Characterization of the extracellular domain of sensor histidine kinase NagS from Paenibacillus sp. str. FPU-7: nagS interacts with oligosaccharide binding protein NagB1 in complexes with N, N'-diacetylchitobiose.

We have previously isolated the Gram-positive chitin-degrading bacterium Paenibacillus sp. str. FPU-7. This bacterium traps chitin disaccharide (GlcNAc)2 on its cell surface using two homologous solute-binding proteins, NagB1 and NagB2. Bacteria use histidine kinase (HK) of the two-component regulatory system as an extracellular environment sensor. In this study, we found that nagS, which encodes a HK, is located next to the nagB1 gene. Biochemical experiments revealed that the NagS sensor domain (NagS30-294) interacts with the NagB1-(GlcNAc)2 complex. However, proof of NagS30-294 interacting with NagB1 without (GlcNAc)2 is currently unavailable. In contrast to NagB1, no complex formation was observed between NagS30-294 and NagB2, even in the presence of (GlcNAc)2. The NagS30-294 crystal structure at 1.8 Å resolution suggested that the canonical tandem-Per-Arnt-Sim fold recognizes the NagB1-(GlcNAc)2 complex. This study provides insight into the recognition of chitin oligosaccharides by bacteria.

Stomatal immunity against fungal invasion comprises not only chitin-induced stomatal closure but also chitosan-induced guard cell death.

Many pathogenic fungi exploit stomata as invasion routes, causing destructive diseases of major cereal crops. Intensive interaction is expected to occur between guard cells and fungi. In the present study, we took advantage of well-conserved molecules derived from the fungal cell wall, chitin oligosaccharide (CTOS), and chitosan oligosaccharide (CSOS) to study how guard cells respond to fungal invasion. In Arabidopsis, CTOS induced stomatal closure through a signaling mediated by its receptor CERK1, Ca2+, and a major S-type anion channel, SLAC1. CSOS, which is converted from CTOS by chitin deacetylases from invading fungi, did not induce stomatal closure, suggesting that this conversion is a fungal strategy to evade stomatal closure. At higher concentrations, CSOS but not CTOS induced guard cell death in a manner dependent on Ca2+ but not CERK1. These results suggest that stomatal immunity against fungal invasion comprises not only CTOS-induced stomatal closure but also CSOS-induced guard cell death.

Simultaneous visualization of callose deposition and plasma membrane for live-cell imaging in plants.

KEY MESSAGE: The appropriate combination of fluorescent probes enabled the simultaneous visualization of callose deposition and plasma membrane in living Arabidopsis and can be useful for the cell biological study of papilla formation in plants. Localized callose deposition at the site of fungal infection is a central part of papilla formation, which creates a barrier between the host plasma membrane and the cell wall and plays an important role in preventing the penetration of fungal hyphae into the host cells. Using chitin-induced callose deposition as a model system, we examined suitable conditions for the simultaneous visualization of callose deposition and plasma membrane dynamics in living Arabidopsis cotyledons. We found that aniline blue fluorochrome (ABF) for callose staining selectively interferes with FM dyes for membrane visualization depending on the structure of the latter compounds and the proper combination of these fluorescent dyes and staining conditions is a key for successful live-cell imaging. The established conditions enabled the live-cell imaging of chitin-induced callose deposition and host membrane systems. The established system/conditions would also be useful for the cell biological studies on the localized callose deposition in other stress/development-associated processes. The finding that the slight difference in the structure of FM dyes affects the interaction with another fluorescent dye, ABF, would also give useful suggestions for the studies where multiple fluorescent dyes are utilized for live-cell imaging.

Characterization of mouse di-N-acetylchitobiase that can degrade chitin-oligosaccharides.

Di-N-acetylchitobiase (Ctbs) degrades ß-1,4 glycoside bonds of the chitobiose core of free asparagine-linked glycan. This study examined whether Ctbs degrades chitin-oligosaccharides to GlcNAc in mammals. We analyzed Ctbs mRNA and protein expression in mouse tissues and characterized enzymatic activity using recombinant mouse Ctbs expressed in Escherichia coli. Ctbs mRNA and protein were expressed in various tissues of mouse, including the stomach. Optimal conditions for recombinant Ctbs were pH 3.0 and 45°C, and the recombinant enzyme was retained more than 94% activity after incubation at pH 3.0-7.0 and below 37°C. The recombinant Ctbs hydrolyzed (GlcNAc)3 and (GlcNAc)6 at pH 3.0 and produced GlcNAc. The K m of Ctbs was lowest with (GlcNAc)3 as a substrate. k cat/K m was fourfold as high with (GlcNAc)3 and (GlcNAc)4 as substrates than with (GlcNAc)2. These results suggest that Ctbs digests chitin-oligosaccharides or (GlcNAc)2 of reducing-end residues of oligosaccharides and produces GlcNAc in mouse tissues.

Chitin Heterodisaccharide, Released from Chitin by Chitinase and Chitin Oligosaccharide Deacetylase, Enhances the Chitin-Metabolizing Ability of Vibrio parahaemolyticus.

Vibrio parahaemolyticus RIMD2210633 secretes both chitinase and chitin oligosaccharide deacetylase and produces ß-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) from chitin. Previously, we reported that GlcNAc-GlcN induces chitinase production by several strains of Vibrio harboring chitin oligosaccharide deacetylase genes (T. Hirano, K. Kadokura, T. Ikegami, Y. Shigeta, et al., Glycobiology 19:1046-1053, 2009). The metabolism of chitin by Vibrio was speculated on the basis of the findings of previous studies, and the role of chitin oligosaccharide produced from chitin has been well studied. However, the role of GlcNAc-GlcN in the Vibrio chitin degradation system, with the exception of the above-mentioned function as an inducer of chitinase production, remains unclear. N,N'-Diacetylchitobiose, a homodisaccharide produced from chitin, is known to induce the expression of genes encoding several proteins involved in chitin metabolism in Vibrio strains (K. L. Meibom, X. B. Li, A. Nielsen, C. Wu, et al., Proc Natl Acad Sci U S A 101:2524-2529, 2004). We therefore hypothesized that GlcNAc-GlcN also affects the expression of enzymes involved in chitin metabolism in the same manner. In this study, we examined the induction of protein expression by several sugars released from chitin using peptide mass fingerprinting and confirmed the expression of genes encoding enzymes involved in chitin metabolism using real-time quantitative PCR analysis. We then confirmed that GlcNAc-GlcN induces the expression of genes encoding many soluble enzymes involved in chitin degradation in Vibrio parahaemolyticus Here, we demonstrate that GlcNAc-GlcN enhances the chitin-metabolizing ability of V. parahaemolyticusIMPORTANCE We demonstrate that ß-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) enhances the chitin-metabolizing ability of V. parahaemolyticus Members of the genus Vibrio are chitin-degrading bacteria, and some species of this genus are associated with diseases affecting fish and animals, including humans (F. L. Thompson, T. Iida, and J. Swings, Microbiol Mol Biol Rev 68:403-431, 2004; M. Y. Ina-Salwany, N. Al-Saari, A. Mohamad, F.-A. Mursidi, et al., J Aquat Anim Health 31:3-22, 2019). Studies on Vibrio are considered important, as they may facilitate the development of solutions related to health, food, and aquaculture problems attributed to this genus. This report enhances the current understanding of chitin degradation by Vibrio bacteria.

Chitin Oligosaccharide Modulates Gut Microbiota and Attenuates High-Fat-Diet-Induced Metabolic Syndrome in Mice.

Gut microbiota has been proved to be an indispensable link between nutrient excess and metabolic syndrome, and chitin oligosaccharide (NACOS) has displayed therapeutic effects on multiple diseases such as cancer and gastritis. In this study, we aim to confirm whether NACOS can ameliorate high-fat diet (HFD)-induced metabolic syndrome by rebuilding the structure of the gut microbiota community. Male C57BL/6J mice fed with HFD were treated with NACOS (1 mg/mL) in drinking water for five months. The results indicate that NACOS improved glucose metabolic disorder in HFD-fed mice and suppressed mRNA expression of the protein regulators related to lipogenesis, gluconeogenesis, adipocyte differentiation, and inflammation in adipose tissues. Additionally, NACOS inhibited the destruction of the gut barrier in HFD-treated mice. Furthermore, 16S ribosome RNA sequencing of fecal samples demonstrates that NACOS promoted the growth of beneficial intestinal bacteria remarkably and decreased the abundance of inflammogenic taxa. In summary, NACOS partly rebuilt the microbial community and improved the metabolic syndrome of HFD-fed mice. These data confirm the preventive effects of NACOS on nutrient excess-related metabolic diseases.

Enzymatic properties of a GH19 chitinase isolated from rice lacking a major loop structure involved in chitin binding.

The catalytic domains of family GH19 chitinases have been found to consist of a conserved, α-helical core-region and different numbers (1-6) of loop structures, located at both ends of the substrate-binding groove and which extend over the glycon- and aglycon-binding sites. We expressed, purified and enzymatically characterized a GH19 chitinase from rice, Oryza sativa L. cv. Nipponbare (OsChia2a), lacking a major loop structure (loop III) connected to the functionally important ß-stranded region. The new enzyme thus contained the five remaining loop structures (loops I, II, IV, V and C-term). The OsChia2a recombinant protein catalyzed hydrolysis of chitin oligosaccharides, (GlcNAc)n (n = 3-6), with inversion of anomeric configuration, indicating that OsChia2a correctly folded without loop III. From thermal unfolding experiments and calorimetric titrations using the inactive OsChia2a mutant (OsChia2a-E68Q), in which the catalytic residue Glu68 was mutated to glutamine, we found that the binding affinities towards (GlcNAc)n (n = 2-6) were almost proportional to the degree of polymerization of (GlcNAc)n, but were much lower than those obtained for a moss GH19 chitinase having only loop III [Ohnuma T, Sørlie M, Fukuda T, Kawamoto N, Taira T, Fukamizo T. 2011. Chitin oligosaccharide binding to a family GH19 chitinase from the moss, Bryum coronatum. FEBS J. 278:3991-4001]. Nevertheless, OsChia2a exhibited significant antifungal activity. It appears that loop III connected to the ß-stranded region is important for (GlcNAc)n binding, but is not essential for antifungal activity.

Hyaluronan synthase assembles hyaluronan on a [GlcNAc(ß1,4)]n-GlcNAc(α1→)UDP primer and hyaluronan retains this residual chitin oligomer as a cap at the nonreducing end.

Class I hyaluronan synthases (HAS) assemble [GlcNAc(ß1,4)GlcUA(ß1,3)]n-UDP at the reducing end and also make chitin. Streptococcus equisimilis HAS (SeHAS) also synthesizes chitin-UDP oligosaccharides, (GlcNAc-ß1,4)n-GlcNAc(α1→)UDP (Weigel et al. 2015). Here we determined if HAS uses chitin-UDPs as primers to initiate HA synthesis, leaving the non-HA primer at the nonreducing (NR) end. HA made by SeHAS membranes was purified, digested with streptomyces lyase, and hydrophobic oligomers were enriched by solid phase extraction and analyzed by MALDI-TOF MS. Jack bean hexosaminidase (JBH) and MS/MS were used to analyze 19 m/z species of possible GnHn ions with clustered GlcNAc (G) residues attached to disaccharide units (H): (GlcNAcß1,4)2-5[GlcUA(ß1,3)GlcNAc]2-6. JBH digestion sequentially removed GlcNAc from the NR-end of GnHn oligomers, producing successively smaller GnH2-3 series members. Since lyase releases dehydro-oligos (dHn; M-18), only the unique NR-end oligo lacks dehydro-GlcUA. Hn oligomers were undetectable in lyase digests, whereas JBH treatment created new H2-6m/z peaks (i.e. HA tetra- through dodeca-oligomers). MS/MS of larger GnHn species produced chitin (2-5 GlcNAcs), HA oligomers and multiple smaller series members with fewer GlcNAcs. All NR-ends (97%) started with GlcNAc, as a chitin trimer (three GlcNAcs), indicating that GlcNAc(ß1,4)2GlcNAc(α1→)-UDP may be optimal for initiation of HA synthesis. Also, HA made by live S. pyogenes cells had G4Hn chitin-oligo NR-ends. We conclude that chitin-UDP functions in vitro and in live cells as a primer to initiate synthesis of all HA chains and these primers remain at the NR-ends of HA chains as residual chitin caps [(GlcNAc-ß1,4)3-4].

Chitin oligosaccharide binding to the lysin motif of a novel type of chitinase from the multicellular green alga, Volvox carteri.

KEY MESSAGE: The chitinase-mediated defense system in higher plants has been intensively studied from physiological and structural viewpoints. However, the defense system in the most primitive plant species, such as green algae, has not yet been elucidated in details. In this study, we solved the crystal structure of a family CBM-50 LysM module attached to the N-terminus of chitinase from Volvox carteri, and successfully analyzed its chitin-binding ability by NMR spectroscopy and isothermal titration calorimetry. Trp96 of the LysM module appeared to make a CH-π stacking interaction with the reducing end sugar residue of the ligand. We believe the data included in this manuscript provide novel insights into the molecular basis of chitinase-mediated defense system in green algae. A chitinase from the multicellular green alga, Volvox carteri, contains two N-terminal lysin motifs (VcLysM1 and VcLysM2), that belong to the CBM-50 family, in addition to a catalytic domain. We produced a recombinant protein of VcLysM2 in order to examine its structure and function. The X-ray crystal structure of VcLysM2 was successfully solved at a resolution of 1.2 Å, and revealed that the protein adopts the ßααß fold typical of members belonging to the CBM-50 family. NMR spectra of 13C- and 15N-labeled proteins were analyzed in order to completely assign the main chain resonances of the 1H,15N-HSQC spectrum in a sequential manner. NMR-based titration experiments of chitin oligosaccharides, (GlcNAc)n (n = 3-6), revealed the ligand-binding site of VcLysM2, in which the Trp96 side chain appeared to interact with the terminal GlcNAc residue of the ligand. We then mutated Trp96 to alanine (VcLysM2-W96A), and the mutant protein was characterized. Based on isothermal titration calorimetry, the affinity of (GlcNAc)6 toward VcLysM2 (-6.9 kcal/mol) was found to be markedly higher than that of (GlcNAc)3 (-4.1 kcal/mol), whereas the difference in affinities between (GlcNAc)6 and (GlcNAc)3 in VcLysM2-W96A (-5.1 and -4.0 kcal/mol, respectively) was only moderate. This suggests that the Trp96 side chain of VcLysM2 interacts with the sugar residue of (GlcNAc)6 not with (GlcNAc)3. VcLysM2 appears to preferentially bind (GlcNAc)n with longer chains and plays a major role in the degradation of the chitinous components of enzyme targets.

A novel analytical procedure for assaying lysozyme activity using an end-blocked chitotetraose derivative as substrate.

An end-modified ß-d-galactosyl chitotetraose derivative [44-O-ß-d-galactosyl-ß-tri-N-acetylchitotriosyl 2-acetamide-2,3-dideoxy-glucopyranose; Gal(GlcN)3D] was designed and synthesized from chitin tetrasaccharide. The derivative was chemically modified by dehydration of the reducing end GlcN and enzymatic addition of a Gal group to the non-reducing end GlcN. Hydrolysis of Gal(GlcN)3D and related compounds using hen egg-white lysozyme was then examined. Gal(GlcN)3D was specifically cleaved to Gal(GlcN)2 and GlcND. Kinetic studies and docking simulations were further conducted to elucidate its mode of binding to lysozyme. These analyses revealed the binding of Gal(GlcN)3D to lysozyme is more favorable than that of (GlcN)4D. We conclude the 4-O-substituted Gal group at the non-reducing end of Gal(GlcN)3D does not prohibit the action of lysozyme, but gives some affinity to the subsite (i.e. equivalent to GlcN). From these results, a new assay method for quantifying lysozyme was established by utilizing the Morgan-Elson reaction based on the generation of product D (2-acetamide-2,3-dideoxy-glucopyranose), which serves as a chromophore, formed from Gal(GlcN)3D by lysozyme through a conjugated reaction involving ß-N-acetylhexosaminidase. The assay system gave a linear dose-response curve in the range of 2-31 µg of lysozyme during a 15 min incubation. This novel assay method for the quantification of lysozyme is highly specific, sensitive, accurate and reproducible.

Chitin oligosaccharide deacetylase from Shewanella baltica ATCC BAA-1091.

Chitin oligosaccharide deacetylase (COD) from bacteria that have been examined so far typically comprise two carbohydrate-binding domains (CBDs) and one polysaccharide deacetylase domain. In contrast, Shewanella baltica ATCC BAA-1091 COD (Sb-COD) has only one CBD, yet exhibits chitin-binding properties and substrate specificities similar to those of other CODs.

Chitin Oligosaccharide (COS) Reduces Antibiotics Dose and Prevents Antibiotics-Caused Side Effects in Adolescent Idiopathic Scoliosis (AIS) Patients with Spinal Fusion Surgery.

Antibiotics are always considered for surgical site infection (SSI) in adolescent idiopathic scoliosis (AIS) surgery. However, the use of antibiotics often causes the antibiotic resistance of pathogens and side effects. Thus, it is necessary to explore natural products as drug candidates. Chitin Oligosaccharide (COS) has anti-inflammation and anti-bacteria functions. The effects of COS on surgical infection in AIS surgery were investigated. A total of 312 AIS patients were evenly and randomly assigned into control group (CG, each patient took one-gram alternative Azithromycin/Erythromycin/Cloxacillin/Aztreonam/Ceftazidime or combined daily), experiment group (EG, each patient took 20 mg COS and half-dose antibiotics daily), and placebo group (PG, each patient took 20 mg placebo and half-dose antibiotics daily). The average follow-up was one month, and infection severity and side effects were analyzed. The effects of COS on isolated pathogens were analyzed. SSI rates were 2%, 3% and 8% for spine wounds and 1%, 2% and 7% for iliac wound in CG, EG and PG (p < 0.05), respectively. COS reduces the side effects caused by antibiotics (p < 0.05). COS improved biochemical indexes and reduced the levels of interleukin (IL)-6 and tumor necrosis factor (TNF) alpha. COS reduced the antibiotics dose and antibiotics-caused side effects in AIS patients with spinal fusion surgery by improving antioxidant and anti-inflammatory activities. COS should be developed as potential adjuvant for antibiotics therapies.

Crystal structures and inhibitor binding properties of plant class V chitinases: the cycad enzyme exhibits unique structural and functional features.

A class V (glycoside hydrolase family 18) chitinase from the cycad Cycas revoluta (CrChiA) is a plant chitinase that has been reported to possess efficient transglycosylation (TG) activity. We solved the crystal structure of CrChiA, and compared it with those of class V chitinases from Nicotiana tabacum (NtChiV) and Arabidopsis thaliana (AtChiC), which do not efficiently catalyze the TG reaction. All three chitinases had a similar (α/ß)8 barrel fold with an (α + ß) insertion domain. In the acceptor binding site (+1, +2 and +3) of CrChiA, the Trp168 side chain was found to stack face-to-face with the +3 sugar. However, this interaction was not found in the identical regions of NtChiV and AtChiC. In the DxDxE motif, which is essential for catalysis, the carboxyl group of the middle Asp (Asp117) was always oriented toward the catalytic acid Glu119 in CrChiA, whereas the corresponding Asp in NtChiV and AtChiC was oriented toward the first Asp. These structural features of CrChiA appear to be responsible for the efficient TG activity. When binding of the inhibitor allosamidin was evaluated using isothermal titration calorimetry, the changes in binding free energy of the three chitinases were found to be similar to each other, i.e. between -9.5 and -9.8 kcal mol(-1) . However, solvation and conformational entropy changes in CrChiA were markedly different from those in NtChiV and AtChiC, but similar to those of chitinase A from Serratia marcescens (SmChiA), which also exhibits significant TG activity. These results provide insight into the molecular mechanism underlying the TG reaction and the molecular evolution from bacterial chitinases to plant class V chitinases.

Functional analyses of chitinases in the moss Physcomitrella patens: chitin oligosaccharide-induced gene expression and enzymatic characterization.

Plant chitinases play diverse roles including defense against pathogenic fungi. Using reverse-transcription quantitative PCR analysis, we found that six chitinase (PpChi) genes and two genes for chitin elicitor receptor kinases (PpCERKs) are expressed at considerable levels in the moss Physcomitrella patens subsp. patens. The expressed PpChis belonged to glycoside hydrolase family 19 (class I: PpChi-Ia and -Ib; class II: PpChi-IIa and -IIc; and class IV: PpChi-IV) and to glycoside hydrolase family 18 (class V: PpChi-Vb). Treatment with chitin tetramer or hexamer increased the expression of class I and IV PpChi genes and decreased that of class II PpChi genes. Recombinant PpChi-Ia, PpChi-IV, and PpChi-Vb were characterized. PpChi-IV exhibited higher activity against chitin tetramer and pentamer than PpChi-Ia did. PpChi-Vb showed transglycosylation activity and PpChi-Ia inhibited fungal growth. These results suggest that chitinases of different classes play different roles in defense mechanism of moss plant against fungal pathogens.

A goose-type lysozyme from ostrich (Struthio camelus) egg white: multiple roles of His101 in its enzymatic reaction.

A goose-type lysozyme from ostrich egg white (OEL) was produced by Escherichia coli expression system, and the role of His101 of OEL in the enzymatic reaction was investigated by NMR spectroscopy, thermal unfolding, and theoretical modeling of the enzymatic hydrolysis of hexa-N-acetylchitohexaose, (GlcNAc)6. Although the binding of tri-N-acetylchitotriose, (GlcNAc)3, to OEL perturbed several backbone resonances in the (1)H-(15)N HSQC spectrum, the chemical shift of the backbone resonance of His101 was not significantly affected. However, apparent pKa values of His101 and Lys102 determined from the pH titration curves of the backbone chemical shifts were markedly shifted by (GlcNAc)3 binding. Thermal unfolding experiments and modeling study of (GlcNAc)6 hydrolysis using a His101-mutated OEL (H101A-OEL) revealed that the His101 mutation affected not only sugar residue affinities at subsites -3 and -2 but also the rate constant for bond cleavage. His101 appears to play multiple roles in the substrate binding and the catalytic reaction.

Crystal structure of a "loopless" GH19 chitinase in complex with chitin tetrasaccharide spanning the catalytic center.

DESCRIPTIONS: The structure of a GH19 chitinase from the moss Bryum coronatum (BcChi-A) in complex with the substrate was examined by X-ray crystallography and NMR spectroscopy in solution. The X-ray crystal structure of the inactive mutant of BcChi-A (BcChi-A-E61A) liganded with chitin tetramer (GlcNAc)4 revealed a clear electron density of the tetramer bound to subsites -2, -1, +1, and +2. Individual sugar residues were recognized by several amino acids at these subsites through a number of hydrogen bonds. This is the first crystal structure of GH19 chitinase liganded with oligosaccharide spanning the catalytic center. NMR titration experiments of chitin oligosaccharides into the BcChi-A-E61A solution showed that the binding mode observed in the crystal structure is similar to that in solution. The C-1 carbon of -1 GlcNAc, the Oε1 atom of the catalytic base (Glu70), and the Oγ atom of Ser102 form a "triangle" surrounding the catalytic water, and the arrangement structurally validated the proposed catalytic mechanism of GH19 chitinases. The glycosidic linkage between -1 and +1 sugars was found to be twisted and under strain. This situation may contribute to the reduction of activation energy for hydrolysis. The complex structure revealed a more refined mechanism of the chitinase catalysis.

Interaction of di-N-acetylchitobiosyl moranoline with a family GH19 chitinase from moss, Bryum coronatum.

Tri-N-acetylchitotriosyl moranoline, (GlcNAc)3-M, was previously shown to strongly inhibit lysozyme (Ogata M, Umemoto N, Ohnuma T, Numata T, Suzuki A, Usui T, Fukamizo T. 2013. A novel transition-state analogue for lysozyme, 4-O-ß-tri-Nacetylchitotriosyl moranoline, provided evidence supporting the covalent glycosyl-enzyme intermediate. J Biol Chem. 288:6072-6082). The findings prompted us to examine the interaction of di-N-acetylchitobiosyl moranoline, (GlcNAc)2-M, with a family GH19 chitinase from moss, Bryum coronatum (BcChi19A). Thermal unfolding experiments using BcChi19A and the catalytic acid-deficient mutant (BcChi19A-E61A) revealed that the transition temperature (Tm) was elevated by 4.3 and 5.8°C, respectively, upon the addition of (GlcNAc)2-M, while the chitin dimer, (GlcNAc)2, elevated Tm only by 1.0 and 1.4°C, respectively. By means of isothermal titration calorimetry, binding free energy changes for the interactions of (GlcNAc)3 and (GlcNAc)2-M with BcChi19A-E61A were determined to be -5.2 and -6.6 kcal/mol, respectively, while (GlcNAc)2 was found to interact with BcChi19A-E61A with markedly lower affinity. nuclear magnetic resonance titration experiments using (15)N-labeled BcChi19A and BcChi19A-E61A revealed that both (GlcNAc)2 and (GlcNAc)2-M interact with the region surrounding the catalytic center of the enzyme and that the interaction of (GlcNAc)2-M is markedly stronger than that of (GlcNAc)2 for both enzymes. However, (GlcNAc)2-M was found to moderately inhibit the hydrolytic reaction of chitin oligosaccharides catalyzed by BcChi19A (IC50 = 130-620 µM). A molecular dynamics simulation of BcChi19A in complex with (GlcNAc)2-M revealed that the complex is quite stable and the binding mode does not significantly change during the simulation. The moranoline moiety of (GlcNAc)2-M did not fit into the catalytic cleft (subsite -1) but was rather in contact with subsite +1. This situation may result in the moderate inhibition toward the BcChi19A-catalyzed hydrolysis.

Characterization of two rice GH18 chitinases belonging to family 8 of plant pathogenesis-related proteins.

Two rice GH18 chitinases, Oschib1 and Oschib2, belonging to family 8 of plant pathogenesis-related proteins (PR proteins) were expressed, purified, and characterized. These enzymes, which have the structural features of class IIIb chitinases, preferentially cleaved the second glycosidic linkage from the non-reducing end of substrate chitin oligosaccharides as opposed to rice class IIIa enzymes, OsChib3a and OsChib3b, which mainly cleaved the fourth linkage from the non-reducing end of chitin hexasaccharide [(GlcNAc)6]. Oschib1 and Oschiab2 inhibited the growth of Fusarium solani, but showed only a weak or no antifungal activity against Aspergillus niger and Trichoderma viride on the agar plates. Structural analysis of Oschib1 and Oschib2 revealed that these enzymes have two large loops extruded from the (ß/α)8 TIM-barrel fold, which are absent in the structures of class IIIa chitinases. The differences in the cleavage site preferences toward chitin oligosaccharides between plant class IIIa and IIIb chitinases are likely attributed to the additional loop structures found in the IIIb enzymes. The class IIIb chitinases, Oschib1 and Oschib2, seem to play important roles for the effective hydrolysis of chitin oligosaccharides released from the cell wall of the pathogenic fungi by the cooperative actions with the extracellular chitinases in rice.

Structural inspection and protein motions modelling of a fungal glycoside hydrolase family 18 chitinase by crystallography depicts a dynamic enzymatic mechanism.

Chitinases degrade chitin into low molecular weight chitooligomers, which have a broad range of industrial, agricultural, and medical functions. Understanding the relationship between the diverse characteristics of chitinases and their functions is necessary for the improvement of functional enzymes that meet specific requirements. We report here a full crystallographic analysis of three complexes obtained from the chitinase Chit42 from Trichoderma harzianum, which represent different states along the enzymatic mechanism. The inactive double mutant D169A/E171A was submitted to soaking/crystallization experiments with hexa-N-acetyl-glucosamine (NAG6) or tetra-N-acetyl-glucosamine (NAG4), trapping the enzyme-substrate complex (Chit42-NAG6), the enzyme-products complex (Chit42-NAG4-NAG2) and a someway intermediate state. Structural comparison among the different complexes depicts the determinants defining the different subsites and revealed a previously unobserved dynamic on-off ligand binding process associated with a motion of its insertion domain, which might be accompanying the role or aromatics in processivity. An ensemble refinement performed to extract dynamic details from the diffraction data elucidates the implication of some highly flexible residues in the productive sliding of the substrate and the product release event. These positions were submitted to mutagenesis and the activity of the variants was investigated in the hydrolysis of NAG6, colloidal chitin and two chitosans with different polymerization and acetylation degree. All the changes affected the Chit42 hydrolytic activity therefore confirming the involvement of these positions in catalysis. Furthermore, we found the variants R295S and E316S improving the apparent catalytic efficiency of chitin and NAG6 and, together with E316A, enhancing the specific activity on chitosan. Therefore, our results provide novel insight into the molecular mechanisms underlying the hydrolysis of chitinous material by fungal chitinases, and suggest new targets to address engineering of these biotechnologically important enzymes.

Structural characterization of two solute-binding proteins for N,N'-diacetylchitobiose/N,N',N''-triacetylchitotoriose of the gram-positive bacterium, Paenibacillus sp. str. FPU-7.

The chitinolytic bacterium Paenibacillus sp. str. FPU-7 efficiently degrades chitin into oligosaccharides such as N-acetyl-D-glucosamine (GlcNAc) and disaccharides (GlcNAc)2 through multiple secretory chitinases. Transport of these oligosaccharides by P. str. FPU-7 has not yet been clarified. In this study, we identified nagB1, predicted to encode a sugar solute-binding protein (SBP), which is a component of the ABC transport system. However, the genes next to nagB1 were predicted to encode two-component regulatory system proteins rather than transmembrane domains (TMDs). We also identified nagB2, which is highly homologous to nagB1. Adjacent to nagB2, two genes were predicted to encode TMDs. Binding experiments of the recombinant NagB1 and NagB2 to several oligosaccharides using differential scanning fluorimetry and surface plasmon resonance confirmed that both proteins are SBPs of (GlcNAc)2 and (GlcNAc)3. We determined their crystal structures complexed with and without chitin oligosaccharides at a resolution of 1.2 to 2.0 Å. The structures shared typical SBP structural folds and were classified as subcluster D-I. Large domain motions were observed in the structures, suggesting that they were induced by ligand binding via the "Venus flytrap" mechanism. These structures also revealed chitin oligosaccharide recognition mechanisms. In conclusion, our study provides insight into the recognition and transport of chitin oligosaccharides in bacteria.