Plant Chitinases Research Articles

Aggressive and defensive roles for chitinases.

Chitinases are produced by a wide variety of pathogenic and parasitic microbes and invertebrates during their attack on chitin-containing organisms. Examples discussed include enzymes of insect and algal viruses, of yeast killer toxin plasmids, of bacterial and fungal pathogens of fungi and insects, and of parasitic protozoa. These chitinases play roles in penetration of fungal cell walls, and of exoskeletons and peritrophic membranes of arthropods. Salivas of some invertebrate predators have chitinolytic activity which may be involved in their attack on their prey. Chitinases play a major defensive role in all plants against attack by fungi, and perhaps also against attack by insect pests. The plant chitinases form a very large and diverse assemblage of enzymes from two families of glycosyl hydrolases. At least some vertebrates, including fish and humans, also may utilise chitinases in their defence against pathogenic fungi and some parasites.

Plant chitinase/lysozyme isoforms show distinct substrate specificity and cleavage site preference towards lipochitooligosaccharide Nod signals

Summary The ability of 16 chitinases from seven different plant species to hydrolyze a collection of several structurally related lipochitooligosaccharides (LCOs) of Rhizobium was analyzed. It was found that the enzymes differed to a large extent in their activity on different LCOs. Differences were attributed to (i) the relative activity on different LCOs as substrate (e.g. sulfated versus non‐sulfated LCOs); (ii) the relative cleavage site preference on a given LCO molecule (hydrolysis of either the second, third or fourth glycosidic bond from the non‐reducing end of the molecule); and (iii) the stereochemistry of the reaction (retention or inversion of the anomeric configuration). A graphic representation of the different substrate specificities resulted in a ‘fingerprint’ that is characteristic for a given enzyme or a family of related enzymes. By comparing the LCO‐fingerprint of unknown enzymes with those obtained for already characterized proteins, it is possible to identify new glycosyl hydrolases. The high diversity of substrate specificity found among plant chitinases may reflect variations in the natural substrates of the enzymes, such as substitutions on the chitin moiety of fungal cell walls or, in plants, the presence of putative endogenous substrates related to LCOs.

Site-directed mutagenesis of active site residues in a class I endochitinase from chestnut seeds.

Despite the intensive research on plant chitinases, largely bolstered by their antifungal properties, little is known at present about the structure-activity relationships of these enzymes. Here we report the identification of essential active site residues in endochitinase Ch3, a class I enzyme abundant in chestnut seeds. Knowledge-based protein modeling as well as structural and sequence comparisons were performed to identify potential catalytic residues. Different mutated proteins were then generated by site-directed mutagenesis, expressed in Escherichia coli , and characterized for their chitinolytic activity. Glu124 and Glu146, the only carboxylic residues properly located into the active site cleft to participate in catalysis, were both mutated to Gln and Asp. Our results suggest that Glu124 functions as the general acid catalyst whereas Glu146 is likely to act as a general base. Other mutations involving three highly conserved active site residues, Gln173, Thr175, and Asn254, also impaired the chitinolytic activity of Ch3. The effects of these variants on the fungus Trichoderma viride revealed that catalysis is not necessary for antifungal activity. Similarly to its homologous nonenzymatic polypeptides hevein and stinging nettle lectin, the N-terminal chitin-binding domain of Ch3 appears to interfere itself with hyphal growth.

Basic chitinases are correlated with the morphogenic response of flax cells

Flax (Linum usitatissimum) hypocotyl protoplasts immobilized in a calcium‐alginate matrix give rise to embryo‐like structures. A direct correlation was established between the presence of a set of ionically‐bound cell wall proteins, which includes the basic polypeptides P184 and P183 with an apparent molecular mass of 25 kDa, and this morphogenic response. Microsequencing of tryptic fragments from P184 and P183 indicated homologies with the chitinase family. These homologies were confirmed by demonstrating that, after renaturation, such proteins express a potential chitinase activity in SDS‐PAGE gel containing glycol chitin as synthetic substrate. Using degenerate primers from P184 internal sequences, we isolated one partial genomic sequence of a chitinase of 626 bp from which a putative 74‐amino acid sequence, disrupted by one intron, was deduced. High degrees of homology with several plant chitinases, including those expressed during somatic embryogenesis or in seeds, were observed. P184 microsequences match the corresponding sequence deduced from the chitinase PCR‐fragment perfectly.

Mutation of either of two essential glutamates converts the catalytic domain of tobacco class I chitinase into a chitin-binding lectin

To study the catalytic mechanism of plant chitinases in a homologous system, we mutated two potential catalytic residues of tobacco chitinase A and transiently expressed the mutants in tobacco protoplasts. The two residues were chosen based on sequence alignment and structural comparison with related glycosyl hydrolases. They were replaced by site-directed mutagenesis in a deletion mutant lacking the chitin-binding domain. Mutation of Glu 122 to Ala and of Glu 144 either to Ala or to Gln all decreased the catalytic activity below background, suggesting that both glutamates are essential for hydrolysis. The result with the mutation of Glu 144 to Gln was unexpected in view of the natural occurrence of Gln at this position in some class II chitinases. The mutant chitinases were found to strongly bind to regenerated chitin despite the deletion of their chitin-binding domain. This result indicates that the catalytic domain of chitinases can be turned into a chitin-binding lectin by the loss of its enzymatic activity.

Substrate specificities of tobacco chitinases.

Ten tobacco chitinases (1,4-N-acetyl-beta-D-glucosaminide glycanhydrolase, EC 3.2.1.14) were purified from tobacco leaves hypersensitively reacting to tobacco mosaic virus. The 10 enzymes, which belong to five distinct structural classes of plant chitinases, were incubated with several potential substrates such as chitin, a beta-1,4 N-acetyl-D-glucosamine (GlcNAc) polymer, chitosan (partially deacetylated chitin), chitin oligomers of variable length and bacterial cell wall. Tobacco chitinases are all endotype enzymes that liberate oligomers from chitin and are capable of processing the chito-oligomers further at differential rates. Chitin reaction products were separated and quantified by HPLC and differential kinetics of oligomer accumulation and degradation were observed with the distinct classes of chitinases. Depending on the substrate to be hydrolysed, each isoform displayed a different spectrum of activity. For example, class I isoforms were the most active on chitin and (GlcNAc)4-6 whereas class III basic isoforms were the most efficient in inducing bacterial lysis. Class V and class VI chitinases were shown to more readily hydrolyse chitin oligomers than the chitin polymer itself. Together, these data indicate that the 10 tobacco chitinases represent complementary enzymes which may have synergistic effects on their substrates. This paper discusses their implication in plant defense by attacking pathogen's structural components and in plant development by maturing signal molecules.

Correct targeting of a vacuolar tobacco chitinase in Saccharomyces cerevisiae – post-translational modifications are dependent on the host strain

The chitinase gene FB7-1 of Nicotiana tabacum cv. samsun line 5 was expressed in the two Saccharomyces cerevisiae strains, INVSC2 and H4, under the control of the GAL1 promoter from S. cerevisiae and a multicopy plasmid vector. Both yeast strains express the plant gene as enzymatic active proteins. In transformants of the strain INVSC2, 94% of the total plant chitinase is contained inside the cells, probably within the vacuole which has been confirmed by subcellular fractionation as well as immunohistochemical experiments. This retention inside the cells is due to the C-terminally located 7 amino acids long vacuolar targeting peptide of the prochitinase. When this sequence was removed, chitinase was transported into the culture medium. Pulse-chase experiments revealed that during translation in transformants of both yeast strains one chitinase polypeptide can be immunoadsorbed with specific antibodies. In the case of INVSC2-transformants, newly formed chitinase is modified in a 60 min chase to slightly increase its molecular mass, whereas in H4-transformants the molecular mass constantly remained 32 kDa. By Western blot analysis, two chitinase corresponding polypeptides of 32 and 37 kDa were accumulated in the culture medium of both transformants carrying the chitinase gene without the vacuolar targeting sequence. The larger one was very likely O-glycosylated. Whereas, both polypepitdes were also detected in cell extracts of the H4-transformant, only the smaller one was found in the INVSC2-transformant. The plant chitinase passed through the endoplasmic reticulum on its way to the vacuole. The N-terminal signal peptide responsible for the uptake into the endoplasmic reticulum is cleaved correctly. However, cleavage of the vacuolar targeting peptide located at the C-terminus, to give the mature chitinase is obviously influenced by the genetic background of the host strain. In INVSC2-transformants chitinase accumulates in its mature form whereas both the polypeptides of H4-transformants retain their vacuolar targeting peptide. Our results demonstrate that in the case of plant class I chitinase, the plant sorting signal is recognized in yeast cells but post-translational modifications are influenced by the host strain.

The abundant 31-kilodalton banana pulp protein is homologous to class-III acidic chitinases

We have identified and characterized the abundant protein from the pulp of banana fruit ( Musa acuminata cv. Grand Nain), and have isolated a cDNA clone encoding this protein. Comparison of the amino terminal sequence of the purified 31 kDa protein (P31) suggests that it is related to plant chitinases. Western analyses utilizing rabbit anti-P31 antiserum demonstrate that this protein is pulp-specific in banana. A full-length cDNA clone homologous to class III acidic chitinase genes has been isolated from a pulp cDNA library by differential screening. The identity of this clone as encoding P31 was verified by comparisons between the amino-terminal peptide sequence and the cDNA sequence and cross-hybridization of the translation product of the cDNA clone with P31 antiserum. Northern and western blot analyses of RNA and protein isolated from banana pulp at different stages of ripening indicate that the cDNA and protein are expressed at high levels in the pulp of unripe fruit, and that their abundance decreases as the fruit ripens. Based on its expression pattern and deduced amino acid sequence and composition, we hypothesize that the physiological role of P31 is not for plant protection, but as a storage protein in banana pulp.

Isolation and characterization of chitinase isoforms from the bulbs of four species of the genus Tulipa.

Six chitinase isoforms, designated TBC-1 to TBC-6, were purified to homogenity from the bulbs of four species (Tulipa bakeri, T. tarda, T. turkestanica, and T. praestans) of the genus Tulipa by CM-cellulose column chromatography, Butyl-Toyopearl 650M hydrophobic column chromatography, gel filtration on Sephadex G-75, and Mono-S fast protein liquid chromatography (FPLC). The chitinases had molecular weights of 30,000 and isoelectric points of 5.2 to 6.1. These chitinases were found to proteins with similar amino acid compositions and N-terminal sequences. The tulip chitinases all had two half-cystine residues, one more than gladiolus bulb class IIIb chitinase, but many fewer than chitinases of plant class I (15-17 Cys residues/mol), II (5-8 Cys residues/mol), or III (6 Cys residues/mol). The N-terminal sequences of tulip chitinases were similar to the sequence of the gladiolus chitinase, but did not resemble sequence of any class of plant chitinase. The optimal pH of these chitinases toward glycolchitin was pH 5. TBC-1 hydrolyzed (GlcNAc)6 into (GlcNAc)2, (GlcNAc)3, and (GlcNAc)4, and hydrolyzed (GlcNAc)5 into (GlcNAc)2 and (GlcNAc)3.

Structural and evolutionary relationships among chitinases of flowering plants.

The analysis of nuclear-encoded chitinase sequences from various angiosperms has allowed the categorization of the chitinases into discrete classes. Nucleotide sequences of their catalytic domains were compared in this study to investigate the evolutionary relationships between chitinase classes. The functionally distinct class III chitinases appear to be more closely related to fungal enzymes involved in morphogenesis than to other plant chitinases. The ordering of other plant chitinases into additional classes mainly relied on the presence of auxiliary domains-namely, a chitin-binding domain and a carboxy-terminal extension-flanking the main catalytic domain. The results of our phylogenetic analyses showed that classes I and IV form discrete and well-supported monophyletic groups derived from a common ancestral sequence that predates the divergence of dicots and monocots. In contrast, other sequences included in classes I* and II, lacking one or both types of auxiliary domains, were nested within class I sequences, indicating that they have a polyphyletic origin. According to phylogenetic analyses and the calculation of evolutionary rates, these chitinases probably arose from different class I lineages by relatively recent deletion events. The occurrence of such evolutionary trends in cultivated plants and their potential involvement in host-pathogen interactions are discussed.

Plant chitinase consensus sequences

Eighty-six plant chitinase sequences from 29 different species and one hybrid were obtained from the on-line GenBank nucleotide database. These sequences were grouped into five gene families based on previously published guidelines (Meins et al., 1994), and the amino-acid and nucleotide sequences of each gene family were aligned. Consensus amino-acid and nucleotide sequences were derived for each gene family based on the alignments. The consensus sequences were analyzed to determine, their amino-acid composition, hydropathy profiles, and codon usage.

Increased resistance toFusarium oxysporumf. sp.radicis-lycopersiciin tomato plants treated with the endophytic bacteriumPseudomonas fluorescensstrain 63-28

The potential ofPseudomonas fluorescensstrain 63-28 for inducing defence reactions in tomato plants inoculated with the vascular fungusFusarium oxysporumf.sp.radicis-lycopersiciwas studied by light and transmission electron microcopy, and further investigated by gold cytochemistry. In non-bacterized tomato plants the pathogen multiplied abundantly through much of the root tissues, causing severe damage including cell disorganization and cell wall alterations. Marked changes in the rate and extent ofFusariumcolonization were observed in bacterized plants. The intensity of colonization was appreciably reduced compared to that observed in control plants, and pathogen growth was restricted to the outer root tissues with preferential localization in the intercellular spaces. The accumulation of an electron-dense material in epidermal and outer cortical cells and the coating of most intercellular spaces with similar substances were among the most typical features of host reactions. Cytochemical labelling of chitin showed that disorganization ofFusariumhyphae colonizing bacterized roots correlated with chitin degradation. These observations provided indirect evidence for the production of plant chitinases. Another noticeable change occurring during pathogen ingress in the outer root cortex was the elaboration of callose-enriched wall appositions at sites of attempted fungal penetration. These appositions were slightly labelled by the gold-complexed β-glucosidase, whereas gold particles were mainly concentrated over the more opaque regions lining the host cell walls and at the periphery of the electron-dense deposits accumulating in the intercellular spaces. The present ultrastructural observations confirm thatP. fluorescensstrain 63-28 may function as an inducer of plant disease resistance.

Heterologous expression and characterization of wild-type and mutant forms of a 26 kDa endochitinase from barley (Hordeum vulgare L.).

To investigate structure-function relationships in plant chitinases, we have developed a heterologous expression system for the 26 kDa endochitinase from Hordeum vulgare L. (barley). Escherichia coli cells harbouring the gene in a T7 RNA polymerase-based expression vector synthesized completely insoluble recombinant protein under standard induction conditions at 37 degrees C. However, a concentration of soluble recombinant protein of approx. 15 mg/l was achieved by inducing bacteria at low temperature (15 degrees C). Recombinant endochitinase was purified to homogeneity and shown to be structurally and functionally identical to the seed protein. An average of three disulphide bonds are present in the recombinant enzyme, consistent with the number found in the natural form. The seed and recombinant proteins showed the same specific activity towards a high-molecular-mass substrate and exhibited similar anti-fungal activity towards Tricoderma reesei. Site-directed mutagenesis was used to replace residues that are likely to be involved in the catalytic event, based on structural similarities with lysozyme and on sequence alignments with related chitinases. The Glu67-->Gln mutation resulted in a protein with undetectable activity, while the Glu89-->Gln mutation yielded an enzyme with 0. 25% of wild-type specific activity. This suggests that two acidic residues are essential for catalytic activity, similar to the situation with many other glycosyl hydrolases. Examination of conserved residues stretching into the proposed substrate binding cleft suggests that Asn124 also plays an important functional role.

Fungal pathogens secrete an inhibitor protein that distinguishes isoforms of plant pathogenesis‐related endo‐β‐1,3‐glucanases

Plant endo‐β‐1,3‐glucanases and chitinases inhibit the growth of some fungi and generate elicitor‐active oligosaccharides while depolymerizing polysaccharides of mycelial walls. Overexpression of the endo‐β‐1,3‐glucanases and/ or chitinases in transgenic plants provides, in some cases, increased protection against fungal pathogens. However, most of the phytopathogenic fungi that have been tested in vitro are resistant to endo‐β‐1,3‐glucanases and chitinases. Furthermore, some phytopathogenic fungi whose growth is inhibited by these enzymes are able to overcome the effect of these enzymes over a period of hours, indicating an ability of those fungi to adapt to the enzymes. Evidence is presented indicating that fungal pathogens secrete proteins that inhibit selective plant endo‐β‐1,3‐glucanases.A glucanase inhibitor protein (GIP‐1) has been purified to homogeneity from the culture fluid of the fungal pathogen of soybeans, Phytophthora sojae f. sp. glycines (Psg), and two basic pathogenesis‐related endo‐β‐1,3‐glucanases (EnGLsoy‐A and EnGLsoy‐B) have been purified from soybean seedlings. GIP‐1 inhibits EnGLsoy‐A but not EnGLsoy‐B. Moreover, GIP‐1 does not inhibit endo‐β‐1,3‐glucanases secreted by Psg itself nor does GIP‐1 inhibit PR‐2c, a pathogenesis‐related endo‐β‐1,3‐glucanase of tobacco. Evidence is presented that Psg secretes other GIPs that inhibit other endo‐β‐1,3‐glucanase(s) of soybean. Furthermore, GIP‐1 does not exhibit proteolytic activity but does appear to physically bind to EnGLsoy‐A. The results reported herein demonstrate specific interactions between gene products of the host and pathogen and establish the need to consider fungal proteins that inhibit plant endo‐β‐1,3‐glucanases when attempting to use the genes encoding endo‐β‐1,3‐glucanases to engineer resistance to fungi in transgenic plants.

Rice class III chitinase homologues isolated by random cloning of rice cDNAs.

We obtained seven kinds of cDNA clones putatively identified as encoding class III chitinases from cDNA libraries constructed from dichlorophenoxyacetic acid (2,4-D)- and benzyl adenine (BA)- treated rice callus. Putative amino acid sequences encoded in these cDNA clones were compared with those of known chitinases of other plants. Two clones coded for homologues that show high similarity to class III chitinases. These clones contained the common glutamic acid at the active site and were classified as true homologues. The other five clones, however, showed relatively low similarity to class III chitinases and their active sites contained aspartic acid instead of glutamic acid. These clones may correspond to relatives of a super family of class III chitinases. The location of the genes coding for these homologues on the rice genome has been determined by genetic linkage analysis with restriction fragment length polymorphism.

Characterization and Inhibitor Studies of Chitinases from a Parasitic Blowfly (Lucilia cuprina), a Tick (Boophilus microplus), an Intestinal Nematode (Haemonchus contortus) and a Bean (Phaseolus vulgaris)

The molecular weight pattern and the stage-specific activities of chitinases from the blowfly Lucilia cuprina, the tick Boophilus microplus and the intestinal nematode Haemonchus contortus were examined. Chitinolytic enzymes could be detected in all parasite species tested, but the activity was different between the stages. Highest chitinolytic titers were found in blowfly pupae (83 kDa, 118 kDa), hatching larvae of ticks (58 kDa, 94 kDa) and nematode eggs (43 kDa). Leaves from ethylene-treated bean plants Phaseolus vulgaris expressed two basic Class I chitinases (Ia, Ib) of 34 kDa, differing in their amino acid sequences at residue 33 and 34 (Ia : glycine, proline ; Ib : lysine, aspartic acid). Inhibitor studies with blowfly pupae revealed that allosamidin (IC 50 = 0.32 (±0.02) μM) was by far the best inhibitor when compared with various amino sugar derivatives. This compound also inhibited chitinases from tick larvae (IC 50 = 0.69(±0.10) μM) and nematode eggs (IC 50 = 0.048(±0.0045) μM) specifically. Whereas Class Ia chitinase from bean leaves was inhibited only up to 18% by 10 μM allosamidin, it had an IC 50 of 1(±0.14) μM for the Ib type, which is the first plant chitinase described to be highly sensitive to allosamidin.

A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037.

The specificity of chitinase C-1 of Streptomyces griseus HUT 6037 for the hydrolysis of the beta-1,4-glycosidic linkages in partially acetylated chitosan is different from that of other microbial chitinases. In order to study the primary structure of this unique chitinase, the chiC gene specifying chitinase C-1 was cloned and its nucleotide sequence was determined. The gene encodes a polypeptide of 294 amino acids with a calculated size of 31.4 kDa. Comparison of the amino acid sequence of the deduced polypeptide with that of other proteins revealed a C-terminal catalytic domain displaying considerable sequence similarity to the catalytic domain of plant class I, II, and IV chitinases which form glycosyl hydrolase family 19. The N-terminal domain of the deduced polypeptide exhibits sequence similarity to substrate-binding domains of several microbial chitinases and cellulases but not to the chitin-binding domains of plant chitinases. The previously purified chitinase C-1 from S. griseus is suggested to be generated by proteolytic removal of the N-terminal chitin-binding domain and corresponds to the catalytic domain of the chitinase encoded by the chiC gene. High-performance liquid chromatography analysis of the hydrolysis products from N-acetyl chitotetraose revealed that chitinase C-1 catalyzes hydrolysis of the glycosidic bond with inversion of the anomeric configuration, in agreement with the previously reported inverting mechanism of plant class I chitinases. This is the first report of a family 19 chitinase found in an organism other than higher plants.

Molecular cloning and primary sequence analysis of a gene encoding a putative chitinase gene in Brassica oleracea vat. capitata

Chitinase, which catalyzes the hydrolysis of the β-1, 4-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin, is involved in inducible plants defense system. By construction of cabbage (Brassica oleracea var. capitata) genomic library and screening the library with pRCH8, a probe of rice chitinase gene fragment, a chitinase genomic sequence was isolated. The complete nucleotide sequence of the putative cabbage chitinase gene (cabch29) was determined, with its longest open reading frame (ORF)encoding a polypeptide of 413 aa. This polypeptide consists of a 21 aa N-terminal signal peptide, two chitin-binding domains different from those of other classes of plant chitinases, and a catalytic domain. Homology analysis illustrated that this cabch29 gene has 58.8% identity at the nucleotide level with the pRCH8 ORF probe and has 50% identity at the amino acid level with the catalytic domains of chitinase from bean,maize and sugar beet. Meanwhile,several kinds of cis-elements, such as TATA box, CAAT box, GATA motif, ASF-1 binding site, wound-response elements and AATAAA, have also been discovered in the flanking region of cabch29 gene.

Plant chitinases use two different hydrolytic mechanisms

Bacterial, fungal, animal, and some plant chitinases form family 18 of glycosyl hydrolases. Most plant chitinases form the family 19. While some chitinases also have lysozyme activity, animal lysozymes belong to different families. For glycosyl hydrolases, two reaction mechanisms are possible, leading to either retention or inversion of the anomeric configuration. We analyzed by HPLC the stereochemical outcome of the hydrolysis catalyzed by cucumber and bean chitinases, belonging to families 18 and 19, respectively. Cucumber chitinase used the retaining mechanism as known for bacterial chitinases and hen egg white lysozyme for which the mechanism has been determined. In contrast, bean chitinase catalyzed the hydrolysis of chitooligosaccharides with overall inversion of anomeric configuration.

Refined structure of the chitinase from barley seeds at 2.0 a resolution.

Chitinase from barley seeds is a monomeric enzyme with 243 amino-acid residues and it plays a role as a defense protein. Its structure, previously determined at 2.8 A resolution by multiple isomorphous replacement method, is mainly alpha-helical [Hart, Monzingo, Ready, Ernst & Robertus, (1993). J. Mol. Biol. 229, 189-193]. The crystallization and preliminary X-ray data of the same enzyme in a different crystal form has been reported independently [Song, Hwang, Kim & Suh, (1993). Proteins, 17, 107-109}, the asymmetric unit of which contains two chitinase molecules. As a step toward understanding the general principles of catalysis, reported here is the structure of chitinase from barley seeds in this crystal form, as determined by molecular replacement and subsequently refined at 2.0 A resolution, with incorporation of partial data to 1.9 A (R factor of 18.9% for 31 038 unique reflections with F(o)> 2sigma(F) in the range 8.0-1.9 A). The r.m.s. deviations from ideal stereochemistry are 0.013 A for bond lengths and 1.32 degrees for bond angles. A superposition of the two independent molecules in the asymmetric unit gives an r.m.s. difference of 0.55 A for all protein atoms (0.43 and 0.74 A for main-chain and side-chain atoms, respectively). When the refined model of each chitinase molecule in the asymmetric unit is superposed with the starting model, the r.m.s. difference for all shared protein atoms is 0.99 A for molecule 1 and 0.85 A for molecule 2, respectively. Through a sequence comparison with homologous plant chitinases as well as a structural comparison with the active sites of other glycosidases, key catalytic residues have been identified and the active site has been located in the three-dimensional structure of the barley chitinase. The present structure, refined at an effective resolution of 2.0 A with incorporation of partial data to 1.9 A, represents a significant improvement in resolution compared to the previously reported model. The improved resolution has enabled the location of solvent atoms, including water molecules near the catalytic residues, in addition to the positioning of protein atoms with greater accuracy.