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.     

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.     

Overview of chitin metabolism enzymes in Manduca sexta: Identification,domain organization,phylogenetic analysis and gene expression

Chitin is one of the most abundant biomaterials in nature. The biosynthesis and degradation of chitin in insects are complex and dynamically regulated to cope with insect growth and development. Chitin metabolism in insects is known to involve numerous enzymes, including chitin synthases (synthesis of chitin), chitin deacetylases (modification of chitin by deacetylation) and chitinases (degradation of chitin by hydrolysis). In this study, we conducted a genome-wide search and analysis of genes encoding these chitin metabolism enzymes in Manduca sexta. Our analysis confirmed that only two chitin synthases are present in M. sexta as in most other arthropods. Eleven chitin deacetylases (encoded by nine genes) were identified, with at least one representative in each of the five phylogenetic groups that have been described for chitin deacetylases to date. Eleven genes encoding for family 18 chitinases (GH18) were found in the M. sexta genome. Based on the presence of conserved sequence motifs in the catalytic sequences and phylogenetic relationships, two of the M. sexta chitinases did not cluster with any of the current eight phylogenetic groups of chitinases: two new groups were created (groups IX and X) and their characteristics are described. The result of the analysis of the Lepidoptera-specific chitinase-h (group h) is consistent with its proposed bacterial origin. By analyzing chitinases from fourteen species that belong to seven different phylogenetic groups, we reveal that the chitinase genes appear to have evolved sequentially in the arthropod lineage to achieve the current high level of diversity observed in M. sexta. Based on the sequence conservation of the catalytic domains and on their developmental stage- and tissue-specific expression, we propose putative functions for each group in each category of enzymes.     

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.     

A novel chitinase from the earthworm,Eisenia andrei

A novel chitinase gene, EaChi, and its expression pattern from the earthworm, E. andrei are demonstrated. Based on a deduced amino acid sequence, in EaChi, two specific domains for GH family 18 are well conserved with two essential amino acid residues for enzyme activity. The phylogenetic analysis shows that earthworm chitinase, EaChi, is evolutionarily close to other lophotrochozoan chitinases. The expression pattern analysis of EaChi indicates that the major expression is localized at intestinal epithelium and epidermis, possibly suggesting that the prime functions of the chitinase activity could be related to not only digestive process but also self-defending immunity as a biochemical barrier to protect the invasion of chitin-containing pathogens, including fungi, nematodes and protozoa.     

细菌几丁质酶结构、功能及分子设计的研究进展

几丁质是仅次于纤维素的第二大天然多糖，由N-乙酰-D-氨基葡萄糖聚合而成，具有重要的应用价值。自然界中几丁质可被细菌高效降解。细菌可分泌多种几丁质降解酶类，主要分布在GH18家族和GH19家族中。细菌中几丁质降解酶基因存在明显的基因扩增及多结构域组合现象，不同家族、不同作用模式的几丁质酶系协同作用打破复杂的抗降解屏障，完成结晶几丁质的高效降解。因此，深入分析细菌几丁质酶结构与功能，对几丁质高效降解与高值转化应用具有重要意义。本文介绍了细菌几丁质酶的分类、结构特点与催化作用机制；总结了不同细菌胞外几丁质降解酶系的协同降解模式；针对几丁质酶家族分子改造的研究进展，展望了以结构生物信息学及大数据深度学习为基础的蛋白质工程设计策略在今后改造中的作用，为几丁质酶的设计与理性改造提供新的视角与思路。     

Molecular and functional evolution of class I chitinases for plant carnivory in the caryophyllales

Proteins produced by the large and diverse chitinase gene family are involved in the hydrolyzation of glycosidic bonds in chitin, a polymer of N-acetylglucosamines. In flowering plants, class I chitinases are important pathogenesis-related proteins, functioning in the determent of herbivory and pathogen attack by acting on insect exoskeletons and fungal cell walls. Within the carnivorous plants, two subclasses of class I chitinases have been identified to play a role in the digestion of prey. Members of these two subclasses, depending on the presence or absence of a C-terminal extension, can be secreted from specialized digestive glands found within the morphologically diverse traps that develop from carnivorous plant leaves. The degree of homology among carnivorous plant class I chitinases and the method by which these enzymes have been adapted for the carnivorous habit has yet to be elucidated. This study focuses on understanding the evolution of carnivory and chitinase genes in one of the major groups of plants that has evolved the carnivorous habit: the Caryophyllales. We recover novel class I chitinase homologs from species of genera Ancistrocladus, Dionaea, Drosera, Nepenthes, and Triphyophyllum, while also confirming the presence of two subclasses of class I chitinases based upon sequence homology and phylogenetic affinity to class I chitinases available from sequenced angiosperm genomes. We further detect residues under positive selection and reveal substitutions specific to carnivorous plant class I chitinases. These substitutions may confer functional differences as indicated by protein structure homology modeling.     

Members of a New Group of Chitinase-Like Genes are Expressed Preferentially in Cotton Cells with Secondary Walls

Two homologous cotton (Gossypium hirsutum L.) genes, GhCTL1 and GhCTL2, encode members of a new group of chitinase-like proteins (called the GhCTL group) that includes other proteins from two cotton species, Arabidopsis, rice, and pea. Members of the GhCTL group are assigned to family GH19 glycoside hydrolases along with numerous authentic chitinases (http://afmb.cnrs-mrs.fr/CAZY/index.html), but the proteins have novel consensus sequences in two regions that are essential for chitinase activity and that were previously thought to be conserved. Maximum parsimony phylogenetic analyses, as well as Neighbor-Joining distance analyses, of numerous chitinases confirmed that the GhCTL group is distinct. A molecular model of GhCTL2 (based on the three-dimensional structure of a barley chitinase) had changes in the catalytic site that are likely to abolish catalytic activity while retaining potential to bind chitin oligosaccharides. RNA blot analysis showed that members of the GhCTL group had preferential expression during secondary wall deposition in cotton lint fiber. Cotton transformed with a fusion of the GhCTL2 promoter to the beta -d-glucuronidase gene showed preferential reporter gene activity in numerous cells during secondary wall deposition. Together with evidence from other researchers that mutants in an Arabidopsis gene within the GhCTL group are cellulose-deficient with phenotypes indicative of altered primary cell walls, these data suggest that members of the GhCTL group of chitinase-like proteins are essential for cellulose synthesis in primary and secondary cell walls. However, the mechanism by which they act is more likely to involve binding of chitin oligosaccharides than catalysis.     

Garlic (Allium sativum) chitinases: characterization and molecular cloning

Leaves and bulbs of garlic ( Allium sativum L.) contain a chitinase which can be separated into three different isoforms with similar molecular structure and N- terminal amino acid sequence. SDS-PAGE of the alkylated chitinase revealed two distinct polypeptides of 32 and 33 kDa. Induction studies of the chitinase in leaves of garlic plants indicated that not only treatment with ethephon or salicylate and wounding but also a temperature shock strongly increased the enzyme level.
cDNA libraries constructed from poly(A)-rich RNA isolated from young garlic shoots and bulbs were screened for chitinase clones using the cDNA clone CCH4 encoding a basic potato chitinase as a probe. Two different cDNA clones (designated CHITAS 1 and CHITAS 2)of ca 1 000 bp were isolated and their sequences analyzed. The amino acid sequences deduced from both cDNA clones were homologous though not identical to the N-terminal sequences of the mature chitinases. Although both clones encode highly homologous chitinases their sequences definitely differ in that they have different signal peptides and one of them contains a glycine-rich domain. The garlic chitinases are apparently translated from an mRNA of 1200 nucleotides which encodes a proprotein of approximately 32 or 33 kDa for CHITAS 1 and CHITAS 2, respectively. Co-translational removal of the signal peptide will result in a 30 (for CHITAS 1) or 31 kDa (for CHITAS 2) protein with an isoelectric point of 4. 94 (for CHITAS 1) or 6. 12 (for CHITAS 2). Garlic chitinases are encoded by a small gene family as shown by Southern blot analysis of genomic DNA isolated from garlic.
The garlic chitinases show a high degree of sequence homology to the previously isolated chitinases from dicotyledonous as well as monocotyledonous species, indicating that these proteins have been conserved from an evolutionary point of view.     

Evolution of mammalian chitinase(-like) members of family 18 glycosyl hydrolases

Family 18 of glycosyl hydrolases encompasses chitinases and so-called chi-lectins lacking enzymatic activity due to amino acid substitutions in their active site. Both types of proteins widely occur in mammals although these organisms lack endogenous chitin. Their physiological function(s) as well as evolutionary relationships are still largely enigmatic. An overview of all family members is presented and their relationships are described. Molecular phylogenetic analyses suggest that both active chitinases (chitotriosidase and AMCase) result from an early gene duplication event. Further duplication events, followed by mutations leading to loss of chitinase activity, allowed evolution of the chi-lectins. The homologous genes encoding chitinase(-like) proteins are clustered in two distinct loci that display a high degree of synteny among mammals. Despite the shared chromosomal location and high homology, individual genes have evolved independently. Orthologs are more closely related than paralogues, and calculated substitution rate ratios indicate that protein-coding sequences underwent purifying selection. Substantial gene specialization has occurred in time, allowing for tissue-specific expression of pH optimized chitinases and chi-lectins. Finally, several family 18 chitinase-like proteins are present only in certain lineages of mammals, exemplifying recent evolutionary events in the chitinase protein family.     

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     

Evolution of family 18 glycoside hydrolases: diversity, domain structures and phylogenetic relationships

Chitin and its derivates have many industrial and medical uses. There is a demand for chitin-modifying enzymes with new or modified properties and as microorganisms are the primary degraders of chitin in the environment, they provide a source of chitin-modifying enzymes with novel properties. We have analyzed the diversity, domain structure and phylogenetic relationships between family 18 chitinases based on complete genome sequences of bacteria, archaea, viruses, fungi, plants and animals. Our study shows that family 18 chitinases are divided into three main clusters, A, B and C. Clusters A and B both contain family 18 chitinases from bacteria, fungi and plants, suggesting that the differentiation of cluster A and B chitinases preceded the appearance of the eukaryotic lineage. Subgroups within clusters can have specific domain structures, as well as specific amino acid replacements in catalytic sites, which imply functional adaptation. This work provides a comprehensive overview of the evolutionary relationships of family 18 chitinases and provides a context for further investigations on functional aspects of family 18 chitinases in ecology and biotechnology.     

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.     

Voltage-gated H+ channels associated with human phagocyte superoxide-generating NADPH oxidases: sequence comparisons,structural predictions,and phylogenetic analyses

The N-terminal domain of the human phagocyte flavocytochrome b558 NADPH oxidase, gp91phox, is believed to be a heme-containing voltage-gated H+ channel. The authors have conducted structural, sequence and phylogenetic analyses of the putative transmembrane channel/heme-binding domains of all homologous proteins in the NCBI GenBank database as of May 2001, as well as of the full-length proteins. Fifty-six homologues were identified, including 26 from animals, 19 from plants, seven from yeast, one from a slime mould and three from bacteria. Six well-defined sub-families were revealed by phylogenetic tree construction, two consisting of animal proteins, two of plant proteins, and one each of yeast and bacterial homologues, with the slime mould protein clustering loosely with one of the animal clusters. Signature sequences for the entire family as well as for the sub-families were determined. Most proteins have six putative TMSs, four of which may comprise the heme-binding H+ channel. The hydrophobic and amphipathic characteristics of each of the putative alpha-helical transmembrane segments were defined, and conserved residues that may be involved in heme binding, channel formation, and/or conformational changes were identified. The analyses lead to the suggestion that the oxidase domain became associated with the channel/heme-binding domain to form a single polypeptide chain early in evolutionary history, before eukaryotes diverged from prokaryotes, and that genetic transmission to present day organisms occurred primarily by vertical descent.     

The GH18 family of chitinases: their domain architectures, functions and evolutions

The glycoside hydrolase 18 (GH18) family of chitinases is a multigene family that plays various roles, such as ecdysis, embryonic development, allergic inflammation and so on. Efforts are still needed to reveal their functional diversification in an evolutionary and systematic manner. We collected 85 GH18 genes from eukaryotic representatives. The domain architectures of GH18 proteins were analyzed and several conserved patterns were identified. It was observed that some (11 proteins) GH18 members in Ecdysozoa or fungi possess repeats of catalytic domains and/or chitin-binding domains (ChtBs). The domain repeats are likely to meet requirements for higher efficiency of chitin degradation in chitin-containing species. On the contrary, all vertebrate GH18 proteins contain no more than one catalytic domain or ChtB. The results from homologous analysis, domain architectures, exon arrangements and synteny loci supported two evolutionary paths for the GH18 family. One path experienced gene expansion and contraction several times during evolution, covering most of GH18 members except CHID1 (stabilin-1 interacting partner) and its homologs. Proteins in this path underwent frequent domain gain and loss, as well as domain recombination, that could achieve versatility in function. The other path is comparatively conserved. The CHID1 gene evolved without gene duplication except in Danio rerio. Domain architectures of CHID1 orthologs are all identical. The diverse phylogeny of the GH18 family in arthropod is also presented.