From bacteria to human: A journey into the world of chitinases

Chitinases, the enzymes responsible for the biological degradation of chitin, are found in a wide range of organisms from bacteria to higher plants and animals. They participate in numerous physiological processes such as nutrition, parasitism, morphogenesis and immunity. Many organisms, in addition to chitinases, produce inactive chitinase-like lectins that despite lacking enzymatic activity are involved in several regulatory functions. Most known chitinases belong to families 18 and 19 of glycosyl hydrolases, however a few chitinases that belong to families 23 and 48 have also been identified in recent years. In this review, different aspects of chitinases and chi-lectins from bacteria, fungi, insects, plants and mammals are discussed.     

A comparative proteomic approach to analyse structure, function and evolution of rice chitinases: a step towards increasing plant fungal resistance

Glycoside hydrolase family 19 chitinases (EC 3.2.1.14) widely distributed in plants, bacteria and viruses catalyse the hydrolysis of chitin and play a major role in plant defense mechanisms and development. Rice possesses several classes of chitinase, out of which a single structure of class I has been reported in PDB to date. In the present study an attempt was made to gain more insight into the structure, function and evolution of class I, II and IV chitinases of GH family 19 from rice. The three-dimensional structures of chitinases were modelled and validated based on available X-ray crystal structures. The structural study revealed that they are highly α-helical and bilobed in nature. These enzymes are single or multi domain and multi-functional in which chitin-binding domain (CBD) and catalytic domain (CatD) are present in class I and IV whereas class II lacks CBD. The CatD possesses a catalytic triad which is thought to be involved in catalytic process. Loop III, which is common in all three classes of chitinases, reflects that it may play a significant role in their function. Our study also confirms that the absence and presence of different loops in GH family 19 of rice may be responsible for various sized products. Molecular phylogeny revealed chitinases in monocotyledons and dicotyledons differed from each other forming two different clusters and may have evolved differentially. More structural study of this enzyme from different plants is required to enhance the knowledge of catalytic mechanism and substrate binding.     

Plant Chitinases and Their Roles in Resistance to Fungal Diseases

Chitinases are enzymes that hydrolyze the N-acetylglucosamine polymer chitin, and they occur in diverse plant tissues over a broad range of crop and noncrop species. The enzymes may be expressed constitutively at low levels but are dramatically enhanced by numerous abiotic agents (ethylene, salicylic acid, salt solutions, ozone, UV light) and by biotic factors (fungi, bacteria, viruses, viroids, fungal cell wall components, and oligosaccharides). Different classes of plant chitinases are distinguishable by molecular, biochemical, and physicochemical criteria. Thus, plant chitinases may differ in substrate-binding characteristics, localization within the cell, and specific activities. Because chitin is a structural component of the cell wall of many phytopathogenic fungi, extensive research has been conducted to determine whether plant chitinases have a role in defense against fungal diseases. Plant chitinases have different degrees of antifungal activity to several fungi in vitro. In vivo, although rapid accumulation and high levels of chitinases (together with numerous other pathogenesis-related proteins) occur in resistant tissues expressing a hypersensitive reaction, high levels also can occur in susceptible tissues. Expression of cloned chitinase genes in transgenic plants has provided further evidence for their role in plant defense. The level of protection observed in these plants is variable and may be influenced by the specific activity of the enzyme, its localization and concentration within the cell, the characteristics of the fungal pathogen, and the nature of the host-pathogen interaction. The expression of chitinase in combination with one or several different antifungal proteins should have a greater effect on reducing disease development, given the complexities of fungal-plant cell interactions and resistance responses in plants. The effects of plant chitinases on nematode development in vitro and in vivo are worthy of investigation.     

Crystal structures of allosamidin derivatives in complex with human macrophage chitinase

The pseudotrisaccharide allosamidin is a potent family 18 chitinase inhibitor with demonstrated biological activity against insects, fungi, and the Plasmodium falciparum life cycle. The synthesis and biological properties of several derivatives have been reported. The structural interactions of allosamidin with several family 18 chitinases have been determined by x-ray crystallography previously. Here, a high resolution structure of chitotriosidase, the human macrophage chitinase, in complex with allosamidin is presented. In addition, complexes of the allosamidin derivatives demethylallosamidin, methylallosamidin, and glucoallosamidin B are described, together with their inhibitory properties. Similar to other chitinases, inhibition of the human chitinase by allosamidin derivatives lacking a methyl group is 10-fold stronger, and smaller effects are observed for the methyl and C3 epimer derivatives. The structures explain the effects on inhibition in terms of altered hydrogen bonding and hydrophobic interactions, together with displaced water molecules. The data reported here represent a first step toward structure-based design of specific allosamidin derivatives.     

Characterization of two antifungal endochitinases from barley grain

A basic chitinase (chitinase T, EC 3.2.1.14, molecular mass 33 kDa, pI 9.8) was isolated and compared with a previously described chitinase (chitinase C, molecular mass 28 kDa, pI 9.7). The two chitinases were isolated in homogeneous form from barley ( Hordeum vulgare L.) Bomi mutant 1508 grains either by two cation exchange steps or by one affinity step followed by cation exchange. Both chitinases are endochitinases with specific activities of 168 and 54 nkat (mg protein)⁻¹ for chitinase T and chitinase C, respectively. Both inhibit the growth of Trichoderma viride efficiently. The lysozyme activity of both chitinases is 10⁴ times lower than that of hen egg-white lysozyme as measured by lysis of cell walls of Micrococcus lysodeikticus . The amino acid composition and two partial amino acid sequences of chitinase T were determined. A 23 residue sequence of the N-terminal domain of chitinase T, which was not present in chitinase C, showed 73% identity with domain B of wheat germ lectin and 65% identity with the N-terminal domain of an endochitinase from bean leaves (deduced from cDNA). A 9 amino acid sequence of a cyanogen bromide fragment of chitinase T was identical with a cDNA deduced sequence of a barley aleurone endochitinase but differed in one residue from chitinase C. Generally, the two grain chitinases have physico-chemical and enzymatic properties similar to the plant leaf chitinases characterized. Both chitinases are localized in the aleurone layer and starchy endosperm of developing and germinating grain, but not in the embryo. The appearance of chitinases T and C at a late state of grain development suggests a role for these enzymes as a defense against fungi in the quiescent and germinating grain.     

Identification,cloning, and characterization of a novel chitinase from leaf-cutting ant Atta sexdens: An enzyme with antifungal and insecticidal activity

Chitinases are enzymes that degrade chitin, a polysaccharide found in the exoskeleton of insects, fungi, yeast, and internal structures of other vertebrates. Although chitinases isolated from bacteria, fungi and plants have been reported to have antifungal or insecticide activities, chitinases from insects with these activities have been seldomly reported. In this study, a leaf-cutting ant Atta sexdens DNA fragment containing 1623 base pairs was amplified and cloned into a vector to express the protein (AsChtII-C4B1) in Pichia pastoris. AsChtII-C4B1, which contains one catalytic domain and one carbohydrate-binding module (CBM), was secreted to the extracellular medium and purified by ammonium sulfate precipitation followed by nickel column chromatography. AsChtII-C4B1 showed maximum activity at pH 5.0 and 55 °C when tested against colloidal chitin substrate and maintained >60% of its maximal activity in different temperatures during 48 h. AsChtII-C4B1 decreased the survival of Spodoptera frugiperda larvae fed with an artificial diet that contained AsChtII-C4B1. Our results have indicated that AsChtII-C4B1 has a higher effect on larva-pupa than larva-larva molts. AsChtII-C4B1 activity targets more specifically the growth of filamentous fungus than yeast. This work describes, for the first time, the obtaining a recombinant chitinase from ants and the characterization of its insecticidal and antifungal activities.     

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.     

Cloning and phylogenetic analysis of the chitinase gene from the facultative pathogen Paecilomyces lilacinus

AIMS: To PCR-amplify the full-length genomic-encoding sequence for one chitinase from the facultative fungal pathogen Paecilomyces lilacinus, analyse the DNA and deduced amino acid sequences and compare the amino acid sequence with chitinases reported from mycopathogens, entomopathogens and nematopathogens. METHODS AND RESULTS: The encoding gene (designated as PLC) was isolated using the degenerate PCR primers and the DNA-Walking method. The gene is 1458 bp in length and contains three putative introns. A number of sequence motifs that might play a role in its regulation and function had also been found. Alignment of the translation product (designated as Plc, molecular mass of 45.783 kDa and pI of 5.65) with homologous sequences from other species showed that Plc belongs to Class V chitinase within the glycosyl hydrolase family 18. The phylogenetic and molecular evolutionary analysis using mega (Molecular Evolutionary Genetics Analysis) indicated that these chitinases from mycopathogens, entomopathogens and nematopathogens, the majority of which belong to glycosyl hydrolase family 18, were clustered into two well-supported subgroups corresponding to ascomycetes fungal and nonfungal chitinases (bacteria, baculoviruses). CONCLUSIONS: Our study showed that chitinases from mycoparasitic, entomopathogenic and nematophagous fungi are closely related to each other and reaffirmed the hypothesis that baculovirus chitinase is most likely to be of a bacterial origin - acquired by gene transfer. Bacterial and baculoviral chitinases in our study are potential pathogenicity factors; however, we still cannot ascribe any specific function to those chitinases from the fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report describing the chitinase gene and its translation product from Paecilomyces lilacinus, which constitutes the largest number of formulated biological nematicides reported so far, this is also the first study to analyse and resolve the phylogenetic and molecular evolutionary relationships among the chitinases produced by mycopathogens, entomopathogens and nematopathogens.     

Human chitotriosidase-catalyzed hydrolysis of chitosan

Chitotriosidase (HCHT) is one of two family 18 chitinases produced by humans, the other being acidic mammalian chitinase (AMCase). The enzyme is thought to be part of the human defense mechanism against fungal parasites, but its precise role and the details of its enzymatic properties have not yet been fully unraveled. We have studied the properties of HCHT by analyzing how the enzyme acts on high-molecular weight chitosans, soluble copolymers of β-1,4-linked N-acetylglucosamine (GlcNAc, A), and glucosamine (GlcN, D). Using methods for in-depth studies of the chitinolytic machinery of bacterial family 18 enzymes, we show that HCHT degrades chitosan primarily via an endoprocessive mechanism, as would be expected on the basis of the structural features of its substrate-binding cleft. The preferences of HCHT subsites for acetylated versus nonacetylated sugars were assessed by sequence analysis of obtained oligomeric products showing a very strong, absolute, and a relative weak preference for an acetylated unit in the -2, -1, and +1 subsites, respectively. The latter information is important for the design of inhibitors that are specific for the human chitinases and also provides insight into what kind of products may be formed in vivo upon administration of chitosan-containing medicines or food products.     

微生物几丁质酶研究进展

微生物几丁质酶不仅在生物降解几丁质方面起着重要作用,而且可通过水解病原真菌的细胞壁而有效地抑制其生长。到目前为止,人们已经分离和克隆出了大量的微生物几丁质酶及其基因。尽管这些几丁质酶各不相同,但它们却具有类同的蛋白质结构域:信号肽、催化结构域和几丁质结合结构域等。本文着重介绍几丁质酶的结构和分子特征、表达和调控机理,并且分析了该酶的应用前景。     

Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences

A phylogenetic analysis of more than 350 multicopper oxidases (MCOs) from fungi, insects, plants, and bacteria provided the basis for a refined classification of this enzyme family into laccases sensu stricto (basidiomycetous and ascomycetous), insect laccases, fungal pigment MCOs, fungal ferroxidases, ascorbate oxidases, plant laccase-like MCOs, and bilirubin oxidases. Within the largest group of enzymes, formed by the 125 basidiomycetous laccases, the gene phylogeny does not strictly follow the species phylogeny. The enzymes seem to group at least partially according to the lifestyle of the corresponding species. Analyses of the completely sequenced fungal genomes showed that the composition of MCOs in the different species can be very variable. Some species seem to encode only ferroxidases, whereas others have proteins which are distributed over up to four different functional clusters in the phylogenetic tree.     

Antifungal proteins from plants. Purification, molecular cloning, and antifungal properties of chitinases from maize seed.

We have purified two 28-kDa chitinases, designated Chitinase A (Chit A) and Chitinase B (Chit B), from maize seeds to homogeneity and isolated cDNA clones encoding these two enzymes using an oligonucleotide probe based on an amino acid sequence of a peptide derived from Chit A. Although these two enzymes share 87% homology in their amino acid sequences, which were deduced from the nucleotide sequences of the isolated cDNA clones, they are significantly different in their biochemical and in vitro antifungal activities. When tested in vitro for antifungal activity against the growth of Trichoderma reesei, Alternaria solani, and Fusarium oxysporum, Chit A showed greater antifungal activity than Chit B. The specific activity of Chit A was determined to be 3-fold higher than that of Chit B. Chit A also had a 10-fold lower binding constant (Kd) against the substrate analogue N,N',N',N'-tetraacetyl chitotetrose than Chit B, indicating that the two enzyme may differ in their affinities for binding to the substrate chitin. Comparison of the amino acid sequences of maize seed chitinases with those of previously published chitinases from monocot and dicot plants indicates that maize seed chitinases have diverged significantly from other chitinases.     

Characterization of chitinase genes from an alkaliphilic actinomycete,Nocardiopsis prasina OPC-131

An alkaliphilic actinomycete, Nocardiopsis prasina OPC-131, secretes chitinases, ChiA, ChiB, and ChiB Delta, in the presence of chitin. The genes encoding ChiA and ChiB were cloned and sequenced. The open reading frame (ORF) of chiA encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed significant similarity to Streptomyces family 19 chitinases. ChiB Delta was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiB Delta were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efficient hydrolysis of chitin and stronger antifungal activity than ChiB Delta, indicating that the ChtBD type 3 of ChiB plays an important role in the efficient hydrolysis of chitin and in antifungal activity. Furthermore, the finding of family 19 chitinase in N. prasina OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus Streptomyces.     

Increased insect virulence in Beauveria bassiana strains overexpressing an engineered chitinase

Entomopathogenic fungi are currently being used for the control of several insect pests as alternatives or supplements to chemical insecticides. Improvements in virulence and speed of kill can be achieved by understanding the mechanisms of fungal pathogenesis and genetically modifying targeted genes, thus improving the commercial efficacy of these biocontrol agents. Entomopathogenic fungi, such as Beauveria bassiana, penetrate the insect cuticle utilizing a plethora of hydrolytic enzymes, including chitinases, which are important virulence factors. Two chitinases (Bbchit1 and Bbchit2) have previously been characterized in B. bassiana, neither of which possesses chitin-binding domains. Here we report the construction and characterization of several B. bassiana hybrid chitinases where the chitinase Bbchit1 was fused to chitin-binding domains derived from plant, bacterial, or insect sources. A hybrid chitinase containing the chitin-binding domain (BmChBD) from the silkworm Bombyx mori chitinase fused to Bbchit1 showed the greatest ability to bind to chitin compared to other hybrid chitinases. This hybrid chitinase gene (Bbchit1-BmChBD) was then placed under the control of a fungal constitutive promoter (gpd-Bbchit1-BmChBD) and transformed into B. bassiana. Insect bioassays showed a 23% reduction in time to death in the transformant compared to the wild-type fungus. This transformant also showed greater virulence than another construct (gpd-Bbchit1) with the same constitutive promoter but lacking the chitin-binding domain. We utilized a strategy where genetic components of the host insect can be incorporated into the fungal pathogen in order to increase host cuticle penetration ability.     

Antifungal proteins and peptides of leguminous and non-leguminous origins

Antifungal proteins and peptides, as their names imply, serve a protective function against fungal invasion. They are produced by a multitude of organisms including leguminous flowering plants, non-leguminous flowering plants, gymnosperms, fungi, bacteria, insects and mammals. The intent of the present review is to focus on the structural and functional characteristics of leguminous, as well as non-leguminous, antifungal proteins and peptides. A spectacular diversity of amino acid sequences has been reported. Some of the antifungal proteins and peptides are classified, based on their structures and/or functions, into groups including chitinases, glucanases, thaumatin-like proteins, thionins, and cyclophilin-like proteins. Some of the well-known proteins such as lectins, ribosome inactivating proteins, ribonucleases, deoxyribonucleases, peroxidases, and protease inhibitors exhibit antifungal activity. Different antifungal proteins may demonstrate different fungal specificities. The mechanisms of antifungal action of only some antifungal proteins including thaumatin-like proteins and chitinases have been elucidated.     

Identification of two group A chitinase genes in Botrytis cinerea which are differentially induced by exogenous chitin

Chitin-degrading enzymes represent potential targets for pesticides in the control of plant pathogenic fungi. Here we describe the cloning, molecular characterization, and expression analysis of two putative chitinases of Botrytis cinerea, a pathogenic fungus infecting a wide range of plants. On the basis of conserved motifs from family 18 of the glycosyl hydrolases and group A of the fungal chitinases, two fragments (BcchiA and BcchiB) were cloned and sequenced. Expression of BcchiA and BcchiB chitinase genes upon growth under different conditions was analysed using RT-PCR. We observed that BcchiA expression was suppressed by glucose, whereas it was strongly stimulated in the presence of chitin or chitin degradation products. Conversely, BcchiB expression was not suppressed by glucose and was not stimulated by chitin or chitin degradation products. The difference in expression regulation is indicative of a functional divergence between the two chitinase paralogous genes.