Chitinolytic Enzymes: Catalysis, Substrate Binding, and their Application

Laboratory of Biophysical Chemistry, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan.
Current Protein and Peptide Science (Impact Factor: 2.33). 07/2000; 1(1):105-24. DOI: 10.2174/1389203003381450
Source: PubMed

ABSTRACT After the epoch-making report on X-ray crystal structure of a lysozyme-N-acetylglucosamine trisaccharide complex in 1967, catalytic mechanisms of glycosyl hydrolases have been discussed with reference to the lysozyme mechanism. From the recent findings of chitinolytic enzymes, however, the enzymes were found to have catalytic and substrate binding mechanisms different from those of lysozyme. Based on the X-ray crystal structures of chitinases and their complexes with substrate analogues, the catalytic mechanisms were discussed considering the relative locations of catalytic residues to the bound substrate analogues. Resembling the lysozyme catalytic center, family 19 chitinases, family 46 chitosanases, and family 23 lysozymes have two carboxyl groups at the catalytic center, which are separated (> 10 +) on either side of the catalytic cleft. The catalytic reaction of the enzymes takes place through a single displacement mechanism. In family 18 chitinases, one can identify only one catalytic carboxylate as a proton donor, but not the second catalytic carboxylate whose function and location are similar to those of Asp52 in lysozyme. The catalytic reaction of family 18 chitinases is most likely to take place through a substrate-assisted mechanism. Hen egg white lysozyme has the binding cleft represented by (-4)(-3)(-2)(-1)(+1)(+2). The binding cleft of family 19 chitinases, family 46 chitosanases, and family 23 lysozymes, however, is represented by (-3)(-2)(-1)(+1)(+2)(+3). Molecular dynamics calculation suggests that family 18 chitinases have the binding cleft, (-4)(-3)(-2)(-1)(+1)(+2). The functional diversity of the chitinolytic enzymes might be related to different physiological functions of the enzymes. The enzymes are now being applied to plant protection from fungal pathogens and insect pests. Structure of the targeted chitinous component was determined by a combination of enzyme digestion and solid state CP/MAS NMR spectroscopy, and have been taken into consideration for efficient application of the enzymes. Recent understanding of the catalytic and substrate binding mechanisms would be helpful as well for arrangement of a powerful strategy in such an application.

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    • "The new cuticle is sclerotized, acquiring hardness and tan color characteristics (Kramer and Muthukrishnan, 2005). The molting cycle is achieved through chitin degradation enzymes, such as chitinases and b-N-acetylglucosaminidases (Fukamizo, 2000; Kramer et al., 1985; Kramer and Koga, 1986; Wilson and Cryan, 1997), and enzymes involved in chitin synthesis, such as CHS (EC:, which is responsible for the last step of chitin polymer formation (Glaser and Brown, 1957a, b; Kramer and Muthukrishnan, 2005; Merzendorfer and Zimoch, 2003). "
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    ABSTRACT: In this study, we provided the demonstration of the presence of a single CHS gene in the Rhodnius prolixus (a blood-sucking insect) non-annotated genome that is expressed in adults (integument and ovary) and in the integument of nymphs during development. This CHS gene appears to be essential for epidermal integrity and egg formation in R. prolixus. Because injection of CHS dsRNA was effective in reducing CHS transcript levels, phenotypic alterations in the normal course of ecdysis occurred. In addition, two phenotypes with severe cuticle deformations were observed, which were associated with loss of mobility and lifetime. The CHS dsRNA treatment in adult females affected oogenesis, reducing the size of the ovary and presenting a greater number of atresic oocytes and a smaller number of chorionated oocytes compared with the control. The overall effect was reduced oviposition. The injection of CHS dsRNA modified the natural course of egg development, producing deformed eggs that were dark in color and unable to hatch, distinct from the viable eggs laid by control females. The ovaries, which were examined under fluorescence microscopy using a probe for chitin detection, showed a reduced deposition on pre-vitellogenic and vitellogenic oocytes compared with control. Taken together, these data suggest that the CHS gene is fundamentally important for ecdysis, oogenesis and egg hatching in R. prolixus and also demonstrated that the CHS gene is a good target for controlling Chagas disease vectors.
    Insect biochemistry and molecular biology 01/2014; 51(1). DOI:10.1016/j.ibmb.2013.12.006 · 3.42 Impact Factor
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    • "In nature degradation of chitin is a slow process and involves a cascade of pathways. Chitinases are reported in the moulting fluid of arthropods and are mainly used to digest the structural polysaccharides in their gut linings and exoskeleton during moulting (Karmer et al. 1985; Fukamizo & Kramer 1985; Karmer & Muthukrishnan 1997; Fukamizo 2000). The present study aimed at understanding the influence of chitinase enzyme on the degradation of nauplii of barnacle, Balanus amphitrite Darwin, 1854 at different time interval . "
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    ABSTRACT: The exoskeleton of most invertebrate larval forms is made of chitin, which is a linear polysaccharide of β (1→4)-linked N-acetylglucosamine (GlcNAc) residues. These larval forms offer extensive body surface for bacterial attachment and colonization. In nature, degradation of chitin involves a cascade of processes brought about by chitinases produced by specific bacteria in the marine environment. Microbial decomposition of larval carcasses serves as an alternate mechanism for nutrient regeneration, elemental cycling and microbial production. The present study was undertaken to assess the influence of chitinase enzyme on the degradation of the nauplii of barnacle, Balanus amphitrite. The survival and abundance of bacteria during the degradation process under different experimental conditions was monitored. To the best of our knowledge, no such study is conducted to understand the degradation of larval exoskeleton using chitinase and its influence on bacteria. An increase in the chitinase activity with increase in temperature was observed. Scanning electron micrographs of chitinase treated nauplii showed scars on the surface of the barnacle nauplii initially and further disruption of the exoskeleton was observed with the increase in the treatment time. Bacterial abundance of the chitinase treated nauplii increased with the increase in enzyme concentration. Pathogenic bacteria such as Vibrio cholerae, V. alginolyticus, V. parahaemolyticus which were initially associated with the exoskeleton were absent after chitinase treatment, however, Bacillus spp. dominated subsequent to chitinase treatment and this might have important implications to marine ecosystem functioning.
    Biologia 08/2013; 68(4). DOI:10.2478/s11756-013-0202-6 · 0.70 Impact Factor
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    • "Based on amino acid sequence similarity of the catalytic domain, chitinases are classified into family-18 and -19 glycosyl hydrolases in the Carbohydrate-Active enZymes CAZy databases ( (Henrissat and Davies, 1997; Fukamizo, 2000). The chitinase 18 family is found in a wide variety of organisms, including bacteria, archaea, fungi, some plants, insects, animals as well as viruses. "
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    ABSTRACT: The small and large intestines contain an abundance of luminal antigens derived from food products and enteric microorganisms. The function of intestinal epithelial cells is tightly regulated by several factors produced by enteric bacteria and the epithelial cells themselves. Epithelial cells actively participate in regulating the homeostasis of intestine, and failure of this function leads to abnormal and host-microbial interactions resulting in the development of intestinal inflammation. Major determinants of host susceptibility against luminal commensal bacteria include genes regulating mucosal immune responses, intestinal barrier function and microbial defense. Of note, it has been postulated that commensal bacterial adhesion and invasion on/into host cells may be strongly involved in the pathogenesis of inflammatory bowel disease (IBD). During the intestinal inflammation, the composition of the commensal flora is altered, with increased population of aggressive and detrimental bacteria and decreased populations of protective bacteria. In fact, some pathogenic bacteria, including Adherent-Invasive Escherichia coli, Listeria monocytogenes and Vibrio cholerae are likely to initiate their adhesion to the host cells by expressing accessory molecules such as chitinases and/or chitin-binding proteins on themselves. In addition, several inducible molecules (e.g., chitinase 3-like 1, CEACAM6) are also induced on the host cells (e.g. epithelial cells, lamina proprial macrophages) under inflammatory conditions, and are actively participated in the host-microbial interactions. In this review, we will summarize and discuss the potential roles of these important molecules during the development of acute and chronic inflammatory conditions.
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