The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation.

The mature form of chitinase A1 from Bacillus circulans WL-12 comprises a C-terminal domain, two type III modules (domains), and a large N-terminal domain which contains the catalytic site of the enzyme. In order to better define the roles of these chitinase domains in chitin degradation, modified chiA genes encoding various deletions of chitinase A1 were constructed. The modified chiA genes were expressed in Escherichia coli, and the gene products were analyzed after purification by high-performance liquid chromatography. Intact chitinase A1 specifically bound to chitin, while it did not show significant binding activity towards partially acetylated chitosan and other insoluble polysaccharides. Chitinases lacking the C-terminal domain lost much of this binding activity to chitin as well as colloidal chitin-hydrolyzing activity. Deletion of the type III domains, on the other hand, did not affect chitin-binding activity but did result in significantly decreased colloidal chitin-hydrolyzing activity. Hydrolysis of low-molecular-weight substrates, soluble high-molecular-weight substrates, and insoluble high-molecular-weight substrates to which chitinase A1 does not bind were not significantly affected by these deletions. Thus, it was concluded that the C-terminal domain is a chitin-binding domain required for the specific binding to chitin and that this chitin-binding activity is important for efficient hydrolysis of the sufficiently acetylated chitin. Type III modules are not directly involved in the chitin binding but play an important functional role in the hydrolysis of chitin by the enzyme bound to chitin.     

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.     

A novel type of family 19 chitinase from Aeromonas sp. No.10S-24. Cloning, sequence, expression, and the enzymatic properties.

A family 19 chitinase gene from Aeromonas sp. No.10S-24 was cloned, sequenced, and expressed in Escherichia coli. From the deduced amino acid sequence, the enzyme was found to possess two repeated N-terminal chitin-binding domains, which are separated by two proline-threonine rich linkers. The calculated molecular mass was 70 391 Da. The catalytic domain is homologous to those of plant family 19 chitinases by about 47%. The enzyme produced alpha-anomer by hydrolyzing beta-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc)6] was hydrolyzed by the chitinase, the second glycosidic linkage from the nonreducing end was predominantly split producing (GlcNAc)2 and (GlcNAc)4. The evidence from this work suggested that the subsite structure of the enzyme was (-2)(-1)(+1)(+2)(+3)(+4), whereas most of plant family 19 chitinases have a subsite structure (-3)(-2)(-1)(+1)(+2)(+3). Thus, the Aeromonas enzyme was found to be a novel type of family 19 chitinase in its structural and functional properties.     

Overexpression,purification, and characterization of Paenibacillus cell surface-expressed chitinase ChiW with two catalytic domains

Paenibacillus sp. strain FPU-7 produces several different chitinases and effectively hydrolyzes robust chitin. Among the P. FPU-7 chitinases, ChiW, a novel monomeric chitinase with a molecular mass of 150?kDa, is expressed as a cell surface molecule. Here, we report that active ChiW lacking the anchoring domains in the N-terminus was successfully overproduced in Escherichia coli and purified to homogeneity. The two catalytic domains at the C-terminal region were classified as typical glycoside hydrolase family 18 chitinases, whereas the N-terminal region showed no sequence similarity to other known proteins. The vacuum-ultraviolet circular dichroism spectrum of the enzyme strongly suggested the presence of a β-stranded-rich structure in the N-terminus. Its biochemical properties were also characterized. Various insoluble chitins were hydrolyzed to N,N’-diacetyl-D-chitobiose as the final product. Based on amino acid sequence similarities and site-directed mutagenesis, Glu691 and Glu1177 in the two GH-18 domains were identified as catalytic residues.     

Properties of catalytic,linker and chitin-binding domains of insect chitinase

Manduca sexta (tobacco hornworm) chitinase is a glycoprotein that consists of an N-terminal catalytic domain, a Ser/Thr-rich linker region, and a C-terminal chitin-binding domain. To delineate the properties of these domains, we have generated truncated forms of chitinase, which were expressed in insect cells using baculovirus vectors. Three additional recombinant proteins composed of the catalytic domain fused with one or two insect or plant chitin-binding domains (CBDs) were also generated and characterized. The catalytic and chitin-binding activities are independent of each other because each activity is functional separately. When attached to the catalytic domain, the CBD enhanced activity toward the insoluble polymer but not the soluble chitin oligosaccharide primarily through an effect on the Km for the former substrate. The linker region, which connects the two domains, facilitates secretion from the cell and helps to stabilize the enzyme in the presence of gut proteolytic enzymes. The linker region is extensively modified by O-glycosylation and the catalytic domain is moderately N-glycosylated. Immunological studies indicated that the linker region, along with elements of the CBD, is a major immunogenic epitope. The results support the hypothesis that the domain structure of insect chitinase evolved for efficient degradation of the insoluble polysaccharide to soluble oligosaccharides during the molting process.     

Comparative study of the reaction mechanism of family 18 chitinases from plants and microbes

Hydrolytic mechanisms of family 18 chitinases from rice (Oryza sativa L.) and Bacillus circulans WL-12 were comparatively studied by a combination of HPLC analysis of the reaction products and theoretical calculation of reaction time-courses. All of the enzymes tested produced beta-anomers from chitin hexasaccharide [(GlcNAc)(6)], indicating that they catalyze the hydrolysis through a retaining mechanism. The rice chitinases hydrolyzed predominantly the fourth and fifth glycosidic linkages from the nonreducing end of (GlcNAc)(6), whereas B. circulans chitinase A1 hydrolyzed the second linkage from the nonreducing end. In addition, the Bacillus enzyme efficiently catalyzed transglycosylation, producing significant amounts of chitin oligomers larger than the initial substrate, but the rice chitinases did not. The time-courses of (GlcNAc)(6) degradation obtained by HPLC were analyzed by theoretical calculation, and the subsite structures of the rice chitinases were identified to be (-4)(-3)(-2)(-1)(+1)(+2). From the HPLC profile of the reaction products previously reported [Terwisscha van Scheltinga et al. (1995) Biochemistry 34, 15619-15623], family 18 chitinase from rubber tree (Hevea brasiliensis) was estimated to have the same type of subsite structure. Theoretical analysis of the reaction time-course for the Bacillus enzyme revealed that the enzyme has (-2)(-1) (+1)(+2)(+3)(+4)-type subsite structure, which is identical to that of fungal chitinase from Coccidioides immitis [Fukamizo et al. (2001) Biochemistry 40, 2448-2454]. The Bacillus enzyme also resembled the fungal chitinase in its transglycosylation activity. Minor structural differences between plant and microbial enzymes appear to result in such functional variations, even though all of these chitinases are classified into the identical family of glycosyl hydrolases.     

Characterization of the First Fungal Glycosyl Hydrolase Family 19 Chitinase (NbchiA) from Nosema bombycis (Nb)

Chitinases (EC 3.2.1.14), as one kind of glycosyl hydrolase, hydrolyze the β‐(1,4) linkages of chitin. According to the sequence similarity, chitinases can be divided into glycoside hydrolase family 18 and family 19. Here, a chitinase from Nosema bombycis (NbchiA) was cloned and purified by metal affinity chromatography and molecular exclusion chromatography. Sequence analysis indicated that NbchiA belongs to glycoside hydrolase family 19 class IV chitinase. The optimal pH and temperature of NbchiA are 7.0 and 40 °C, respectively. This purified chitinase showed high activity toward soluble substrates such as ethylene glycol chitin and soluble chitosan. The degradation of chitin oligosaccharides (GlcNAc)_2–5 detected by high‐performance liquid chromatography showed that NbchiA hydrolyzed mainly the second glycosidic linkage from the reducing end of (GlcNAc)_3‐5. On the basis of structure‐based multiple‐sequence alignment, Glu51 and Glu60 are believed to be the key catalytic residues. The site‐directed mutation analysis revealed that the enzymatic activity was decreased upon mutation of Glu60, whereas mutation of Glu51 totally abolished the enzymatic activity. This is the first report of a GH19 chitinase in fungi and in Microsporidia.     

Characterization of chondroitin sulfate lyase ABC from Bacteroides thetaiotaomicron WAL2926

Chondroitin sulfate ABC lyase (ChonABC) is an enzyme with broad specificity that depolymerizes via beta-elimination chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans (GAGs). ChonABC eliminates the glycosidic bond of its GAG substrates on the nonreducing end of their uronic acid component. This lyase possesses the unusual ability to act on both epimers of uronic acid, either glucuronic acid present in CS or iduronic acid in DS. Recently, we cloned, purified, and determined the three-dimensional structure of a broad specificity chondroitin sulfate ABC lyase from Bacteroides thetaiotaomicron (BactnABC) and identified two sets of catalytic residues. Here, we report the detailed biochemical characterization of BactnABC together with extensive site-directed mutagenesis resulting in characterization of the previously identified active site residues. BactnABC's catalysis is stimulated by Ca(2+) and Mg(2+) cations, particularly against DS. It displays extremely low activity toward hyaluronic acid and no activity toward heparin/heparan sulfate. Degradation of CS and DS by BactnABC yields only disaccharide products, pointing to an exolytic mode of action. The kinetic evaluations of the active-site mutants indicate that CS and DS substrates bind in the same active site, which is accompanied by a conformational change bringing the two sets of active site residues together. Conservative replacements of key residues suggest that His345 plays the role of a general base, initiating the degradation by abstracting the C5 bound proton from DS substrates, whereas either Tyr461 or His454 perform the equivalent role for CS substrates. Tyr461 is proposed, as well, to serve as general acid, completing the degradation of both CS and DS by protonating the leaving group.     

Cloning and biochemical characterization of the fucanase FcnA: definition of a novel glycoside hydrolase family specific for sulfated fucans

Sulfated fucans are matrix polysaccharides from marine brown algae, consisting of an alpha-L-fucose backbone substituted by sulfate-ester groups, masked with ramifications, and containing other monosaccharide residues. We here report on the characterization of a novel glycoside hydrolase (FcnA) specific for the degradation of sulfated fucans. This glycoside hydrolase was purified to electrophoretic homogeneity from a Flavobacteriaceae referred to as SW5. The gene fcnA was cloned and sequenced (3021 nucleotides), and the protein (1007 amino acids) was produced in Escherichia coli. FcnA exhibited a modular architecture consisting of a 400-residue-long N-terminal domain followed by three repeated domains predicted to adopt an immunoglobulin fold and by an 80-amino acid-long C-terminal domain. A truncated recombinant protein encompassing the N-terminal domain and the immunoglobulin-like repeats was shown to retain the enzyme activity. The N-terminal catalytic domain shared approximately 25% of sequence identity with two patented fucanase genes, and these three fucanases delineate a new family of glycoside hydrolases. As shown by size-exclusion chromatography (SEC) and 1H-NMR analyses, the fucanase FcnA proceeds according to an endolytic mode of action and cleaves the alpha-(1-->4) glycosidic linkages within the blocks of repeating motifs [-->4)-alpha-L-fucopyranosyl-2,3-disulfate-(1-->3)-alpha-L-fucopyranosyl-2-sulfate-(1-->]n.     

Characterization of an exo-beta-D-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

We previously clarified that the chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 produces diacetylchitobiose (GlcNAc(2)) as an end product from chitin. Here we sought to identify enzymes in T. kodakaraensis that were involved in the further degradation of GlcNAc(2). Through a search of the T. kodakaraensis genome, one candidate gene identified as a putative beta-glycosyl hydrolase was found in the near vicinity of the chitinase gene. The primary structure of the candidate protein was homologous to the beta-galactosidases in family 35 of glycosyl hydrolases at the N-terminal region, whereas the central region was homologous to beta-galactosidases in family 42. The purified protein from recombinant Escherichia coli clearly showed an exo-beta-D-glucosaminidase (GlcNase) activity but not beta-galactosidase activity. This GlcNase (GlmA(Tk)), a homodimer of 90-kDa subunits, exhibited highest activity toward reduced chitobiose at pH 6.0 and 80 degrees C and specifically cleaved the nonreducing terminal glycosidic bond of chitooligosaccharides. The GlcNase activity was also detected in T. kodakaraensis cells, and the expression of GlmA(Tk) was induced by GlcNAc(2) and chitin, strongly suggesting that GlmA(Tk) is involved in chitin catabolism in T. kodakaraensis. These results suggest that T. kodakaraensis, unlike other organisms, possesses a novel chitinolytic pathway where GlcNAc(2) from chitin is first deacetylated and successively hydrolyzed to glucosamine. This is the first report that reveals the primary structure of GlcNase not only from an archaeon but also from any organism.     

Chitinase B of "Microbulbifer degradans" 2-40 contains two catalytic domains with different chitinolytic activities

Chitinase B of "Microbulbifer degradans" 2-40 is a modular protein that is predicted to contain two glycoside hydrolase family 18 (GH18) catalytic domains, two polyserine domains, and an acidic repeat domain. Each of the GH18 domains was shown to be catalytically active against chitin. Activity assays reveal that the amino-terminal catalytic domain (GH18(N)) releases methylumbelliferone from 4'-methylumbelliferyl-N,N'-diacetylchitobiose 13.6-fold faster than the carboxy-terminal catalytic domain (GH18(C)) and releases chitobiose from the nonreducing end of chitooligosaccharides, therefore functioning as an exochitinase. GH18(C) releases methylumbelliferone from 4'-methylumbelliferyl-N,N',N"-triacetylchitotriose 2.7-fold faster than GH18(N) and cleaves chitooligosaccharides at multiple bonds, consistent with endochitinolytic activity. Each domain was maximally active from 30 to 37 degrees C and from pH 7.2 to 8.0 and was not affected by Mg(2+), Mn(2+), Ca(2+), K(+), EDTA, EGTA, or 1.0 M NaCl. The activity of each domain was moderately inhibited by Ni(2+), Sr(2+), and Cu(2+), while Hg(2+) completely abolished activity. When the specific activities of various recombinant portions of ChiB were calculated by using native chitin as a substrate, the polypeptide containing the endo-acting domain was twofold more active on native chitin than the other containing the exo-acting domain. The presence of both domains in a single reaction increased the amount of reducing sugars released from native chitin to 140% above the theoretical combined rate, indicating that the domains function cooperatively to degrade chitin. These data demonstrate that the GH18 domains of ChiB have different activities on the same substrate and function cooperatively to enhance chitin depolymerization.     

New evidence for the role of calcium in the glycosidase reaction of GH43 arabinanases

Endo-1,5-α-L-arabinanases are glycosyl hydrolases that are able to cleave the glycosidic bonds of α-1,5-L-arabinan, releasing arabino-oligosaccharides and L-arabinose. Two extracellular endo-1,5-α-L-arabinanases have been isolated from Bacillus subtilis, BsArb43A and BsArb43B (formally named AbnA and Abn2, respectively). BsArb43B shows low sequence identity with previously characterized 1,5-α-L-arabinanases and is a much larger enzyme. Here we describe the 3D structure of native BsArb43B, biochemical and structure characterization of two BsArb43B mutant proteins (H318A and D171A), and the 3D structure of the BsArb43B D171A mutant enzyme in complex with arabinohexose. The 3D structure of BsArb43B is different from that of other structurally characterized endo-1,5-α-L-arabinanases, as it comprises two domains, an N-terminal catalytic domain, with a 3D fold similar to that observed for other endo-1,5-α-L-arabinanases, and an additional C-terminal domain. Moreover, this work also provides experimental evidence for the presence of a cluster containing a calcium ion in the catalytic domain, and the importance of this calcium ion in the enzymatic mechanism of BsArb43B.     

Catalytic Efficiency of Chitinase-D on Insoluble Chitinous Substrates Was Improved by Fusing Auxiliary Domains

Chitin is an abundant renewable polysaccharide, next only to cellulose. Chitinases are important for effective utilization of this biopolymer. Chitinase D from Serratia proteamaculans (SpChiD) is a single domain chitinase with both hydrolytic and transglycosylation (TG) activities. SpChiD had less of hydrolytic activity on insoluble polymeric chitin substrates due to the absence of auxiliary binding domains. We improved catalytic efficiency of SpChiD in degradation of insoluble chitin substrates by fusing with auxiliary domains like polycystic kidney disease (PKD) domain and chitin binding protein 21 (CBP21). Of the six different SpChiD fusion chimeras, two C-terminal fusions viz. ChiD+PKD and ChiD+CBP resulted in improved hydrolytic activity on α- and β-chitin, respectively. Time-course degradation of colloidal chitin also confirmed that these two C-terminal SpChiD fusion chimeras were more active than other chimeras. More TG products were produced for a longer duration by the fusion chimeras ChiD+PKD and PKD+ChiD+CBP.     

Kinetics of hydrolysis of chitin/chitosan oligomers in concentrated hydrochloric acid

The kinetics of hydrolysis in concentrated hydrochloric acid (12.07 M) of the fully N-acetylated chitin tetramer (GlcNAc(4)) and the fully N-deacetylated chitosan tetramer (GlcN(4)) were followed by determining the amounts of the lower DP oligomers as a function of time. A theoretical model was developed to simulate the kinetics of hydrolysis of the three different glycosidic linkages in the tetramers. The model uses two different rate constants for the hydrolysis of the glycosidic bonds in the oligomers, assuming that the glycosidic bond next to one of the end residues are hydrolysed faster than the two other glycosidic linkages. The two rate constants were estimated by fitting model data to experimental results. The results show that the hydrolysis of the tetramers is a nonrandom process as the glycosidic bonds next to one of the end residues are hydrolysed 2.5 and 2.0 times faster as compared to the other glycosidic linkages in the fully N-acetylated and fully N-deacetylated tetramer, respectively. From previous results on other oligomers and the reaction mechanism, it is likely that the glycosidic bond that is hydrolysed fastest is the one next to the nonreducing end. The absolute values for the rate constants for the hydrolysis of the glycosidic linkages in GlcNAc(4) were found to be 50 times higher as compared to the glycosidic linkages in GlcN(4), due to the catalytic role of the N-acetyl group and the presence of the positively charged amino-group on the N-deacetylated sugar residue.     

Structural Insights into the Catalytic Mechanism of Bacterial Guanosine-diphospho-d-mannose Pyrophosphorylase and Its Regulation by Divalent Ions

GMP catalyzes the formation of GDP-Man, a fundamental precursor for protein glycosylation and bacterial cell wall and capsular polysaccharide biosynthesis. Crystal structures of GMP from the thermophilic bacterium Thermotoga maritima in the apo form, in complex with the substrates mannose-1-phosphate or GTP and bound with the end product GDP-Man in the presence of the essential divalent cation Mg²⁺, were solved in the 2.1–2.8 Å resolution range. The T. maritima GMP molecule is organized in two separate domains: a N-terminal Rossman fold-like domain and a C-terminal left-handed β-helix domain. Two molecules associate into a dimer through a tail-to-tail arrangement of the C-terminal domains. Comparative analysis of the structures along with characterization of enzymatic parameters reveals the bases of substrate specificity of this class of sugar nucleotidyltransferases. In particular, substrate and product binding are associated with significant changes in the conformation of loop regions lining the active center and in the relative orientation of the two domains. Involvement of both the N- and C-terminal domains, coupled to the catalytic role of a bivalent metal ion, highlights the catalytic features of bacterial GMPs compared with other members of the pyrophosphorylase superfamily.     

Active-site mutations which change the substrate specificity of the Clostridium stercorarium cellulase CelZ implications for synergism.

CelZ from the cellulolytic thermophile Clostridium stercorarium has been described as a 'monomeric' cellulase able to effect both the endoglucanolytic hydrolysis of internal glycosidic linkages and the exoglucanolytic degradation from the chain ends in a processive mode of action. The putative catalytic residues of this family 9 cellulase, Asp84 and Glu447 located within the N-terminal domain of the modular protein, were replaced by site-directed mutagenesis. A minimized CelZ derivative (CelZC') comprising the catalytic domain and the adjacent cellulose-binding domain (CBD) family IIIc domain C' was used as target for mutagenesis. Six mutant enzymes and the unmodified CelZC' protein were purified to homogeneity and compared with respect to thermoactivity, substrate specificity, product profile and synergism. CD studies revealed that no major changes to the overall structure of the proteins had occurred. Replacement of either one or both catalytic residues completely eliminated the ability of CelZ to attack insoluble Avicel preparations indicative of the exo-activity, whereas the endo-activity measured via hydrolysis of CM-cellulose was retained upon substitution of the catalytic base Asp84. Thus, endo-active CelZ mutants defective in the exo-activity were available for co-operativity studies with the C. stercorarium exoglucanase CelY. Synergism was found to be dependent on the endo-activity of CelZ. Mutants Asp84Gly and Asp84Glu were able to enhance the degradation of crystalline cellulose significantly, although no products could be released from this substrate by individual action of the mutants.     

Crystal structure of heparinase II from Pedobacter heparinus and its complex with a disaccharide product

Heparinase II depolymerizes heparin and heparan sulfate glycosaminoglycans, yielding unsaturated oligosaccharide products through an elimination degradation mechanism. This enzyme cleaves the oligosaccharide chain on the nonreducing end of either glucuronic or iduronic acid, sharing this characteristic with a chondroitin ABC lyase. We have determined the first structure of a heparin-degrading lyase, that of heparinase II from Pedobacter heparinus (formerly Flavobacterium heparinum), in a ligand-free state at 2.15 A resolution and in complex with a disaccharide product of heparin degradation at 2.30 A resolution. The protein is composed of three domains: an N-terminal alpha-helical domain, a central two-layered beta-sheet domain, and a C-terminal domain forming a two-layered beta-sheet. Heparinase II shows overall structural similarities to the polysaccharide lyase family 8 (PL8) enzymes chondroitin AC lyase and hyaluronate lyase. In contrast to PL8 enzymes, however, heparinase II forms stable dimers, with the two active sites formed independently within each monomer. The structure of the N-terminal domain of heparinase II is also similar to that of alginate lyases from the PL5 family. A Zn2+ ion is bound within the central domain and plays an essential structural role in the stabilization of a loop forming one wall of the substrate-binding site. The disaccharide binds in a long, deep canyon formed at the top of the N-terminal domain and by loops extending from the central domain. Based on structural comparison with the lyases from the PL5 and PL8 families having bound substrates or products, the disaccharide found in heparinase II occupies the "+1" and "+2" subsites. The structure of the enzyme-product complex, combined with data from previously characterized mutations, allows us to propose a putative chemical mechanism of heparin and heparan-sulfate degradation.     

Cloning,expression and biocharacterization of OfCht5, the chitinase from the insect Ostrinia furnacalis

Abstract Chitinase catalyzes β‐1,4‐glycosidic linkages in chitin and has attracted research interest due to it being a potential pesticide target and an enzymatic tool for preparation of N‐acetyl‐β‐D‐glucosamine. An individual insect contains multiple genes encoding chitinases, which vary in domain architectures, expression patterns, physiological roles and biochemical properties. Herein, OfCht5, the glycoside hydrolase family 18 chitinase from the widespread lepidopteran pest Ostrinia furnacalis, was cloned, expressed in the yeast Pichia pastoris and biochemically characterized in an attempt to facilitate both pest control and biomaterial preparation. Complementary DNA sequence analysis indicated that OfCHT5 consisted of an open reading frame of 1 665‐bp nucleotides. Phylogenic analysis suggested OfCht5 belongs to the Group I insect chitinases. Expression of OfCht5 in Pichia pastoris resulted in highest specific activity after 120 h of induction with methanol. Through two steps of purification, consisting of ammonium sulfate precipitation and metal chelating chromatography, about 7 mg of the recombinant OfCht5 was purified to homogeneity from 1 L culture supernatant. OfCht5 effectively converted colloidal chitin into chitobiose, but had relatively low activity toward α‐chitin. When chitooligosaccharides [(GlcNAc)_n, n= 3–6] were used as substrates, OfCht5 was observed to possess the highest catalytic efficiency parameter toward (GlcNAc)₄ and predominantely hydrolyzed the second glycosidic bond from the non‐reducing end. Together with β‐N‐acetyl‐D‐hexosaminidase OfHex1, OfCht5 achieved its highest efficiency in chitin degradation that yielded N‐acetyl‐β‐D‐glucosamine, a valuable pharmacological reagent and food supplement, within a molar concentration ratio of OfCht5 versus OfHex1 in the range of 9 : 1–15 : 1. This work provides an alternative to existing preparation of chitinase for pesticides and other applications.     

The C-terminal module of Chi1 from Aeromonas caviae CB101 has a function in substrate binding and hydrolysis

The chitinase gene chi1 of Aeromonas caviae CB101 encodes an 865-amino-acid protein (with signal peptide) composed of four domains named from the N-terminal as an all-beta-sheet domain ChiN, a triosephosphate isomerase (TIM) catalytic domain, a function-unknown A region, and a putative chitin-binding domain (ChBD) composed of two repeated sequences. The N-terminal 563-amino-acid segment of Chi1 (Chi1DeltaADeltaChBD) shares 74% identity with ChiA of Serratia marcescens. By the homology modeling method, the three-dimensional (3D) structure of Chi1DeltaADeltaChBD was constructed. It fit the structure of ChiA very well. To understand fully the function of the C-terminal module of Chi1 (from 564 to 865 amino acids), two different C-terminal truncates, Chi1DeltaChBD and Chi1DeltaADeltaChBD, were constructed, based on polymerase chain reaction (PCR). Comparison studies of the substrate binding, hydrolysis capacity, and specificity among Chi1 and its two truncates showed that the C-terminal putative ChBD contributed to the insoluble substrate-protein binding and hydrolysis; the A region did not have any function in the insoluble substrate-protein binding, but it did have a role in the chitin hydrolysis: Deletion of the A region caused the enzyme to lose 30-40% of its activity toward amorphous colloidal chitin and soluble chitin, and around 50% toward p-nitrophenyl (pNP)-chitobiose pNP-chitotriose, and its activity toward low-molecular-weight chitooligomers (GlcNAc)3-6 also dropped, as shown by analysis of its digestion processes. This is the first clear demonstration that a domain or segment without a function in insoluble substrate-chitinase binding has a role in the digestion of a broad range of chitin substrates, including low-molecular-weight chitin oligomers. The reaction mode of Chi1 is also described and discussed.     

Structure of the N-terminal calcium sensor domain of centrin reveals the biochemical basis for domain-specific function

Centrin is an essential component of microtubule-organizing centers in organisms ranging from algae and yeast to humans. It is an EF-hand calcium-binding protein with homology to calmodulin but distinct calcium binding properties. In a previously proposed model, the C-terminal domain of centrin serves as a constitutive anchor to target proteins, and the N-terminal domain serves as the sensor of calcium signals. The three-dimensional structure of the N-terminal domain of Chlamydomonas rheinhardtii centrin has been determined in the presence of calcium by solution NMR spectroscopy. The domain is found to occupy an open conformation typical of EF-hand calcium sensors. Comparison of the N- and C-terminal domains of centrin reveals a structural and biochemical basis for the domain specificity of interactions with its cellular targets and the distinct nature of centrin relative to other EF-hand proteins. An NMR titration of the centrin N-terminal domain with a fragment of the known centrin target Sfi1 reveals binding of the peptide to a discrete site on the protein, which supports the proposal that the N-terminal domain serves as a calcium sensor in centrin.