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. Received: 5 July 1996 / Accepted: 9 January 1997     

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.     

Structure,heterologous expression,and properties of rice (Oryza sativa L.) family 19 chitinases

We identified four new family 19 chitinases in Oryza sativa L. cv. Nipponbare: one class I (OsChia1d), two class II (OsChia2a and OsChia2b), and one class IV (OsChia4a). OsChia2a resembled (about 60% identity) the catalytic domains of class I chitinases, but OsChia2b was almost identical (95% identity) to that of the class IV enzyme. OsChia1c, OsChia1c deltaCBD (a deletion of OsChia1c lacking a chitin-binding domain, CBD), and OsChia2b were separately expressed and purified in Pichia pastoris. OsChia1c inhibited fungal growth significantly more than OsChia1c deltaCBD or OsChia2b. The activities of these enzymes on chitin polymers were similar, but they acted differently on N-acetylchitooligosaccharides, (GlcNAC)n. OsChia1c slowly hydrolyzed (GlcNAC)6 and very poorly hydrolyzed (GlcNAC)4 and (GlcNAC)5. In contrast, OsChia2b efficiently hydrolyzed these oligosaccharides. The high antifungal activity and low hydrolytic activity of the class I enzyme towards (GlcNAC)n imply that it participates in the generation of N-acetylchitooligosaccharide elicitors from the cell walls of infecting fungi.     

Distribution and phylogenetic analysis of family 19 chitinases in Actinobacteria

In organisms other than higher plants, family 19 chitinase was first discovered in Streptomyces griseus HUT6037, and later, the general occurrence of this enzyme in Streptomyces species was demonstrated. In the present study, the distribution of family 19 chitinases in the class Actinobacteria and the phylogenetic relationship of Actinobacteria family 19 chitinases with family 19 chitinases of other organisms were investigated. Forty-nine strains were chosen to cover almost all the suborders of the class Actinobacteria, and chitinase production was examined. Of the 49 strains, 22 formed cleared zones on agar plates containing colloidal chitin and thus appeared to produce chitinases. These 22 chitinase-positive strains were subjected to Southern hybridization analysis by using a labeled DNA fragment corresponding to the catalytic domain of ChiC, and the presence of genes similar to chiC of S. griseus HUT6037 in at least 13 strains was suggested by the results. PCR amplification and sequencing of the DNA fragments corresponding to the major part of the catalytic domains of the family 19 chitinase genes confirmed the presence of family 19 chitinase genes in these 13 strains. The strains possessing family 19 chitinase genes belong to 6 of the 10 suborders in the order Actinomycetales, which account for the greatest part of the Actinobacteria: Phylogenetic analysis suggested that there is a close evolutionary relationship between family 19 chitinases found in Actinobacteria and plant class IV chitinases. The general occurrence of family 19 chitinase genes in Streptomycineae and the high sequence similarity among the genes found in Actinobacteria suggest that the family 19 chitinase gene was first acquired by an ancestor of the Streptomycineae and spread among the Actinobacteria through horizontal gene transfer.     

The first crystal structures of a family 19 class IV chitinase: the enzyme from Norway spruce

Chitinases help plants defend themselves against fungal attack, and play roles in other processes, including development. The catalytic modules of most plant chitinases belong to glycoside hydrolase family 19. We report here x-ray structures of such a module from a Norway spruce enzyme, the first for any family 19 class IV chitinase. The bi-lobed structure has a wide cleft lined by conserved residues; the most interesting for catalysis are Glu113, the proton donor, and Glu122, believed to be a general base that activate a catalytic water molecule. Comparisons to class I and II enzymes show that loop deletions in the class IV proteins make the catalytic cleft shorter and wider; from modeling studies, it is predicted that only three N-acetylglucosamine-binding subsites exist in class IV. Further, the structural comparisons suggest that the family 19 enzymes become more closed on substrate binding. Attempts to solve the structure of the complete protein including the associated chitin-binding module failed, however, modeling studies based on close relatives indicate that the binding module recognizes at most three N-acetylglucosamine units. The combined results suggest that the class IV enzymes are optimized for shorter substrates than the class I and II enzymes, or alternatively, that they are better suited for action on substrates where only small regions of chitin chain are accessible. Intact spruce chitinase is shown to possess antifungal activity, which requires the binding module; removing this module had no effect on measured chitinase activity.     

Evolution, Homology Conservation, and Identification of Unique Sequence Signatures in GH19 Family Chitinases

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.     

Functional analysis of the chitin-binding domain of a family 19 chitinase from Streptomyces griseus HUT6037: substrate-binding affinity and cis-dominant increase of antifungal function

Chitinase C (ChiC) is the first bacterial family 19 chitinase discovered in Streptomyces griseus HUT6037. While it shares significant similarity with the plant family 19 chitinases in the catalytic domain, its N-terminal chitin-binding domain (ChBD(ChiC)) differs from those of the plant enzymes. ChBD(ChiC) and the catalytic domain (CatD(ChiC)), as well as intact ChiC, were separately produced in E. coli and purified to homogeneity. Binding experiments and isothermal titration calorimetry assays demonstrated that ChBD(ChiC) binds to insoluble chitin, soluble chitin, cellulose, and N-acetylchitohexaose (roughly in that order). A deletion of ChBD(ChiC) resulted in moderate (about 50%) reduction of the hydrolyzing activity toward insoluble chitin substrates, but most (about 90%) of the antifungal activity against Trichoderma reesei was abolished by this deletion. Thus, this domain appears to contribute more importantly to antifungal properties than to catalytic activities. ChBD(ChiC) itself did not have antifungal activity or a synergistic effect on the antifungal activity of CatD(ChiC) in trans.     

Structure of full‐length class I chitinase from rice revealed by X‐ray crystallography and small‐angle X‐ray scattering

The rice class I chitinase OsChia1b, also referred to as RCC2 or Cht‐2, is composed of an N‐terminal chitin‐binding domain (ChBD) and a C‐terminal catalytic domain (CatD), which are connected by a proline‐ and threonine‐rich linker peptide. Because of the ability to inhibit fungal growth, the OsChia1b gene has been used to produce transgenic plants with enhanced disease resistance. As an initial step toward elucidating the mechanism of hydrolytic action and antifungal activity, the full‐length structure of OsChia1b was analyzed by X‐ray crystallography and small‐angle X‐ray scattering (SAXS). We determined the crystal structure of full‐length OsChia1b at 2.00‐Å resolution, but there are two possibilities for a biological molecule with and without interdomain contacts. The SAXS data showed an extended structure of OsChia1b in solution compared to that in the crystal form. This extension could be caused by the conformational flexibility of the linker. A docking simulation of ChBD with tri‐N‐acetylchitotriose exhibited a similar binding mode to the one observed in the crystal structure of a two‐domain plant lectin complexed with a chitooligosaccharide. A hypothetical model based on the binding mode suggested that ChBD is unsuitable for binding to crystalline α‐chitin, which is a major component of fungal cell walls because of its collisions with the chitin chains on the flat surface of α‐chitin. This model also indicates the difference in the binding specificity of plant and bacterial ChBDs of GH19 chitinases, which contribute to antifungal activity. Proteins 2010. © 2010 Wiley‐Liss,Inc.     

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.     

Crystal Structures of the Catalytic Domain of a Novel Glycohydrolase Family 23 Chitinase from Ralstonia sp. A-471 Reveals a Unique Arrangement of the Catalytic Residues for Inverting Chitin Hydrolysis

Chitinase C from Ralstonia sp. A-471 (Ra-ChiC) has a catalytic domain sequence similar to goose-type (G-type) lysozymes and, unlike other chitinases, belongs to glycohydrolase (GH) family 23. Using NMR spectroscopy, however, Ra-ChiC was found to interact only with the chitin dimer but not with the peptidoglycan fragment. Here we report the crystal structures of wild-type, E141Q, and E162Q of the catalytic domain of Ra-ChiC with or without chitin oligosaccharides. Ra-ChiC has a substrate-binding site including a tunnel-shaped cavity, which determines the substrate specificity. Mutation analyses based on this structural information indicated that a highly conserved Glu-141 acts as a catalytic acid, and that Asp-226 located at the roof of the tunnel activates a water molecule as a catalytic base. The unique arrangement of the catalytic residues makes a clear contrast to the other GH23 members and also to inverting GH19 chitinases.     

Unique GH18 chitinase from Euglena gracilis: full-length cDNA cloning and characterization of its catalytic domain

A cDNA of putative chitinase from Euglena gracilis, designated EgChiA, encoded 960 amino acid residues, which is arranged from N-terminus in the order of signal peptide, glycoside hydrolase family 18 (GH18) domain, carbohydrate binding module family 18 (CBM18) domain, GH18 domain, CBM18 domain, and transmembrane helix. It is likely that EgChiA is anchored on the cell surface. The recombinant second GH18 domain of EgChiA, designated as CatD2, displayed optimal catalytic activity at pH 3.0 and 50 °C. The lower the polymerization degree of the chitin oligosaccharides [(GlcNAc)_4–6] used as the substrates, the higher was the rate of degradation by CatD2. CatD2 degraded chitin nanofibers as an insoluble substrate, and it produced only (GlcNAc)₂ and GlcNAc. Therefore, we speculated that EgChiA localizes to the cell surface of E. gracilis and is involved in degradation of chitin polymers into (GlcNAc)₂ or GlcNAc, which are easily taken up by the cells.     

Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects

A bioinformatics-based investigation of three insect species with completed genome sequences has revealed that insect chitinase-like proteins (glycosylhydrolase family 18) are encoded by a rather large and diverse group of genes. We identified 16, 16 and 13 putative chitinase-like genes in the genomic databases of the red flour beetle, Tribolium castaneum, the fruit fly, Drosophila melanogaster, and the malaria mosquito, Anopheles gambiae, respectively. Chitinase-like proteins encoded by this gene family were classified into five groups based on phylogenetic analyses. Group I chitinases are secreted proteins that are the most abundant such enzymes in molting fluid and/or integument, and represent the prototype enzyme of the family, with a single copy each of the catalytic domain and chitin-binding domain (ChBD) connected by an S/T-rich linker polypeptide. Group II chitinases are unusually larger-sized secreted proteins that contain multiple catalytic domains and ChBDs. Group III chitinases contain two catalytic domains and are predicted to be membrane-anchored proteins. Group IV chitinases are the most divergent. They usually lack a ChBD and/or an S/T-rich linker domain, and are known or predicted to be secreted proteins found in gut or fat body. Group V proteins include the putative chitinase-like imaginal disc growth factors (IDGFs). In each of the three insect genomes, multiple genes encode group IV and group V chitinase-like proteins. In contrast, groups I-III are each represented by only a singe gene in each species.     

Brittle culm15 encodes a membrane-associated chitinase-like protein required for cellulose biosynthesis in rice

Plant chitinases, a class of glycosyl hydrolases, participate in various aspects of normal plant growth and development, including cell wall metabolism and disease resistance. The rice (Oryza sativa) genome encodes 37 putative chitinases and chitinase-like proteins. However, none of them has been characterized at the genetic level. In this study, we report the isolation of a brittle culm mutant, bc15, and the map-based cloning of the BC15/OsCTL1 (for chitinase-like1) gene affected in the mutant. The gene encodes the rice chitinase-like protein BC15/OsCTL1. Mutation of BC15/OsCTL1 causes reduced cellulose content and mechanical strength without obvious alterations in plant growth. Bioinformatic analyses indicated that BC15/OsCTL1 is a class II chitinase-like protein that is devoid of both an amino-terminal cysteine-rich domain and the chitinase activity motif H-E-T-T but possesses an amino-terminal transmembrane domain. Biochemical assays demonstrated that BC15/OsCTL1 is a Golgi-localized type II membrane protein that lacks classical chitinase activity. Quantitative real-time polymerase chain reaction and β-glucuronidase activity analyses indicated that BC15/OsCTL1 is ubiquitously expressed. Investigation of the global expression profile of wild-type and bc15 plants, using Illumina RNA sequencing, further suggested a possible mechanism by which BC15/OsCTL1 mediates cellulose biosynthesis and cell wall remodeling. Our findings provide genetic evidence of a role for plant chitinases in cellulose biosynthesis in rice, which appears to differ from their roles as revealed by analysis of Arabidopsis (Arabidopsis thaliana).     

Structural features of plant chitinases and chitin-binding proteins

Structural features of plant chitinases and chitin-binding proteins are discussed. Many of these proteins consist of multiple domains, of which the chitin-binding hevein domain is a predominant one. X-ray and NMR structures of representatives of the major classes of these proteins are available now, and are used to describe the structures of the other ones. Conserved positions of Cys residues can be taken as evidence for identically located disulfide bridges or cysteine residues. The current classification of chitinases is unsatisfactory and needs to be replaced by an evolutionarily more correct one. As the currently known three-dimensional structures of chitinases are those from barley and the rubber tree, Hevea brasiliensis, it is proposed to adopt the designation b-type (classes I, II and IV) and h-type (classes III and V) chitinases, respectively.     

Mutational analysis of a CBM family 5 chitin-binding domain of an alkaline chitinase from Bacillus sp. J813

Chitinase J from alkaliphilic Bacillus sp. J813 comprises a glycoside hydrolase (GH) family 18 catalytic domain (CatD), a fibronectin type III like domain, and a carbohydrate-binding module (CBM) family 5 chitin-binding domain (ChBD). It has been suggested that the ChBD binds to insoluble chitin and enhances its degradation by the CatD. To investigate the roles of two aromatic residues (Trp541 and Trp542), which are exposed on the surface of the ChBD, mutational analysis was performed. Single and double mutations of the two aromatic residues decreased binding and hydrolyzing abilities toward insoluble chitin. This result suggests that the ChBD binds to chitin by hydrophobic interactions via two surface-exposed aromatic residues. However, the double mutant, which has no such aromatic residue, bound to chitin at pH 5.2, probably by electrostatic interactions. Moreover, the ChBD bound to insoluble chitosan by electrostatic interactions.