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: 3.15). 07/2000; 1(1):105-24. DOI: 10.2174/1389203003381450
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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- "Recombinant human SLLP1 (hSLLP1) was prepared by the same protocol of protein production and purification for mSLLP1 as described above. The bindings of recombinant mSLLP1, recombinant hSLLP1 and chicken lysozyme (Sigma, St. Louis, Missouri, USA) to chitin were compared (in a qualitative and not quantitative manner) by applying each protein to a column gravity-packed with crab chitin powder (Sigma) (Fukamizo, 2000). The column was attached to an AKTA FPLC system and pre-equilibrated with 200 mM NaCl, 50 mM Tris pH 8.5. "
ABSTRACT: Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization. © 2015 American Society of Andrology and European Academy of Andrology.Andrology 07/2015; 3(4):756-71. DOI:10.1111/andr.12057 · 2.30 Impact Factor
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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: 126.96.36.199), which is responsible for the last step of chitin polymer formation (Glaser and Brown, 1957a, b; Kramer and Muthukrishnan, 2005; Merzendorfer and Zimoch, 2003). "
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 · 3.45 Impact Factor
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- "Chitin can be degraded by chitinases, which are enzymes that are generally divided into two categories: endochitinases and exochitinases211. Endochitinases cleave chitin polymers at random internal sites, whereas exochitinases progressively cleave chitin beginning at the non-reducing end of the chitin chain, releasing N-acetyl-D-glucosamine monomers and diacetyl-chitobiose in the process12. "
ABSTRACT: The mature cDNA of endochitinase from Trichoderma viride sp. was optimised based on the codon bias of Pichia pastoris GS115 and synthesised by successive PCR; the sequence was then transformed into P. pastoris GS115 via electroporation. The transformant with the fastest growth rate on YPD plates containing 4 mg/mL G418 was screened and identified. This transformant produced 23.09 U/mL of the recombinant endochitinase, a 35% increase compared to the original strain bearing the wild-type endochitinase cDNA. The recombinant endochitinase was sequentially purified by ammonia sulphate precipitation, DE-52 anion-exchange chromatography and Sephadex G-100 size-exclusion chromatography. Thin-layer chromatography indicated that the purified endochitinase could hydrolyse chito-oligomers or colloidal chitin to generate diacetyl-chitobiose (GlcNAc)2 as the main product. This study demonstrates (1) a means for high expression of Trichoderma viride sp. endochitinase in P. pastoris using codon optimisation and (2) the preparation of chito-oligomers using endochitinase.Scientific Reports 10/2013; 3:3043. DOI:10.1038/srep03043 · 5.58 Impact Factor
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