, and expressed in Escherichia coli. From the deduced amino acid sequence, the enzyme was found to possess two repeated N-terminal chitin-binding domains, which are separated by two proline-threonine rich linkers. The calculated molecular mass was 70 391 Da. The catalytic domain is homologous to those of plant family 19 chitinases by about 47%. The enzyme produced alpha-anomer by hydrolyzing beta-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc)6] was hydrolyzed by the chitinase, the second glycosidic linkage from the nonreducing end was predominantly split producing (GlcNAc)2 and (GlcNAc)4. The evidence from this work suggested that the subsite structure of the enzyme was (-2)(-1)(+1)(+2)(+3)(+4), whereas most of plant family 19 chitinases have a subsite structure (-3)(-2)(-1)(+1)(+2)(+3). Thus, the Aeromonas enzyme was found to be a novel type of family 19 chitinase in its structural and functional properties.     

The Fission Yeast Mitotic Regulator win1+ Encodes an MAP Kinase Kinase Kinase That Phosphorylates and Activates Wis1 MAP Kinase Kinase in Response to High Osmolarity

The Schizosaccharomyces pombe win1-1 mutant has a defect in the G2-M transition of the cell cycle. Although the defect is suppressed by wis1⁺ and wis4⁺, which are components of a stress-activated MAP kinase pathway that links stress response and cell cycle control, the molecular identity of Win1 has not been known. We show here that win1⁺ encodes a polypeptide of 1436 residues with an apparent molecular size of 180 kDa and demonstrate that Win1 is a MAP kinase kinase kinase that phosphorylates and activates Wis1. Despite extensive similarities between Win1 and Wis4, the two MAP kinase kinase kinases have distinct functions. Wis4 is able to compensate for loss of Win1 only under unstressed conditions to maintain basal Wis1 activity, but it fails to suppress the osmosignaling defect conferred by win1 mutations. The win1-1 mutation is a spontaneous duplication of 16 nucleotides, which leads to a frameshift and production of a truncated protein lacking the kinase domain. We discuss the cell cycle phenotype of the win1-1 cdc25-22 wee1-50 mutant and its suppression by wis genes.     

Membrane association, mechanism of action, and structure of Arabidopsis embryonic factor 1 (FAC1)

Embryonic factor 1 (FAC1) is one of the earliest expressed plant genes and encodes an AMP deaminase (AMPD), which is also an identified herbicide target. This report identifies an N-terminal transmembrane domain in Arabidopsis FAC1, explores subcellular fractionation, and presents a 3.3-A globular catalytic domain x-ray crystal structure with a bound herbicide-based transition state inhibitor that provides the first glimpse of a complete AMPD active site. FAC1 contains an (alpha/beta)(8)-barrel characterized by loops in place of strands 5 and 6 that places it in a small subset of the amidohydrolase superfamily with imperfect folds. Unlike tetrameric animal orthologs, FAC1 is a dimer and each subunit contains an exposed Walker A motif that may be involved in the dramatic combined K(m) (25-80-fold lower) and V(max) (5-6-fold higher) activation by ATP. Normal mode analysis predicts a hinge motion that flattens basic surfaces on each monomer that flank the dimer interface, which suggests a reversible association between the FAC1 globular catalytic domain and intracellular membranes, with N-terminal transmembrane and disordered linker regions serving as the anchor and attachment to the globular catalytic domain, respectively.     

Molecular cloning of multiple chitinase genes in Japanese flounder, Paralichthys olivaceus

Three different chitinase genes (fChi1, fChi2 and fChi3) were identified from Japanese flounder, Paralichthys olivaceus. The deduced amino-acid sequences of flounder chitinases revealed a typical chitinase structure containing a catalytic glyco-18 domain, a hinge region and a chitin binding domain type 2. The fChi1 and fChi2 mRNAs were predominantly expressed in the gastric glands of stomach. In contrast, expression of fChi3 was found in spleen, pancreas, stomach, intestine, liver, kidney and gonads of adult flounder by RT-PCR. The expression level of fChi3 in the adult tissues was below the detection limit of in situ hybridization (ISH) analysis; however, ISH signals were detected in the liver of flounder larvae. These results suggest that fChi1 and fChi2 are acidic chitinases that digest dietary chitin and that fChi3 probably is a macrophage specific chitinase (chitotriosidase) for biodefense and has an important unknown role in the liver during larval stages.     

High-level expression and characterization of two chitinases, ChiCH and ChiCW, of Bacillus cereus 28-9 in Escherichia coli

Many chitinase genes have been cloned and sequenced from prokaryotes and eukaryotes but overexpression of chitinases in Escherichia coli cells was less reported. ChiCH and ChiCW of Bacillus cereus 28-9 belong to two distinct groups based on their amino acid sequences of catalytic domains, and in addition, domain structures of two enzymes are different. In this study, we established an ideal method for high-level expression of chitinases in E. coli as glutathione-S-transferase fusion proteins using pGEX-6P-1 vector. Both ChiCH and ChiCW were successfully highly expressed in E. coli cells as soluble GST-chitinase fusion proteins, and recombinant native ChiCH and ChiCW could be purified after cleavage with PreScission protease to remove GST tag. Purified chitinases were used for biochemical characterization of kinetics, hydrolysis products, and binding activities. The results indicate that ChiCW is an endo-chitinase and effectively hydrolyzes chitin and chito-multimers to chito-oligomers and the end product chitobiose, and ChiCH is an exo-chitinase and degrades chito-oligomers to produce chitobiose. Furthermore, due to higher affinity of ChiCW toward colloidal chitin than Avicel, C-terminal domain of ChiCW should be classified as a chitin-binding domain not a cellulose-binding domain although that was revealed as a cellulose-binding domain by conserved domain analysis. Therefore, the method of high-level expression of chitinases is helpful to studies and applications of chitinases.     

Domain identification of hormone-sensitive lipase by circular dichroism and fluorescence spectroscopy, limited proteolysis, and mass spectrometry.

Structure-function relationship analyses of hormone-sensitive lipase (HSL) have suggested that this metabolically important enzyme consists of several functional and at least two structural domains (Osterlund, T., Danielsson, B., Degerman, E., Contreras, J. A., Edgren, G., Davis, R. C., Schotz, M. C., and Holm, C. (1996) Biochem. J. 319, 411-420; Contreras, J. A., Karlsson, M., Osterlund, T., Laurell, H., Svensson, A., and Holm, C. (1996) J. Biol. Chem. 271, 31426-31430). To analyze the structural domain composition of HSL in more detail, we applied biophysical methods. Denaturation of HSL was followed by circular dichroism measurements and fluorescence spectroscopy, revealing that the unfolding of HSL is a two-step event. Using limited proteolysis in combination with mass spectrometry, several proteolytic fragments of HSL were identified, including one corresponding exactly to the proposed N-terminal domain. Major cleavage sites were found in the predicted hinge region between the two domains and in the regulatory module of the C-terminal, catalytic domain. Analyses of a hinge region cleavage mutant and calculations of the hydropathic pattern of HSL further suggest that the hinge region and regulatory module are exposed parts of HSL. Together, these data support our previous hypothesis that HSL consists of two major structural domains, encoded by exons 1-4 and 5-9, respectively, of which the latter contains an exposed regulatory module outside the catalytic alpha/beta-hydrolase fold core.     

Properties of catalytic,linker and chitin-binding domains of insect chitinase

Manduca sexta (tobacco hornworm) chitinase is a glycoprotein that consists of an N-terminal catalytic domain, a Ser/Thr-rich linker region, and a C-terminal chitin-binding domain. To delineate the properties of these domains, we have generated truncated forms of chitinase, which were expressed in insect cells using baculovirus vectors. Three additional recombinant proteins composed of the catalytic domain fused with one or two insect or plant chitin-binding domains (CBDs) were also generated and characterized. The catalytic and chitin-binding activities are independent of each other because each activity is functional separately. When attached to the catalytic domain, the CBD enhanced activity toward the insoluble polymer but not the soluble chitin oligosaccharide primarily through an effect on the Km for the former substrate. The linker region, which connects the two domains, facilitates secretion from the cell and helps to stabilize the enzyme in the presence of gut proteolytic enzymes. The linker region is extensively modified by O-glycosylation and the catalytic domain is moderately N-glycosylated. Immunological studies indicated that the linker region, along with elements of the CBD, is a major immunogenic epitope. The results support the hypothesis that the domain structure of insect chitinase evolved for efficient degradation of the insoluble polysaccharide to soluble oligosaccharides during the molting process.     

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.     

Methyl jasmonate induces expression of a novel Brassica juncea chitinase with two chitin-binding domains

We have cloned a 1.3 kb Brassica juncea cDNA encoding BjCHI1, a novel acidic chitinase with two chitin-binding domains that shows 62% identity to Nicotiana tabacum Chia1 chitinase. BjCHI1 is structurally unlike Chia1 that has one chitin-binding domain, but resembles Chia5 chitinase UDA1, the precursor of Urtica dioica agglutinin; however there is only 36.9% identity between them. We propose that BjCHI1 should be classified under a new class, Chia7. The spacer and the hinge region of BjCHI1 are proline-rich, like that of Beta vulgaris Ch1, a Chia6 chitinase with half a chitin-binding domain. Northern blot analysis showed that the 1.3 kb BjCHI1 mRNA is induced by wounding and methyl jasmonate (MeJA) treatment but is unaffected by ethylene, salicylic acid (SA) or abscisic acid (ABA). This is the first report on MeJA induction of chitinase gene expression and further suggests that wound-related JA-mediated signal transduction is independent of that involving SA. Western blot analysis using polyclonal antibodies against BjCHI1 showed a cross-reacting band with an apparent molecular mass of 37 kDa in wounded tissues of B. juncea, revealing that, unlike UDA1, BjCHI1 is not cleaved post-translationally at the hinge. Expression of recombinant BjCHI1 in Escherichia coli BL21(DE3) inhibited its growth while crude extracts from E. coli JM109 expressing recombinant BjCHI1 showed chitinase activity. Results from polymerase chain reaction (PCR) suggest that genes encoding chitinases with single or double chitin-binding domains exist in B. juncea.     

Purification, cloning, and DNA sequence analysis of a chitinase from an overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis

Extracellular chitinases of Streptomyces peucetius and a chitinase overproducing mutant, SPVI, were purified to homogeneity by ion exchange and gel filtration chromatography. The purified enzyme has a molecular mass of 42 kDa on SDS-PAGE, and the N-terminal amino acid sequence of the protein from the wild type showed homology to catalytic domains (Domain IV) of several other Streptomyces chitinases such as S. lividans 66, S. coelicolor A3(2), S. plicatus, and S. thermoviolaceus OPC-520. Purified SPVI chitinase cross-reacted to anti-chitinase antibodies of wild-type S. peucetius chitinase. A genomic library of SPVI constructed in E. coli using lambda DASH II was probed with chiC of S. lividans 66 to screen for the chitinase gene. A 2.7 kb fragment containing the chitinase gene was subcloned from a lambda DASH II clone, and sequenced. The deduced protein had a molecular mass of 68 kDa, and showed domain organization similar to that of S. lividans 66 chiC. The N-terminal amino acid sequence of the purified S. peucetius chitinase matched with the N-terminus of the catalytic domain, indicating the proteolytic processing of 68 kDa chitinase precursor protein to 42 kDa mature chitinase containing the catalytic domain only. A putative chiR sequence of a two-component regulatory system was found upstream of the chiC sequence.