NORMAL GENOMIC

• Dey T, Basu R, Ghosh SK
• Entamoeba invadens: cloning and molecular characterization of chitinases.
• Exp Parasitol. 2009; 123: 244-9
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• Entamoeba histolytica, the causative agent of amebiasis infects throughits cyst form and this transmission may be blocked using encystationspecific protein as drug target. In this study, we have characterized theenzyme chitinase which express specifically during encystation. Thereptilian parasite Entamoeba invadens, used as a model for encystationstudy contain three chitinases. We report the molecular cloning,over-expression and biochemical characterization of all three E. invadenschitinase. Cloned chitinases were over-expressed in bacterial system andpurified by affinity chromatography. Their enzymatic profiles andsubstrate cleaving patterns were characterized. All of them showed bindingaffinity towards insoluble chitin though two of them lack the chitinbinding domain. All the chitinases cleaved and released dimmers from theinsoluble substrate and act as an exochitinase. Homology modeling was alsodone to understand the substrate binding and cleavage pattern.

• Maddi A, Bowman SM, Free SJ
• Trifluoromethanesulfonic acid-based proteomic analysis of cell wall andsecreted proteins of the ascomycetous fungi Neurospora crassa and Candidaalbicans.
• Fungal Genet Biol. 2009; 46: 768-81
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• Cell wall proteins from purified Candida albicans and Neurospora crassacell walls were released using trifluoromethanesulfonic acid (TFMS) whichcleaves the cell wall glucan/chitin matrix and deglycosylates theproteins. The cell wall proteins were then characterized by SDS-PAGE andidentified by proteomic analysis. The analyses for C. albicans identified15 cell wall proteins and six secreted proteins. For N. crassa, theanalyses identified 26 cell wall proteins and nine secreted proteins. Mostof the C. albicans cell wall proteins are found in the cell walls of bothyeast and hyphae cells, but some cell type-specific cell wall proteinswere observed. The analyses showed that the pattern of cell wall proteinspresent in N. crassa vegetative hyphae and conidia (asexual spores) arequite different. Almost all of the cell wall proteins identified in N.crassa have close homologs in the sequenced fungal genomes, suggestingthat these proteins have important conserved functions within the cellwall.

• Pantoom S, Songsiriritthigul C, Suginta W
• The effects of the surface-exposed residues on the binding and hydrolyticactivities of Vibrio carchariae chitinase A.
• BMC Biochem. 2008; 9: 2-2
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• BACKGROUND: Vibrio carchariae chitinase A (EC3.2.1.14) is a family-18glycosyl hydrolase and comprises three distinct structural domains: i) theamino terminal chitin binding domain (ChBD); ii) the (alpha/beta)8 TIMbarrel catalytic domain (CatD); and iii) the alpha + beta insertiondomain. The predicted tertiary structure of V. carchariae chitinase A haslocated the residues Ser33 & Trp70 at the end of ChBD and Trp231 & Tyr245at the exterior of the catalytic cleft. These residues are surface-exposedand presumably play an important role in chitin hydrolysis. RESULTS: Pointmutations of the target residues of V. carchariae chitinase A weregenerated by site-directed mutagenesis. With respect to their bindingactivity towards crystalline alpha-chitin and colloidal chitin, chitinbinding assays demonstrated a considerable decrease for mutants W70A andY245W, and a notable increase for S33W and W231A. When the specifichydrolyzing activity was determined, mutant W231A displayed reducedhydrolytic activity, whilst Y245W showed enhanced activity. This suggestedthat an alteration in the hydrolytic activity was not correlated with achange in the ability of the enzyme to bind to chitin polymer. A mutationof Trp70 to Ala caused the most severe loss in both the binding andhydrolytic activities, which suggested that it is essential forcrystalline chitin binding and hydrolysis. Mutations varied neither thespecific hydrolyzing activity against pNP-[GlcNAc]2, nor the catalyticefficiency against chitohexaose, implying that the mutated residues arenot important in oligosaccharide hydrolysis. CONCLUSION: Our data providedirect evidence that the binding as well as hydrolytic activities of V.carchariae chitinase A to insoluble chitin are greatly influenced by Trp70and less influenced by Ser33. Though Trp231 and Tyr245 are involved inchitin hydrolysis, they do not play a major role in the binding process ofcrystalline chitin and the guidance of the chitin chain into the substratebinding cleft of the enzyme.

• Vandevenne M et al.
• The Bacillus licheniformis BlaP beta-lactamase as a model protein scaffoldto study the insertion of protein fragments.
• Protein Sci. 2007; 16: 2260-71
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• Using genetic engineering technologies, the chitin-binding domain (ChBD)of the human macrophage chitotriosidase has been inserted into the hostprotein BlaP, a class A beta-lactamase produced by Bacillus licheniformis.The product of this construction behaved as a soluble chimeric proteinthat conserves both the capacity to bind chitin and to hydrolyzebeta-lactam moiety. Here we describe the biochemical and biophysicalproperties of this protein (BlaPChBD). This work contributes to a betterunderstanding of the reciprocal structural and functional effects of theinsertion on the host protein scaffold and the heterologous structuredprotein fragments. The use of BlaP as a protein carrier represents anefficient approach to the functional study of heterologous proteinfragments.

• Akagi K et al.
• Identification of the substrate interaction region of the chitin-bindingdomain of Streptomyces griseus chitinase C.
• J Biochem. 2006; 139: 483-93
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• Chitinase C from Streptomyces griseus HUT6037 was discovered as the firstbacterial chitinase in family 19 other than chitinases found in higherplants. Chitinase C comprises two domains: a chitin-binding domain(ChBD(ChiC)) for attachment to chitin and a chitin-catalytic domain fordigesting chitin. The structure of ChBD(ChiC) was determined by means of13C-, 15N-, and 1H-resonance nuclear magnetic resonance (NMR)spectroscopy. The conformation of its backbone comprised two beta-sheetscomposed of two and three antiparallel beta-strands, respectively, thisbeing very similar to the backbone conformations of the cellulose-bindingdomain of endoglucanase Z from Erwinia chrysanthemi (CBD(EGZ)) and thechitin-binding domain of chitinase A1 from Bacillus circulans WL-12(ChBD(ChiA1)). The interaction between ChBD(ChiC) andhexa-N-acetyl-chitohexaose was monitored through chemical shiftperturbations, which showed that ChBD(ChiC) interacted with the substratethrough two aromatic rings exposed to the solvent as CBD(EGZ) interactswith cellulose through three characteristic aromatic rings. Comparison ofthe conformations of ChBD(ChiA1), ChBD(ChiC), and other typical chitin-and cellulose-binding domains, which have three solvent-exposed aromaticresidues responsible for binding to polysaccharides, has suggested thatthey have adopted versatile binding site conformations depending on thesubstrates, with almost the same backbone conformations being retained.

• Andronopoulou E, Vorgias CE
• Multiple components and induction mechanism of the chitinolytic system ofthe hyperthermophilic archaeon Thermococcus chitonophagus.
• Appl Microbiol Biotechnol. 2004; 65: 694-702
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• Thermococcus chitonophagus produces several, cellular and extracellularchitinolytic enzymes following induction with various types of chitin andchitin oligomers, as well as cellulose. Factors affecting the anaerobicculture of this archaeon, such as optimal temperature, agitation speed andtype of chitin, were investigated. A series of chitinases, co-isolatedwith the major, cell membrane-associated endochitinase (Chi70), and aperiplasmic chitobiase (Chi90) were subsequently isolated. In addition, adistinct chitinolytic activity was detected in the culture supernatant andpartially purified. This enzyme exhibited an apparent molecular mass of 50kDa (Chi50) and was optimally active at 80 degrees C and pH 6.0. Chi50 wasclassified as an exochitinase based on its ability to release chitobioseas the exclusive hydrolysis product of colloidal chitin. A multi-componentenzymatic apparatus, consisting of an extracellular exochitinase (Chi50),a periplasmic chitobiase (Chi90) and at least one cell-membrane-anchoredendochitinase (Chi70), seems to be sufficient for effective synergistic invivo degradation of chitin. Induction with chitin stimulates thecoordinated expression of a combination of chitinolytic enzymes exhibitingdifferent specificities for polymeric chitin and its degradation products.Among all investigated potential inducers and nutrient substrates,colloidal chitin was the strongest inducer of chitinase synthesis, whereasthe highest growth rate was obtained following the addition of yeastextract and/or peptone to the minimal, mineralic culture medium in theabsence of chitin. In rich medium, chitin monomer acted as a repressor oftotal chitinolytic activity, indicating the presence of a negativefeedback regulatory mechanism. Despite the undisputable fact that themulti-component chitinolytic system of this archaeon is strongly inducedby chitin, it is clear that, even in the absence of any chitinoussubstrates, there is low-level, basal, constitutive production ofchitinolytic enzymes, which can be attributed to the presence of traces ofchito-oligosaccharides and other structurally related molecules (in theundefined, rich, non-inducing medium) that act as potential inducers ofchitinolytic activity. The low, basal and constitutive levels of chitinasegene expression may be sufficient to initiate chitin degradation and torelease soluble oligomers, which, in turn, induce chitinase synthesis.

• Arakane Y, Zhu Q, Matsumiya M, Muthukrishnan S, Kramer KJ
• Properties of catalytic, linker and chitin-binding domains of insectchitinase.
• Insect Biochem Mol Biol. 2003; 33: 631-48
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• Manduca sexta (tobacco hornworm) chitinase is a glycoprotein that consistsof an N-terminal catalytic domain, a Ser/Thr-rich linker region, and aC-terminal chitin-binding domain. To delineate the properties of thesedomains, we have generated truncated forms of chitinase, which wereexpressed in insect cells using baculovirus vectors. Three additionalrecombinant proteins composed of the catalytic domain fused with one ortwo insect or plant chitin-binding domains (CBDs) were also generated andcharacterized. The catalytic and chitin-binding activities are independentof each other because each activity is functional separately. Whenattached to the catalytic domain, the CBD enhanced activity toward theinsoluble polymer but not the soluble chitin oligosaccharide primarilythrough an effect on the Km for the former substrate. The linker region,which connects the two domains, facilitates secretion from the cell andhelps to stabilize the enzyme in the presence of gut proteolytic enzymes.The linker region is extensively modified by O-glycosylation and thecatalytic domain is moderately N-glycosylated. Immunological studiesindicated that the linker region, along with elements of the CBD, is amajor immunogenic epitope. The results support the hypothesis that thedomain structure of insect chitinase evolved for efficient degradation ofthe insoluble polysaccharide to soluble oligosaccharides during themolting process.

• Kobayashi DY, Reedy RM, Bick J, Oudemans PV
• Characterization of a chitinase gene from Stenotrophomonas maltophiliastrain 34S1 and its involvement in biological control.
• Appl Environ Microbiol. 2002; 68: 1047-54
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• A chitinase gene was cloned on a 2.8-kb DNA fragment from Stenotrophomonasmaltophilia strain 34S1 by heterologous expression in Burkholderiacepacia. Sequence analysis of this fragment identified an open readingframe encoding a deduced protein of 700 amino acids. Removal of the signalpeptide sequence resulted in a predicted protein that was 68 kDa in size.Analysis of the sequence indicated that the chitinase contained acatalytic domain belonging to family 18 of glycosyl hydrolases. Threeputative binding domains, a chitin binding domain, a novel polycystickidney disease (PKD) domain, and a fibronectin type III domain, were alsoidentified within the sequence. Pairwise comparisons of each domain to themost closely related sequences found in database searches clearlydemonstrated variation in gene sources and the species from which relatedsequences originated. A 51-kDa protein with chitinolytic activity waspurified from culture filtrates of S. maltophilia strain 34S1 byhydrophobic interaction chromatography. Although the protein wassignificantly smaller than the size predicted from the sequence, theN-terminal sequence verified that the first 15 amino acids were identicalto the deduced sequence of the mature protein encoded by chiA. Markerexchange mutagenesis of chiA resulted in mutant strain C5, which wasdevoid of chitinolytic activity and lacked the 51-kDa protein in culturefiltrates. Strain C5 was also reduced in the ability to suppress summerpatch disease on Kentucky bluegrass, supporting a role for the enzyme inthe biocontrol activity of S. maltophilia.

• Watanabe T et al.
• Trp122 and Trp134 on the surface of the catalytic domain are essential forcrystalline chitin hydrolysis by Bacillus circulans chitinase A1.
• FEBS Lett. 2001; 494: 74-8
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• From the 3D-structural analysis of the catalytic domain of chitinase A1,two exposed tryptophan residues (W122 and W134) are proposed to play animportant role in guiding a chitin chain into the catalytic cleft duringthe crystalline chitin hydrolysis. Mutation of either W122 or W134 toalanine significantly reduced the hydrolyzing activity against highlycrystalline beta-chitin microfibrils. Double mutation almost completelyabolished the hydrolyzing activity. On the other hand, the hydrolyzingactivity against either soluble or amorphous substrate was not reduced.These mutations slightly impaired the binding activity of this enzyme.These results clearly demonstrated that the two exposed aromatic residuesplay a critical role in hydrolyzing the chitin chain in crystallinechitin.

• Zakowska Z, Gabara B, Kusewicz D
• Cell wall analysis in Aspergillus niger strains characterized by differenttolerance to toxic compounds of beet molasses.
• Acta Microbiol Pol. 1997; 46: 27-36
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• Aspergillus niger strains, sensitive or resistant to toxic compounds ofbeet molasses, were the object of the present studies. Between the studiedstrains differences existed in the cell wall dry mass and wall componentscontent, chitin synthesis, activity of enzymes involved in cell wallsynthesis, and in the wall ultrastructure. Higher content of proteins andlipids but lower of glucan and chitin; less or lack of fibrillarcomponents, thinner cell wall as well as lower level of glucanase andchitinase, diminished [3H] glucosamine incorporation into cytoplasm andcell wall characterized the sensitive strains of A. niger.

• Hanzlikova A, Jandera A
• Chitinase and changes of microbial community in soil.
• Folia Microbiol (Praha). 1993; 38: 159-60
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• Enrichment of soil with chitin (0.6%) significantly stimulated growth ofchitinolytic microorganisms (the relative proportion was increased from1.7 to 26.5%) and the formation of chitinase in soil. In a soil enrichedwith chitin and glucose (0.6%), the proportion of chitinolyticmicroorganisms remained similar to that in the nonenriched soil (1.4%),the enzyme formation was negatively affected.

• Barrett-Bee K, Hamilton M
• The detection and analysis of chitinase activity from the yeast form ofCandida albicans.
• J Gen Microbiol. 1984; 130: 1857-61
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• Chitinase activity was detected in the supernatant fraction of ahigh-speed centrifugation preparation of broken Candida albicans yeastcells. The enzyme showed peak activity during the rapid budding phase ofgrowth and was found to parallel the chitin synthase activity. The optimumconditions for the hydrolysis of chitin, regenerated from acetylation ofchitosan, were determined. Analysis of the kinetics of theenzyme-substrate interaction and a measurement of their binding suggeststhat an equilibrium binding situation exists and that the kinetics followa Langmuir isotherm interaction.