Molecular Basis for Auto- and Hetero-catalytic Maturation of a Thermostable Subtilase from Thermophilic Bacillus sp. WF146

The proform of the WF146 protease, an extracellular subtilase produced by thermophilic Bacillus sp. WF146, matures efficiently at high temperatures. Here we report that the proform, which contains an N-terminal propeptide composed of a core domain (N*) and a linker peptide, is intrinsically able to mature via multiple pathways. One autocatalytic pathway is initiated by cis-processing of N* to generate an autoprocessed complex N*-I^WT, and this step is followed by truncation of the linker peptide and degradation of N*. Another autocatalytic pathway is initiated by trans-processing of the linker peptide followed by degradation of N*. Unlike most reported subtilases, the maturation of the WF146 protease occurs not only autocatalytically but also hetero-catalytically whereby heterogeneous proteases accelerate the maturation of the WF146 protease via trans-processing of the proform and N*-I^WT. Although N* acts as an intramolecular chaperone and an inhibitor of the mature enzyme, the linker peptide is susceptible to proteolysis, allowing the trans-processing reaction to occur auto- and hetero-catalytically. These studies also demonstrate that the WF146 protease undergoes subtle structural adjustments during the maturation process and that the binding of Ca²⁺ is required for routing the proform to mature properly at high temperatures. Interestingly, under Ca²⁺-free conditions, the proform is cis-processed into a unique propeptide-intermediate complex (N*-I^E) capable of re-synthesis of the proform. Based on the basic catalytic principle of serine proteases and these experimental results, a mechanism for the cis-processing/re-synthesis equilibrium of the proform and the role of the linker peptide in regulation of this equilibrium has been proposed.     

Protein Engineering of Chit42 Towards Improvement of Chitinase and Antifungal Activities

The antagonism of Trichoderma strains usually correlates with the secretion of fungal cell wall degrading enzymes such as chitinases. Chitinase Chit42 is believed to play an important role in the biocontrol activity of Trichoderma strains as a biocontrol agent against phytopathogenic fungi. Chit42 lacks a chitin-binding domain (ChBD) which is involved in its binding activity to insoluble chitin. In this study, a chimeric chitinase with improved enzyme activity was produced by fusing a ChBD from T. atroviride chitinase 18–10 to Chit42. The improved chitinase containing a ChBD displayed a 1.7-fold higher specific activity than chit42. This increase suggests that the ChBD provides a strong binding capacity to insoluble chitin. Moreover, Chit42-ChBD transformants showed higher antifungal activity towards seven phytopathogenic fungal species.     

Immobilization of nisin producer Lactococcus lactis strains to chitin with surface-displayed chitin-binding domain

In this study, nisin producer Lactococcus lactis strains displaying cell surface chitin-binding domain (ChBD) and capable of immobilizing to chitin flakes were constructed. To obtain ChBD-based cell immobilization, Usp45 signal sequence with ChBD of chitinase A1 enzyme from Bacillus circulans was fused with different lengths of PrtP (153, 344, and 800 aa) or AcmA (242 aa) anchors derived from L. lactis. According to the whole cell ELISA analysis, ChBD was successfully expressed on the surface of L. lactis cells. Scanning electron microscope observations supported the conclusion of the binding analysis that L. lactis cells expressing the ChBD with long PrtP anchor (800 aa) did bind to chitin surfaces more efficiently than cells with the other ChBD anchors. The attained binding affinity of nisin producers for chitin flakes retained them in the fermentation during medium changes and enabled storage for sequential productions. Initial nisin production was stably maintained with many cycles. These results demonstrate that an efficient immobilization of L. lactis cells to chitin is possible for industrial scale repeated cycle or continuous nisin fermentation.     

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.     

Immobilization of Cells with Surface-Displayed Chitin-Binding Domain

To explore chitin-binding domain (ChBD)-based cell immobilization, a tripartite gene fusion consisting of an in-frame fusion of ChBD to lpp and ompA was constructed and expressed in Escherichia coli. ChBD-displayed cells exhibited highly specific and stable binding to chitin within a wide range of pHs (5 to 8) and temperatures (15 to 37°C). These results illustrate the promising use of this approach for engineering applications.     

Designing a new chitinase with more chitin binding and antifungal activity

Chitinases have the ability of chitin digestion that constitutes a main compound of the cell wall in many of the phytopathogens such as fungi. Chitinase Chit42 from Trichoderma atroviride PTCC5220 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin binding domain (ChBD). We have produced a chimeric chitinase with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Serratia marcescens Chitinase B. The fusion of ChBD improved the affinity to crystalline and colloidal chitin and also the enzyme activity of the chimeric chitinase when compared with the native Chit42. The chimeric chitinase showed higher antifungal activity toward phytopathogenic fungi.     

Structure of full‐length class I chitinase from rice revealed by X‐ray crystallography and small‐angle X‐ray scattering

The rice class I chitinase OsChia1b, also referred to as RCC2 or Cht‐2, is composed of an N‐terminal chitin‐binding domain (ChBD) and a C‐terminal catalytic domain (CatD), which are connected by a proline‐ and threonine‐rich linker peptide. Because of the ability to inhibit fungal growth, the OsChia1b gene has been used to produce transgenic plants with enhanced disease resistance. As an initial step toward elucidating the mechanism of hydrolytic action and antifungal activity, the full‐length structure of OsChia1b was analyzed by X‐ray crystallography and small‐angle X‐ray scattering (SAXS). We determined the crystal structure of full‐length OsChia1b at 2.00‐Å resolution, but there are two possibilities for a biological molecule with and without interdomain contacts. The SAXS data showed an extended structure of OsChia1b in solution compared to that in the crystal form. This extension could be caused by the conformational flexibility of the linker. A docking simulation of ChBD with tri‐N‐acetylchitotriose exhibited a similar binding mode to the one observed in the crystal structure of a two‐domain plant lectin complexed with a chitooligosaccharide. A hypothetical model based on the binding mode suggested that ChBD is unsuitable for binding to crystalline α‐chitin, which is a major component of fungal cell walls because of its collisions with the chitin chains on the flat surface of α‐chitin. This model also indicates the difference in the binding specificity of plant and bacterial ChBDs of GH19 chitinases, which contribute to antifungal activity. Proteins 2010. © 2010 Wiley‐Liss,Inc.     

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.     

Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus

A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both α and β crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded β-sheets, was composed of a typical β-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass.     

Nisin production in a chitin-including continuous fermentation system with Lactococcus lactis displaying a cell wall chitin-binding domain

The limiting factors in the continuous production of nisin are high amount of biomass loss and low dilution rate application. In this study, a chitin-including continuous nisin fermentation system (CICON-FER) was constructed for high volumetric nisin production using nisin producer L. lactis displaying cell wall chitin-binding domain (ChBD) together with chitin in the reactor. In this respect, the highest binding conditions of relevant L. lactis cells to chitin were determined. Then the chitin flakes carrying nisin-producing L. lactis cells were used within the CICON-FER system at different dilution rates (0.1–0.9 h^?1) and initial glucose concentrations (20–60 g l^?1). The results revealed that the pH 7 conditions and the use of 100 mM sodium phosphate buffer with 0.1 % Tween 20 and Triton X-100 significantly increased the binding capacity of ChBD displaying L. lactis cells to chitin. The constructed CICON-FER system maintained the presence of the ChBD surface displaying L. lactis cells in the reactor system until 0.9 h^?1 dilution rate that resulted in a considerably high level of volumetric nisin production and productivity (10,500 IU ml^?1 and 9,450 IU ml^?1 h^?1, respectively) with the combination of a 0.9-h^?1 dilution rate and a 40-g l^?1 initial glucose concentration. In conclusion, an innovative nisin fermentation system that yielded the highest nisin production thus far and that was feasible for industrial application was created.     

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.     

Identification and Characterization of the Three Chitin-Binding Domains within the Multidomain Chitinase Chi92 from Aeromonas hydrophila JP101

The gene (chi92) encoding the extracellular chitinase of Aeromonas hydrophila JP101 has been cloned and expressed in Escherichia coli. The mature form of Chi92 is an 842-amino-acid (89.830-kDa) modular enzyme comprised of a family 18 catalytic domain, an unknown-function region (the A region), and three chitin-binding domains (ChBDs; Chi92-N, ChBD_CI, and ChBD_CII). The C-terminally repeated ChBDs, ChBD_CI and ChBD_CII, were grouped into family V of cellulose-binding domains on the basis of sequence homology. Chitin binding and enzyme activity studies with C-terminally truncated Chi92 derivatives lacking ChBDs demonstrated that the ChBDs are responsible for its adhesion to unprocessed and colloidal chitins. Further adsorption experiments with glutathione S-transferase (GST) fusion proteins (GST-CI and GST-CICII) demonstrated that a single ChBD (ChBD_CI) could promote efficient chitin and cellulose binding. In contrast to the two C-terminal ChBDs, the Chi92-N domain is similar to ChiN of Serratia marcescens ChiA, which has been proposed to participate in chitin binding. A truncated derivative of Chi92 that contained only a catalytic domain and Chi92-N still exhibited insoluble-chitin-binding and hydrolytic activities. Thus, it appears that Chi92 contains Chi92-N as the third ChBD in addition to two ChBDs (ChBD_CI and ChBD_CII).     

Interaction force of chitin-binding domains onto chitin surface

Interaction force of chitin-binding domains (ChBD1 and ChBD2) from a thermostable chitinase onto chitin surface was directly measured by atomic force microscopy (AFM) in a buffer solution. In the force curve measurement, multiple pull-off events were observed for the AFM tips functionalized with either ChBD1 or ChBD2, whereas the AFM tips terminated with nitrilotriacetic acid groups without ChBD showed no interaction peak, suggesting that the detected forces are derived from the binding functions of ChBDs onto the chitin surface. The force curve analyses indicate that the binding force of ChBD2 is stronger than that of ChBD1. This result suggests that ChBD1 and ChBD2 play different roles in adsorption onto chitin surface.     

Identification and characterization of the three chitin-binding domains within the multidomain chitinase Chi92 from Aeromonas hydrophila JP101.

The gene (chi92) encoding the extracellular chitinase of Aeromonas hydrophila JP101 has been cloned and expressed in Escherichia coli. The mature form of Chi92 is an 842-amino-acid (89.830-kDa) modular enzyme comprised of a family 18 catalytic domain, an unknown-function region (the A region), and three chitin-binding domains (ChBDs; Chi92-N, ChBD(CI), and ChBD(CII)). The C-terminally repeated ChBDs, ChBD(CI) and ChBD(CII), were grouped into family V of cellulose-binding domains on the basis of sequence homology. Chitin binding and enzyme activity studies with C-terminally truncated Chi92 derivatives lacking ChBDs demonstrated that the ChBDs are responsible for its adhesion to unprocessed and colloidal chitins. Further adsorption experiments with glutathione S-transferase (GST) fusion proteins (GST-CI and GST-CICII) demonstrated that a single ChBD (ChBD(CI)) could promote efficient chitin and cellulose binding. In contrast to the two C-terminal ChBDs, the Chi92-N domain is similar to ChiN of Serratia marcescens ChiA, which has been proposed to participate in chitin binding. A truncated derivative of Chi92 that contained only a catalytic domain and Chi92-N still exhibited insoluble-chitin-binding and hydrolytic activities. Thus, it appears that Chi92 contains Chi92-N as the third ChBD in addition to two ChBDs (ChBD(CI) and ChBD(CII)).     

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.     

Immobilization of cells with surface-displayed chitin-binding domain

To explore chitin-binding domain (ChBD)-based cell immobilization, a tripartite gene fusion consisting of an in-frame fusion of ChBD to lpp and ompA was constructed and expressed in Escherichia coli. ChBD-displayed cells exhibited highly specific and stable binding to chitin within a wide range of pHs (5 to 8) and temperatures (15 to 37 degrees C). These results illustrate the promising use of this approach for engineering applications.     

Molecular analysis of the gene encoding a novel chitin-binding protease from Alteromonas sp. strain O-7 and its role in the chitinolytic system

Alteromonas sp. strain O-7 secretes several proteins in response to chitin induction. We have found that one of these proteins, designated AprIV, is a novel chitin-binding protease involved in chitinolytic activity. The gene encoding AprIV (aprIV) was cloned in Escherichia coli. DNA sequencing analysis revealed that the open reading frame of aprIV encoded a protein of 547 amino acids with a calculated molecular mass of 57,104 Da. AprIV is a modular enzyme consisting of five domains: the signal sequence, the N-terminal proregion, the family A subtilase region, the polycystic kidney disease domain (PkdD), and the chitin-binding domain type 3 (ChtBD3). Expression plasmids coding for PkdD or both PkdD and ChtBD (PkdD-ChtBD) were constructed. The PkdD-ChtBD but not PkdD exhibited strong binding to alpha-chitin and beta-chitin. Western and Northern analyses demonstrated that aprIV was induced in the presence of N-acetylglucosamine, N-acetylchitobiose, or chitin. Native AprIV was purified to homogeneity from Alteromonas sp. strain O-7 and characterized. The molecular mass of mature AprIV was estimated to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature of AprIV were pH 11.5 and 35 degrees C, respectively, and even at 10 degrees C the enzyme showed 25% of the maximum activity. Pretreatment of native chitin with AprIV significantly promoted chitinase activity.     

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.     

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.     

Growth of Hyperthermophilic Archaeon Pyrococcus furiosus on Chitin Involves Two Family 18 Chitinases

Pyrococcus furiosus was found to grow on chitin, adding this polysacharide to the inventory of carbohydrates utilized by this hyperthermophilic archaeon. Accordingly, two open reading frames (chiA [Pf1234] and chiB [Pf1233]) were identified in the genome of P. furiosus, which encodes chitinases with sequence similarity to proteins from the glycosyl hydrolase family 18 in less-thermophilic organisms. Both enzymes contain multiple domains that consist of at least one binding domain and one catalytic domain. ChiA (ca. 39 kDa) contains a putative signal peptide, as well as a binding domain (ChiA_BD), that is related to binding domains associated with several previously studied bacterial chitinases. chiB, separated by 37 nucleotides from chiA and in the same orientation, encodes a polypeptide with two different proline-threonine-rich linker regions (6 and 3 kDa) flanking a chitin-binding domain (ChiB_BD [11 kDa]), followed by a catalytic domain (ChiB_cat [35 kDa]). No apparent signal peptide is encoded within chiB. The two chitinases share little sequence homology to each other, except in the catalytic region, where both have the catalytic glutamic acid residue that is conserved in all family 18 bacterial chitinases. The genes encoding ChiA, without its signal peptide, and ChiB were cloned and expressed in Escherichia coli. ChiA exhibited no detectable activity toward chitooligomers smaller than chitotetraose, indicating that the enzyme is an endochitinase. Kinetic studies showed that ChiB followed Michaelis-Menten kinetics toward chitotriose, although substrate inhibition was observed for larger chitooligomers. Hydrolysis patterns on chitooligosaccharides indicated that ChiB is a chitobiosidase, processively cleaving off chitobiose from the nonreducing end of chitin or other chitooligomers. Synergistic activity was noted for the two chitinases on colloidal chitin, indicating that these two enzymes work together to recruit chitin-based substrates for P. furiosus growth. This was supported by the observed growth on chitin as the sole carbohydrate source in sulfur-free media.