Mutational Analysis of a CBM Family 5 Chitin-Binding Domain of an Alkaline Chitinase from Bacillus sp. J813

Chitinase J from alkaliphilic Bacillus sp. J813 comprises a glycoside hydrolase (GH) family 18 catalytic domain (CatD), a fibronectin type III like domain, and a carbohydrate-binding module (CBM) family 5 chitin-binding domain (ChBD). It has been suggested that the ChBD binds to insoluble chitin and enhances its degradation by the CatD. To investigate the roles of two aromatic residues (Trp541 and Trp542), which are exposed on the surface of the ChBD, mutational analysis was performed. Single and double mutations of the two aromatic residues decreased binding and hydrolyzing abilities toward insoluble chitin. This result suggests that the ChBD binds to chitin by hydrophobic interactions via two surface-exposed aromatic residues. However, the double mutant, which has no such aromatic residue, bound to chitin at pH 5.2, probably by electrostatic interactions. Moreover, the ChBD bound to insoluble chitosan by electrostatic interactions.     

Structural studies of a two-domain chitinase from Streptomyces griseus HUT6037

Chitinase C (ChiC) from Streptomyces griseus HUT6037 was the first glycoside hydrolase family 19 chitinase that was found in an organism other than higher plants. An N-terminal chitin-binding domain and a C-terminal catalytic domain connected by a linker peptide constitute ChiC. We determined the crystal structure of full-length ChiC, which is the only representative of the two-domain chitinases in the family. The catalytic domain has an alpha-helix-rich fold with a deep cleft containing a catalytic site, and lacks three loops on the domain surface compared with the catalytic domain of plant chitinases. The chitin-binding domain is an all-beta protein with two tryptophan residues (Trp59 and Trp60) aligned on the surface. We suggest the binding mechanism of tri-N-acetylchitotriose onto the chitin-binding domain on the basis of molecular dynamics (MD) simulations. In this mechanism, the ligand molecule binds well on the surface-exposed binding site through two stacking interactions and two hydrogen bonds and only Trp59 and Trp60 are involved in the binding. Furthermore, the flexibility of the Trp60 side-chain, which may be involved in adjusting the binding surface to fit the surface of crystalline chitin by the rotation of chi2 angle, is shown.     

Solution structure of the chitin-binding domain of Bacillus circulans WL-12 chitinase A1

The three-dimensional structure of the chitin-binding domain (ChBD) of chitinase A1 (ChiA1) from a Gram-positive bacterium, Bacillus circulans WL-12, was determined by means of multidimensional heteronuclear NMR methods. ChiA1 is a glycosidase that hydrolyzes chitin and is composed of an N-terminal catalytic domain, two fibronectin type III-like domains, and C-terminal ChBD(ChiA1) (45 residues, Ala(655)-Gln(699)), which binds specifically to insoluble chitin. ChBD(ChiA1) has a compact and globular structure with the topology of a twisted beta-sandwich. This domain contains two antiparallel beta-sheets, one composed of three strands and the other of two strands. The core region formed by the hydrophobic and aromatic residues makes the overall structure rigid and compact. The overall topology of ChBD(ChiA1) is similar to that of the cellulose-binding domain (CBD) of Erwinia chrysanthemi endoglucanase Z (CBD(EGZ)). However, ChBD(ChiA1) lacks the three aromatic residues aligned linearly and exposed to the solvent, which probably interact with cellulose in CBDs. Therefore, the binding mechanism of a group of ChBDs including ChBD(ChiA1) may be different from that proposed for CBDs.     

Mutational analysis of a CBM family 5 chitin-binding domain of an alkaline chitinase from Bacillus sp. J813

Chitinase J from alkaliphilic Bacillus sp. J813 comprises a glycoside hydrolase (GH) family 18 catalytic domain (CatD), a fibronectin type III like domain, and a carbohydrate-binding module (CBM) family 5 chitin-binding domain (ChBD). It has been suggested that the ChBD binds to insoluble chitin and enhances its degradation by the CatD. To investigate the roles of two aromatic residues (Trp541 and Trp542), which are exposed on the surface of the ChBD, mutational analysis was performed. Single and double mutations of the two aromatic residues decreased binding and hydrolyzing abilities toward insoluble chitin. This result suggests that the ChBD binds to chitin by hydrophobic interactions via two surface-exposed aromatic residues. However, the double mutant, which has no such aromatic residue, bound to chitin at pH 5.2, probably by electrostatic interactions. Moreover, the ChBD bound to insoluble chitosan by electrostatic interactions.     

Identification of the substrate interaction region of the chitin-binding domain of Streptomyces griseus chitinase C

Chitinase C from Streptomyces griseus HUT6037 was discovered as the first bacterial chitinase in family 19 other than chitinases found in higher plants. Chitinase C comprises two domains: a chitin-binding domain (ChBD(ChiC)) for attachment to chitin and a chitin-catalytic domain for digesting chitin. The structure of ChBD(ChiC) was determined by means of 13C-, 15N-, and 1H-resonance nuclear magnetic resonance (NMR) spectroscopy. The conformation of its backbone comprised two beta-sheets composed of two and three antiparallel beta-strands, respectively, this being very similar to the backbone conformations of the cellulose-binding domain of endoglucanase Z from Erwinia chrysanthemi (CBD(EGZ)) and the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12 (ChBD(ChiA1)). The interaction between ChBD(ChiC) and hexa-N-acetyl-chitohexaose was monitored through chemical shift perturbations, which showed that ChBD(ChiC) interacted with the substrate through two aromatic rings exposed to the solvent as CBD(EGZ) interacts with cellulose through three characteristic aromatic rings. Comparison of the conformations of ChBD(ChiA1), ChBD(ChiC), and other typical chitin- and cellulose-binding domains, which have three solvent-exposed aromatic residues responsible for binding to polysaccharides, has suggested that they have adopted versatile binding site conformations depending on the substrates, with almost the same backbone conformations being retained.     

Expression and characterization of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12

Chitinase A1 from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III-like domains, and a C-terminal chitin-binding domain (ChBD). In order to study the biochemical properties and structure of the ChBD, ChBD(ChiA1) was produced in Escherichia coli using a pET expression system and purified by chitin affinity column chromatography. Purified ChBD(ChiA1) specifically bound to various forms of insoluble chitin but not to other polysaccharides, including chitosan, cellulose, and starch. Interaction of soluble chitinous substrates with ChBD(ChiA1) was not detected by means of nuclear magnetic resonance and isothermal titration calorimetry. In addition, the presence of soluble substrates did not interfere with the binding of ChBD(ChiA1) to regenerated chitin. These observations suggest that ChBD(ChiA1) recognizes a structure which is present in insoluble or crystalline chitin but not in chito-oligosaccharides or in soluble derivatives of chitin. ChBD(ChiA1) exhibited binding activity over a wide range of pHs, and the binding activity was enhanced at pHs near its pI and by the presence of NaCl, suggesting that the binding of ChBD(ChiA1) is mediated mainly by hydrophobic interactions. Hydrolysis of beta-chitin microcrystals by intact chitinase A1 and by a deletion derivative lacking the ChBD suggested that the ChBD is not absolutely required for hydrolysis of beta-chitin microcrystals but greatly enhances the efficiency of degradation.     

Mutations of Trp275 and Trp397 altered the binding selectivity of Vibrio carchariae chitinase A

Point mutations of the active-site residues Trp168, Tyr171, Trp275, Trp397, Trp570 and Asp392 were introduced to Vibrio carchariae chitinase A. The modeled 3D structure of the enzyme illustrated that these residues fully occupied the substrate binding cleft and it was found that their mutation greatly reduced the hydrolyzing activity against pNP-[GlcNAc]₂ and colloidal chitin. Mutant W397F was the only exception, as it instead enhanced the hydrolysis of the pNP substrate to 142% and gave no activity loss towards colloidal chitin. The kinetic study with the pNP substrate demonstrated that the mutations caused impaired K_m and k_cat values of the enzyme. A chitin binding assay showed that mutations of the aromatic residues did not change the binding equilibrium. Product analysis by thin layer chromatography showed higher efficiency of W275G and W397F in G4–G6 hydrolysis over the wild type enzyme. Though the time course of colloidal chitin hydrolysis displayed no difference in the cleavage behavior of the chitinase variants, the time course of G6 hydrolysis exhibited distinct hydrolytic patterns between wild-type and mutants W275G and W397F. Wild type initially hydrolyzed G6 to G4 and G2, and finally G2 was formed as the major end product. W275G primarily created G2–G5 intermediates, and later G2 and G3 were formed as stable products. In contrast, W397F initially produced G1–G5, and then the high-M_r intermediates (G3–G5) were broken down to G1 and G2 end products. This modification of the cleavage patterns of chitooligomers suggested that residues Trp275 and Trp397 are involved in defining the binding selectivity of the enzyme to soluble substrates.     

A single surface tryptophan in the chitin-binding domain from Bacillus circulans chitinase A1 plays a pivotal role in binding chitin and can be modified to create an elutable affinity tag

Site-directed mutagenesis was carried out to investigate the roles of a number of highly conserved residues of the chitin-binding domain (ChBD) of Bacillus circulans chitinase A1 (ChiA1) in the binding of chitin. Analysis of single alanine replacement mutants showed that mutation of an exposed tryptophan residue (Trp(687)) impaired the binding to chitin, while mutation of other highly conserved residues, most carrying aromatic or hydrophobic side chains, did not significantly affect the binding activity. Interestingly, replacement of Trp(687) with phenylalanine significantly reduced chitin-binding activity at lower salt concentrations (0-1 M NaCl) but allowed strong binding to chitin at 2 M NaCl. Since Trp(687) is conserved among the ChBDs belonging to the bacterial ChiA1 subfamily, the data presented suggest a general mechanism in which this exposed tryptophan, which is located in the cleft formed between two beta-sheets as revealed by the solution structure [J. Biol. Chem. 275 (2000) 13654], makes a major contribution to ligand binding presumably through hydrophobic interactions. Furthermore, modulation of the chitin-binding activity by the conserved amino acid replacement (W687F) and a shift in the ionic strength of buffer has led to the development of an elutable affinity tag for single column purification of recombinant proteins.     

Molecular analysis of the gene encoding a new chitinase from the marine psychrophilic bacterium <Emphasis Type="Italic">Moritella marina</Emphasis> and biochemical characterization of the recombinant enzyme

The marine psychrophilic bacterium Moritella marina, isolated from a sample raised from a depth of 1,200 m in the northern Pacific Ocean, secretes several chitinases in response to chitin induction. A gene coding for an extracellular chitinolytic enzyme was cloned and its nucleotide sequence was determined. The chitinase gene consists of an open reading frame of 1,650 nucleotides and encodes a protein of 550 amino acids with a calculated molecular weight of 60.788 kDa, named MmChi60. MmChi60 has a modular structure consisting of a glycosyl-hydrolase family 18 N-terminal catalytic region as well as a C-terminal chitin-binding domain (ChBD). The new chitinase was purified to homogeneity from the intracellular fraction of Escherichia coli. The optimum pH and temperature of the recombinant MmChi60 were 5.0 and 28 degrees C, respectively. The mode of action of the new enzyme on N-acetylchitooligomers, chitin polymers, and other substrates was examined, and MmChi60 was classified as an endochitinase. Thermal unfolding of MmChi60 was studied using differential scanning microcalorimetry and revealed that the protein unfolds reversibly at 65 degrees C. On the basis of the crystal structure of the chitinase C of Streptomyces griseus, a homology-based 3-D model of the ChBD of the MmChi60 was calculated.     

Molecular cloning,sequencing and expression of the gene encoding a novel chitinase A from a marine bacterium, <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. PE2, and its domain structure

The pchA gene encoding chitinase A (PchA) from a Pythium porphyrae cell-wall-degrading marine bacterium, Pseudomonas sp. PE2, was cloned and characterized. The deduced PchA was a modular enzyme composed of an N-terminal signal peptide, a glycoside hydrolase family 18 catalytic domain that was responsible for the chitinase activity, the chitin-binding domains (ChBDs), and the carbohydrate-binding modules (CBM). The amino acid sequence of ChBD(PchA) was highly conserved in the CBM family 12 that also accommodates ChBDs without an AKWWTQG motif, a domain commonly found in bacterial chitinase and Streptomyces griseus protease C. Interestingly, CBM(PchA) showed significant sequence homology to the C-terminal region of endoglucanase B from Cellvibrio mixtus, which is a member of CBM family 6. This is the first report of a chitinase possessing a domain with high similarity to CBM family 6. Deletion analysis indicated clearly that ChBD(PchA) might play an important role in the binding of native chitin and chitosan, but not processed chitin. CBM(PchA) also appeared to play such a role in the binding of xylan and Avicel. These results suggest that the C-terminal region of PchA might be a key component in the binding of chitin in the cell walls of P. porphyrae or other structural components of marine organisms.     

Crystal structure of crotoxin reveals key residues involved in the stability and toxicity of this potent heterodimeric β-neurotoxin

The crystal structure of crotoxin, a potent presynaptic neurotoxin from Crotalus durissusterrificus, was solved at 1.35 Å resolution. It shows the architecture of the three disulfide-linked polypeptide chains (α, β, and γ) of the acidic subunit CA noncovalently complexed with the basic phospholipase A₂ (PLA₂) subunit CB. The unique structural scaffold of the association of the CA and CB subunits indicates that posttranslational cleavage of the pro-CA precursor is a prerequisite for the assembly of the CA-CB complex. These studies provide novel structural insights to explain the role of the CA subunit in the mechanism of action of crotoxin. The crystal structure of the highly toxic and stable CA₂CBb complex crystallized here allows us to identify key amino acid residues responsible for significant differences in the pharmacological activities of the two classes of crotoxin complexes. In particular, we show that critical residues Trp31 and Trp70 of the CBb subunit establish intermolecular polar contacts with Asp99 and Asp89, respectively, of the β-chain of CA₂ and contribute to the stability and toxicity of the CA₂CBb complex. These interactions also lead to decreased PLA₂ activity by partially blocking substrate access to the catalytic dyad and by masking several interfacial binding surface residues important for PLA₂ interaction with phospholipids.Identification of the binding interface between the CA subunits and the CB subunits of crotoxin is important for the structure-based design of antineurotoxic inhibitors. Since crotoxin displays numerous physiological functions, including antitumoral properties, knowledge of its three-dimensional structure will be useful for the understanding of these diverse effects.     

Additional Carbohydrate-Binding Modules Enhance the Insoluble Substrate-Hydrolytic Activity of β-1,3-Glucanase from Alkaliphilic Nocardiopsis sp. F96

β-1,3-Glucanase (BglF) from Nocardiopsis sp. F96 is composed of only a catalytic domain. To improve the enzymatic properties of BglF, we attempted to construct chimeric enzymes consisting of BglF and some carbohydrate-binding modules, such as the C-terminal additional domain (CAD) and the N-terminal additional domain (NAD) of β-1,3-glucanase H from Bacillus circulans IAM1165 and the chitin-binding domain (ChBD) of chitinase from alkaliphilic Bacillus sp. J813. CAD-fused BglF (BglF-CAD), NAD-fused BglF (NAD-BglF), both NAD- and CAD-fused BglF (NAD-BglF-CAD) and ChBD-fused BglF (BglF-ChBD) were constructed and characterized. The addition of CAD caused increases in binding abilities and hydrolytic activities toward insoluble β-1,3-glucans. As well as BglF-CAD, the binding ability and hydrolytic activity of BglF-ChBD toward pachyman were also increased. The hydrolytic activity of BglF-CAD at pH 9–10 was higher than that of BglF. The relative activities of BglF-CAD and BglF-ChBD at around 50–70 °C were higher than that of BglF.     

Molecular cloning and functional expression of chitinase-encoding cDNA from the cabbage moth, Mamestra brassicae

Chitinase is a rate-limiting and endo-splitting enzyme involved in the bio-degradation of chitin, an important component of the cuticular exoskeleton and peritrophic matrix in insects. We isolated a cDNA-encoding chitinase from the last larval integument of the cabbage moth, Mamestra brassicae (Lepidoptera; Noctuidae), cloned the ORF cDNA into E. coli to confirm its functionality, and analyzed the deduced amino acid sequence in comparison with previously described lepidopteran chitinases. M. brassicae chitinase expressed in the transformed E. coli cells with the chitinase-encoding cDNA enhanced cell proliferation to about 1.6 times of the untransformed wild type strain in a colloidal chitin-including medium with only a very limited amount of other nutrients. Compared with the wild type strain, the intracellular levels of chitin degradation derivatives, glucosamine and N-acetylglucosamine were about 7.2 and 2.3 times higher, respectively, while the extracellular chitinase activity was about 2.2 times higher in the transformed strain. The ORF of M. brassicae chitinaseencoding cDNA consisted of 1686 nucleotides (562 amino acid residues) except for the stop codon, and its deduced amino acid composition revealed a calculated molecular weight of 62.7 and theoretical pI of 5.3. The ORF was composed of N-terminal leading signal peptide (AA 1-20), catalytic domain (AA 21-392), linker region (AA 393-498), and C-terminal chitin-binding domain (AA 499-562) showing its characteristic structure as a molting fluid chitinase. In phylogenetic analysis, the enzymes from 6 noctuid species were grouped together, separately from a group of 3 bombycid and 1 tortricid enzymes, corresponding to their taxonomic relationships at both the family and genus levels.     

High-level expression and characterization of two chitinases, ChiCH and ChiCW, of Bacillus cereus 28-9 in Escherichia coli

Many chitinase genes have been cloned and sequenced from prokaryotes and eukaryotes but overexpression of chitinases in Escherichia coli cells was less reported. ChiCH and ChiCW of Bacillus cereus 28-9 belong to two distinct groups based on their amino acid sequences of catalytic domains, and in addition, domain structures of two enzymes are different. In this study, we established an ideal method for high-level expression of chitinases in E. coli as glutathione-S-transferase fusion proteins using pGEX-6P-1 vector. Both ChiCH and ChiCW were successfully highly expressed in E. coli cells as soluble GST-chitinase fusion proteins, and recombinant native ChiCH and ChiCW could be purified after cleavage with PreScission protease to remove GST tag. Purified chitinases were used for biochemical characterization of kinetics, hydrolysis products, and binding activities. The results indicate that ChiCW is an endo-chitinase and effectively hydrolyzes chitin and chito-multimers to chito-oligomers and the end product chitobiose, and ChiCH is an exo-chitinase and degrades chito-oligomers to produce chitobiose. Furthermore, due to higher affinity of ChiCW toward colloidal chitin than Avicel, C-terminal domain of ChiCW should be classified as a chitin-binding domain not a cellulose-binding domain although that was revealed as a cellulose-binding domain by conserved domain analysis. Therefore, the method of high-level expression of chitinases is helpful to studies and applications of chitinases.     

Tobacco-expressed Brassica juncea chitinase BjCHI1 shows antifungal activity in vitro

We have previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains, and have shown that its mRNA is induced by wounding and methyl jasmonate treatment (K.-J. Zhao and M.-L. Chye, Plant Mol. Biol. 40 (1999) 1009–1018). By the presence of two chitin-binding domains, BjCHI1 resembles the precursor of UDA (Urtica dioica agglutinin) but, unlike UDA, BjCHI1 retains its chitinase catalytic domain after post-translational processing. Here, we indicate the role of BjCHI1 in plant defense by demonstrating its mRNA induction upon Aspergillus niger infection or caterpillar Pieris rapae (L.) feeding. To further investigate the biological properties of BjCHI1, we transformed tobacco with a construct expressing the BjCHI1 cDNA from the CaMV 35S promoter. Subsequently, we purified BjCHI1 from the resultant transgenic R₀ plants using a regenerated chitin column followed by fast protein liquid chromatography (FPLC). Also, the significance of the second chitin-binding domain in BjCHI1 was investigated by raising transgenic tobacco plants expressing BjCHI2, a deletion derivative of BjCHI1 lacking one chitin-binding domain. Colorimetric chitinase assays at 25 °C, pH 5, showed no significant differences between the activities of BjCHI1 and BjCHI2, suggesting that chitinase activity, due to the catalytic domain, is not enhanced by the presence of a second chitin-binding domain. Both BjCHI1 and BjCHI2 show in vitro anti-fungal activity toward Trichoderma viride, causing reductions in hyphal diameter, hyphal branching and conidia size.     

Structure and mode of action of a mosquitocidal holotoxin

The crystal structure of the full mosquitocidal toxin from Bacillus sphaericus (MTX_holo) has been determined at 2.5 Å resolution by the molecular replacement method. The resulting structure revealed essentially the complete chain consisting of four ricin B-type domains curling around the catalytic domain in a hedgehog-like assembly. As the structure was virtually identical in three different crystal packings, it is probably not affected by packing contacts. The structure of MTX_holo explains earlier autoinhibition data. An analysis of published complexes comprising ricin B-type lectin domains and sugar molecules shows that the general construction principle applies to all four lectin domains of MTX_holo, indicating 12 putative sugar-binding sites. These sites are sequence-related to those of the cytotoxin pierisin from cabbage butterfly, which are known to bind glycolipids. It seems therefore likely that MTX_holo also binds glycolipids. The seven contact interfaces between the five domains are predominantly polar and not stronger than common crystal contacts so that in an appropriate environment, the multidomain structure would likely uncurl into a string of single domains. The structure of the isolated catalytic domain plus an extended linker was established earlier in three crystal packings, two of which showed a peculiar association around a 7-fold axis. The catalytic domain of the reported MTX_holo closely resembles all three published structures, except one with an appreciable deviation of the 40 N-terminal residues. A comparison of all structures suggests a possible scenario for the translocation of the toxin into the cytosol.     

Crystal structure of Aspergillus niger isopullulanase, a member of glycoside hydrolase family 49

An isopullulanase (IPU) from Aspergillus niger ATCC9642 hydrolyzes α-1,4-glucosidic linkages of pullulan to produce isopanose. Although IPU does not hydrolyze dextran, it is classified into glycoside hydrolase family 49 (GH49), major members of which are dextran-hydrolyzing enzymes. IPU is highly glycosylated, making it difficult to obtain its crystal. We used endoglycosidase H_f to cleave the N-linked oligosaccharides of IPU, and we here determined the unliganded and isopanose-complexed forms of IPU, both solved at 1.7-Å resolution. IPU is composed of domains N and C joined by a short linker, with electron density maps for 11 or 12 N-acetylglucosamine residues per molecule. Domain N consists of 13 β-strands and forms a β-sandwich. Domain C, where the active site is located, forms a right-handed β-helix, and the lengths of the pitches of each coil of the β-helix are similar to those of GH49 dextranase and GH28 polygalacturonase. The entire structure of IPU resembles that of a GH49 enzyme, Penicillium minioluteum dextranase (Dex49A), despite a difference in substrate specificity. Compared with the active sites of IPU and Dex49A, the amino acid residues participating in subsites + 2 and + 3 are not conserved, and the glucose residues of isopanose bound to IPU completely differ in orientation from the corresponding glucose residues of isomaltose bound to Dex49A. The shape of the catalytic cleft characterized by the seventh coil of the β-helix and a loop from domain N appears to be critical in determining the specificity of IPU for pullulan.