Design of Protease-Responsive Antifungal Liposomal Formulation Decorated with a Lipid-Modified Chitin-Binding Domain.

Candida albicans is a prevalent fungal pathogen that displays antibiotic resistance. The polyene antifungal amphotericin B (AmB) has been the gold standard because of its broad antifungal spectra, and its liposomal formulation, AmBisome, has been used widely and clinically in treating fungal infections. Herein, we explored enhancing the antifungal activity of AmBisome by integrating a small chitin-binding domain (LysM) of chitinase A derived from Pteris ryukyuensis. LysM conjugated with a lipid (LysM-lipid) was initially prepared through microbial transglutaminase (MTG)-mediated peptide tag-specific conjugation of LysM with a lipid-peptide substrate. The AmBisome formulation modified with LysM-lipid conjugates had a size distribution that was comparable to the native liposomes but an increased zeta potential, indicating that LysM-lipid conjugates were anchored to AmBisome. LysM-lipid-modified AmBisome exhibited long-term stability at 4 °C while retaining the capacity to bind chitin. Nevertheless, the antifungal efficacy of LysM-lipid-modified AmBisome against C. albicans was modest. We then redesigned a new LysM-lipid conjugate by introducing a peptide linker containing a thrombin digestion (TD) site at the C-terminus of LysM (LysM-TD linker-lipid), thereby facilitating the liberation of the LysM domain from AmBisome upon the addition of thrombin. This new AmBisome formulation anchored with LysM-TD linker-lipid exhibited superior performance in suppressing C. albicans growth in the presence of thrombin compared with the LysM-lipid formulation. These results provide a platform to design stimuli-responsive AmBisome formulations that respond to external environments and thus advance the treatment of pathogenic fungi infections.

Characterization of an antifungal ß-1,3-glucanase from Ficus microcarpa latex and comparison of plant and bacterial ß-1,3-glucanases for fungal cell wall ß-glucan degradation.

MAIN CONCLUSION: Each ß-1,3-glucanase with antifungal activity or yeast lytic activity hydrolyzes different structures of ß-1,3-glucans in the fungal cell wall, respectively. Plants express several glycoside hydrolases that target chitin and ß-glucan in fungal cell walls and inhibit pathogenic fungal infection. An antifungal ß-1,3-glucanase was purified from gazyumaru (Ficus microcarpa) latex, designated as GlxGluA, and the corresponding gene was cloned and expressed in Escherichia coli. The sequence shows that GlxGluA belongs to glycoside hydrolase family 17 (GH17). To investigate how GlxGluA acts to degrade fungal cell wall ß-glucan, it was compared with ß-1,3-glucanase with different substrate specificities. We obtained recombinant ß-1,3-glucanase (designated as CcGluA), which belongs to GH64, from the bacterium Cellulosimicrobium cellulans. GlxGluA inhibited the growth of the filamentous fungus Trichoderma viride but was unable to lyse the yeast Saccharomyces cerevisiae. In contrast, CcGluA lysed yeast cells but had a negligible inhibitory effect on the growth of filamentous fungi. GlxGluA degraded the cell wall of T. viride better than CcGluA, whereas CcGluA degraded the cell wall of S. cerevisiae more efficiently than GlxGluA. These results suggest that the target substrates in fungal cell walls differ between GlxGluA (GH17 class I ß-1,3-glucanase) and CcGluA (GH64 ß-1,3-glucanase).

Structural Analysis and Construction of a Thermostable Antifungal Chitinase.

Chitin is a biopolymer of N-acetyl-d-glucosamine with ß-1,4-bond and is the main component of arthropod exoskeletons and the cell walls of many fungi. Chitinase (EC 3.2.1.14) is an enzyme that hydrolyzes the ß-1,4-bond in chitin and degrades chitin into oligomers. It has been found in a wide range of organisms. Chitinase from Gazyumaru (Ficus microcarpa) latex exhibits antifungal activity by degrading chitin in the cell wall of fungi and is expected to be used in medical and agricultural fields. However, the enzyme's thermostability is an important factor; chitinase is not thermostable enough to maintain its activity under the actual application conditions. In addition to the fact that thermostable chitinases exhibiting antifungal activity can be used under various conditions, they have some advantages for the production process and long-term preservation, which are highly demanded in industrial use. We solved the crystal structure of chitinase to explore the target sites to improve its thermostability. We rationally introduced proline residues, a disulfide bond, and salt bridges in the chitinase using protein-engineering methods based on the crystal structure and sequence alignment among other chitinases. As a result, we successfully constructed the thermostable mutant chitinases rationally with high antifungal and specific activities. The results provide a useful strategy to enhance the thermostability of this enzyme family. IMPORTANCE We solved the crystal structure of the chitinase from Gazyumaru (Ficus microcarpa) latex exhibiting antifungal activity. Furthermore, we demonstrated that the thermostable mutant enzyme with a melting temperature (Tm) 6.9°C higher than wild type (WT) and a half-life at 60°C that is 15 times longer than WT was constructed through 10 amino acid substitutions, including 5 proline residues substitutions, making disulfide bonding, and building a salt bridge network in the enzyme. These mutations do not affect its high antifungal activity and chitinase activity, and the principle for the construction of the thermostable chitinase was well explained by its crystal structure. Our results provide a useful strategy to enhance the thermostability of this enzyme family and to use the thermostable mutant as a seed for antifungal agents for practical use.

Liposomal Amphotericin B Formulation Displaying Lipid-Modified Chitin-Binding Domains with Enhanced Antifungal Activity.

Fungal infections affect more than one billion people worldwide and cause more than one million deaths per year. Amphotericin B (AmB), a polyene antifungal drug, has been used as the gold standard for many years because of its broad antifungal spectrum, high activity, and low tendency of drug resistance. However, the side effects of AmB, such as nephrotoxicity and hepatotoxicity, have hampered its widespread use, leading to the development of a liposome-type AmB formulation, AmBisome. Herein, we report a simple but highly effective strategy to enhance the antifungal activity of AmBisome with a lipid-modified protein. The chitin-binding domain (LysM) of the antifungal chitinase, Pteris ryukyuensis chitinase A (PrChiA), a small 5.3 kDa protein that binds to fungal cell wall chitin, was engineered to have a glutamine-containing peptide tag at the C-terminus for the microbial transglutaminase (MTG)-catalyzed crosslinking reaction (LysM-Q). LysM-Q was site-specifically modified with a lysine-containing lipid peptide substrate of MTG with a palmitoyl moiety (Pal-K). The resulting palmitoylated LysM (LysM-Pal) exhibited negligible cytotoxicity to mammalian cells and can be easily anchored to yield LysM-presenting AmBisome (LysM-AmBisome). LysM-AmBisome exhibited a dramatic enhancement of antifungal activity toward Trichoderma viride and Cryptococcus neoformans, demonstrating the marked impact of displaying a cell-wall binder protein on the targeting ability of antifungal liposomal formulations. Our simple strategy with enzymatic protein lipidation provides a potent approach to upgrade other types of lipid-based drug formulations.

cDNA cloning, expression, and antifungal activity of chitinase from Ficus microcarpa latex: difference in antifungal action of chitinase with and without chitin-binding domain.

MAIN CONCLUSION: A chitin-binding domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions. Complementary DNA encoding the antifungal chitinase of gazyumaru (Ficus microcarpa), designated GlxChiB, was cloned and expressed in Escherichia coli cells. The results of cDNA cloning showed that the precursor of GlxChiB has an N-terminal endoplasmic reticulum targeting signal and C-terminal vacuolar targeting signal, whereas mature GlxChiB is composed of an N-terminal carbohydrate-binding module family-18 domain (CBM18) and a C-terminal glycoside hydrolase family-19 domain (GH19) with a short linker. To clarify the role of the CBM18 domain in the antifungal activity of chitinase, the recombinant GlxChiB (wild type) and its catalytic domain (CatD) were used in quantitative antifungal assays under different ionic strengths and microscopic observations against the fungus Trichoderma viride. The antifungal activity of the wild type was stronger than that of CatD under all ionic strength conditions used in this assay; however, the antifungal activity of CatD became weaker with increasing ionic strength, whereas that of the wild type was maintained. The results at high ionic strength further verified the contribution of the CBM18 domain to the antifungal ability of GlxChiB. The microscopic observations clearly showed that the wild type acted on both the tips and the lateral wall of fungal hyphae, while CatD acted only on the tips. These results suggest that the CBM18 domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions.

Identification of Genes Involved in the Synthesis of the Fungal Cell Wall Component Nigeran and Regulation of Its Polymerization in Aspergillus luchuensis.

Certain Aspergillus and Penicillium spp. produce the fungal cell wall component nigeran, an unbranched d-glucan with alternating α-1,3- and α-1,4-glucoside linkages, under nitrogen starvation. The mechanism underlying nigeran biosynthesis and the physiological role of nigeran in fungal survival are not clear. We used RNA sequencing (RNA-seq) to identify genes involved in nigeran synthesis in the filamentous fungus Aspergillus luchuensis when grown under nitrogen-free conditions. agsB, which encodes a putative α-1,3-glucan synthase, and two adjacent genes (agtC and gnsA) were upregulated under conditions of nitrogen starvation. Disruption of agsB in A. luchuensis (ΔagsB) resulted in the complete loss of nigeran synthesis. Furthermore, the overexpression of agsB in an Aspergillus oryzae strain that cannot produce nigeran resulted in nigeran synthesis. These results indicated that agsB encodes a nigeran synthase. Therefore, we have renamed the A. luchuensis agsB gene the nigeran synthase gene (nisA). Nigeran synthesis in an agtC mutant (ΔagtC) increased to 121%; conversely, those in the ΔgnsA and ΔagtC ΔgnsA strains decreased to 64% and 63%, respectively, compared to that in the wild-type strain. Our results revealed that AgtC and GnsA play an important role in regulating not only the quantity of nigeran but also its polymerization. Collectively, our results demonstrated that nisA (agsB) is essential for nigeran synthesis in A. luchuensis, whereas agtC and gnsA contribute to the regulation of nigeran synthesis and its polymerization. This research provides insights into fungal cell wall biosynthesis, specifically the molecular evolution of fungal α-glucan synthase genes and the potential utilization of nigeran as a novel biopolymer. IMPORTANCE The fungal cell wall is composed mainly of polysaccharides. Under nitrogen-free conditions, some Aspergillus and Penicillium spp. produce significant levels of nigeran, a fungal cell wall polysaccharide composed of alternating α-1,3/1,4-glucosidic linkages. The mechanisms regulating the biosynthesis and function of nigeran are unknown. Here, we performed RNA sequencing of Aspergillus luchuensis cultured under nitrogen-free or low-nitrogen conditions. A putative α-1,3-glucan synthase gene, whose transcriptional level was upregulated under nitrogen-free conditions, was demonstrated to encode nigeran synthase. Furthermore, two genes encoding an α-glucanotransferase and a hypothetical protein were shown to be involved in controlling the nigeran content and molecular weight. This study reveals genes involved in the synthesis of nigeran, a potential biopolymer, and provides a deeper understanding of fungal cell wall biosynthesis.

Orthogonal Enzymatic Conjugation Reactions Create Chitin Binding Domain Grafted Chitinase Polymers with Enhanced Antifungal Activity.

Enzymatic reaction offers site-specific conjugation of protein units to form protein conjugates or protein polymers with intrinsic functions. Herein, we report horseradish peroxidase (HRP)- and microbial transglutaminase (MTG)-catalyzed orthogonal conjugation reactions to create antifungal protein polymers composed of Pteris ryukyuensis chitinase-A (ChiA) and its two domains, catalytic domain, CatD, and chitin-binding domain, LysM2. We engineered the ChiA and CatD by introducing a peptide tag containing tyrosine (Y-tag) at N-termini and a peptide tag containing lysine and tyrosine (KY-tag) at C-termini to construct Y-ChiA-KY and Y-CatD-KY. Also, LysM2 with Y-tag and KY-tag (Y-LysM2-KY) or with a glutamine-containing peptide tag (Q-tag) (LysM2-Q) were constructed. The proteins with Y-tag and KY-tag were efficiently polymerized by HRP reaction through the formation of dityrosine bonds at the tyrosine residues in the peptide tags. The Y-CatD-KY polymer was further treated by MTG to orthogonally graft LysM2-Q to the KY-tag via isopeptide formation between the side chains of the glutamine and lysine residues in the peptide tags to form LysM2-grafted CatD polymer. The LysM2-grafted CatD polymer exhibited significantly higher antifungal activity than the homopolymer of Y-ChiA-KY and the random copolymer of Y-CatD-KY and Y-LysM2-KY, demonstrating that the structural differences of artificial chitinase polymers have a significant impact on the antifungal activity. This strategy of polymerization and grafting reaction of protein can contribute to the further research and development of functional protein polymers for specific applications in various fields in biotechnology.

Mode of action and specificity of a chitinase from unicellular microalgae, Euglena gracilis.

KEY MESSAGE: Euglena gracilis is a unicellular microalga showing characteristics of both plants and animals, and extensively used as a model organism in the research works of biochemistry and molecular biology. Biotechnological applications of E. gracilis have been conducted for production of numerous important compounds. However, chitin-mediated defense system intensively studied in higher plants remains to be investigated in this microalga. Recently, Taira et al. (Biosci Biotechnol Biochem 82:1090-1100, 2018) isolated a unique chitinase gene, comprising two catalytic domains almost homologous to each other (Cat1 and Cat2) and two chitin-binding domains (CBD1 and CBD2), from E. gracilis. We herein examined the mode of action and the specificity of the recombinant Cat2 by size exclusion chromatography and NMR spectroscopy. Both Cat1 and Cat2 appeared to act toward chitin substrate with non-processive/endo-splitting mode, recognizing two contiguous N-acetylglucosamine units at subsites - 2 and - 1. This is the first report on a chitinase having two endo-splitting catalytic domains. A cooperative action of two different endo-splitting domains may be advantageous for defensive action of the E. gracilis chitinase. The unicellular alga, E. gracilis, produces a chitinase consisting of two GH18 catalytic domains (Cat1 and Cat2) and two CBM18 chitin-binding domains (CBD1 and CBD2). Here, we produced a recombinant protein of the Cat2 domain to examine its mode of action as well as specificity. Cat2 hydrolyzed N-acetylglucosamine (A) oligomers (An, n = 4, 5, and 6) and partially N-acetylated chitosans with a non-processive/endo-splitting mode of action. NMR analysis of the product mixture from the enzymatic digestion of chitosan revealed that the reducing ends were exclusively A-unit, and the nearest neighbors of the reducing ends were mostly A-unit but not exclusively. Both A-unit and D-unit were found at the non-reducing ends and the nearest neighbors. These results indicated strong and absolute specificities for subsites - 2 and - 1, respectively, and no preference for A-unit at subsites + 1 and + 2. The same results were obtained from sugar sequence analysis of the individual enzymatic products from the chitosans. The subsite specificities of Cat2 are similar to those of GH18 human chitotriosidase, but differ from those of plant GH18 chitinases. Since the structures of Cat1 and Cat2 resemble to each other (99% similarity in amino acid sequences), Cat1 may hydrolyze the substrate with the same mode of action. Thus, the E. gracilis chitinase appears to act toward chitin polysaccharide chain through a cooperative action of the two endo-splitting catalytic domains, recognizing two contiguous A-units at subsites - 2 and - 1.

Comparative multi-omics analysis reveals diverse latex-based defense strategies against pests among latex-producing organs of the fig tree (Ficus carica).

MAIN CONCLUSION: Latexes in immature fruit, young petioles and lignified trunks of fig trees protect the plant using toxic proteins and metabolites in various organ-dependent ways. Latexes from plants contain high amounts of toxic proteins and metabolites, which attack microbes and herbivores after exudation at pest-induced wound sites. The protein and metabolite constituents of latexes are highly variable, depending on the plant species and organ. To determine the diversity of latex-based defense strategies in fig tree (Ficus carica) organs, we conducted comparative proteomic, transcriptomic and metabolomic analyses on latexes isolated from immature fruit, young petioles and lignified trunks of F. carica after constructing a unigene sequence library using RNA-seq data. Trypsin inhibitors were the most abundant proteins in petiole latex, while cysteine proteases ("ficins") were the most abundant in immature fruit and trunk latexes. Galloylglycerol, a possible defense-related metabolite, appeared to be highly accumulated in all three latexes. The expression levels of pathogenesis-related proteins were highest in the latex of trunk, suggesting that this latex had adapted a defensive role against microbe attacks. Although young petioles and immature fruit are both unlignified soft organs, and potential food for herbivorous insects, unigenes for the sesquiterpenoid pathway, which likely produces defense-associated volatiles, and the phenylpropanoid pathway, which produces toxic furanocoumarins, were expressed less in immature fruit latex. This difference may indicate that while petioles and fruit protect the plant from attack by herbivores, the fruit must also attract insect pollinators at younger stages and animals after ripening. We also suggest possible candidate transcription factors and signal transduction proteins that are involved in the differential expression of the unigenes.

Unique GH18 chitinase from Euglena gracilis: full-length cDNA cloning and characterization of its catalytic domain.

A cDNA of putative chitinase from Euglena gracilis, designated EgChiA, encoded 960 amino acid residues, which is arranged from N-terminus in the order of signal peptide, glycoside hydrolase family 18 (GH18) domain, carbohydrate binding module family 18 (CBM18) domain, GH18 domain, CBM18 domain, and transmembrane helix. It is likely that EgChiA is anchored on the cell surface. The recombinant second GH18 domain of EgChiA, designated as CatD2, displayed optimal catalytic activity at pH 3.0 and 50 °C. The lower the polymerization degree of the chitin oligosaccharides [(GlcNAc)4-6] used as the substrates, the higher was the rate of degradation by CatD2. CatD2 degraded chitin nanofibers as an insoluble substrate, and it produced only (GlcNAc)2 and GlcNAc. Therefore, we speculated that EgChiA localizes to the cell surface of E. gracilis and is involved in degradation of chitin polymers into (GlcNAc)2 or GlcNAc, which are easily taken up by the cells.

Purification, cDNA cloning, and characterization of plant chitinase with a novel domain combination from lycophyte Selaginella doederleinii.

Chitinase-A from a lycophyte Selaginella doederleinii (SdChiA), having molecular mass of 53 kDa, was purified to homogeneity by column chromatography. The cDNA encoding SdChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1477 nucleotides and its open reading frame encoded a polypeptide of 467 amino acid residues. The deduced amino acid sequence indicated that SdChiA consisted of two N-terminal chitin-binding domains and a C-terminal plant class V chitinase catalytic domain, belonging to the carbohydrate-binding module family 18 (CBM18) and glycoside hydrolase family 18 (GH18), respectively. SdChiA had chitin-binding ability. The time-dependent cleavage pattern of (GlcNAc)4 by SdChiA showed that SdChiA specifically recognizes the ß-anomer in the + 2 subsite of the substrate (GlcNAc)4 and cleaves the glycoside bond at the center of the substrate. This is the first report of the occurrence of a family 18 chitinase containing CBM18 chitin-binding domains. ABBREVIATIONS: AtChiC: Arabidopsis thaliana class V chitinase; CBB: Coomassie brilliant blue R250; CBM: carbohydrate binding module family; CrChi-A: Cycas revolute chitinase-A; EaChiA: Equisetum arvense chitinase-A; GH: glycoside hydrolase family, GlxChi-B: gazyumaru latex chitinase-B; GlcNAc: N-acetylglucosamine; HPLC: high performance liquid chromatography; LysM; lysin motif; MtNFH1: Medicago truncatula ecotypes R108-1 chitinase; NCBI: national center for biotechnology information; NF: nodulation factor; NtChiV: Nicotiana tabacum class V chitinase; PCR: polymerase chain reaction; PrChi-A: Pteris ryukyuensis chitinase-A; RACE: rapid amplification of cDNA ends; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SdChiA: Selaginella doederleinii chitinase-A.

Crystal structure and thermodynamic dissection of chitin oligosaccharide binding to the LysM module of chitinase-A from Pteris ryukyuensis.

We determined the crystal structure of a LysM module from Pteris ryukyuensis chitinase-A (PrLysM2) at a resolution of 1.8 Å. Structural and binding analysis of PrLysM2 indicated that this module recognizes chitin oligosaccharides in a shallow groove comprised of five sugar-binding subsites on one side of the molecule. The free energy changes (ΔGr°) for binding of (GlcNAc)6, (GlcNAc)5, and (GlcNAc)4 to PrLysM2 were determined to be -5.4, -5,4 and -4.6 kcal mol-1, respectively, by ITC. Thermodynamic dissection of the binding energetics of (GlcNAc)6 revealed that the driving force is the enthalpy change (ΔHr° = -11.7 ± 0.2 kcal/mol) and the solvation entropy change (-TΔSsolv° = -5.9 ± 0.6 kcal/mol). This is the first description of thermodynamic signatures of a chitin oligosaccharide binding to a LysM module.

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.

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.

A class III chitinase without disulfide bonds from the fern, Pteris ryukyuensis: crystal structure and ligand-binding studies.

MAIN CONCLUSION: We first solved the crystal structure of class III catalytic domain of a chitinase from fern (PrChiA-cat), and found a structural difference between PrChiA-cat and hevamine. PrChiA-cat was found to have reduced affinities to chitin oligosaccharides and allosamidin. Plant class III chitinases are subdivided into enzymes with three disulfide bonds and those without disulfide bonds. We here referred to the former enzymes as class IIIa chitinases and the latter as class IIIb chitinases. In this study, we solved the crystal structure of the class IIIb catalytic domain of a chitinase from the fern Pteris ryukyuensis (PrChiA-cat), and compared it with that of hevamine, a class IIIa chitinase from Hevea brasiliensis. PrChiA-cat was found to adopt an (α/ß)8 fold typical of GH18 chitinases in a similar manner to that of hevamine. However, PrChiA-cat also had two large loops that extruded from the catalytic site, and the corresponding loops in hevamine were markedly smaller than those of PrChiA-cat. An HPLC analysis of the enzymatic products revealed that the mode of action of PrChiA-cat toward chitin oligosaccharides, (GlcNAc) n (n = 4-6), differed from those of hevamine and the other class IIIa chitinases. The binding affinities of (GlcNAc)3 and (GlcNAc)4 toward the inactive mutant of PrChiA-cat were determined by isothermal titration calorimetry, and were markedly lower than those toward other members of the GH18 family. The affinity and the inhibitory activity of allosamidin toward PrChiA-cat were also lower than those toward the GH18 chitinases investigated to date. Several hydrogen bonds found in the crystal structure of hevamine-allosamidin complex were missing in the modeled structure of PrChiA-cat-allosamidin complex. The structural findings for PrChiA-cat successfully interpreted the functional data presented.

Purification, cDNA cloning, and characterization of LysM-containing plant chitinase from horsetail (Equisetum arvense).

Chitinase-A (EaChiA), molecular mass 36 kDa, was purified from the vegetative stems of a horsetail (Equisetum arvense) using a series of column chromatography. The N-terminal amino acid sequence of EaChiA was similar to the lysin motif (LysM). A cDNA encoding EaChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1320 nucleotides and encoded an open reading frame of 361 amino acid residues. The deduced amino acid sequence indicated that EaChiA is composed of a N-terminal LysM domain and a C-terminal plant class IIIb chitinase catalytic domain, belonging to the glycoside hydrolase family 18, linked by proline-rich regions. EaChiA has strong chitin-binding activity, however, no antifungal activity. This is the first report of a chitinase from Equisetopsida, a class of fern plants, and the second report of a LysM-containing chitinase from a plant.

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.

The mannobiose-forming exo-mannanase involved in a new mannan catabolic pathway in Bacteroides fragilis.

We have proposed a new mannan catabolic pathway in Bacteroides fragilis NCTC 9343 that involves a putative mannanase ManA in glycoside hydrolase family 26 (BF0771), a mannobiose and/or sugar transporter (BF0773), mannobiose 2-epimerase (BF0774), and mannosylglucose phosphorylase (BF0772). If this hypothesis is correct, ManA has to generate mannobiose from mannans as the major end product. In this study, the BF0771 gene from the B. fragilis genome was cloned and expressed in Escherichia coli cells. The expressed protein was found to produce mannobiose exclusively from mannans and initially from manno-oligosaccharides. Production of 4-O-ß-D-glucopyranosyl-D-mannose or 4-O-ß-D-mannopyranosyl-D-glucose from mannans was not detectable. The results indicate that this enzyme is a novel mannobiose-forming exo-mannanase, consistent with the new microbial mannan catabolic pathway we proposed.

Crystallization and preliminary X-ray diffraction analysis of an active-site mutant of `loopless' family GH19 chitinase from Bryum coronatum in a complex with chitotetraose.

The catalytic mechanism of family GH19 chitinases is not well understood owing to insufficient information regarding the three-dimensional structures of enzyme-substrate complexes. Here, the crystallization and preliminary X-ray diffraction analysis of a selenomethionine-labelled active-site mutant of `loopless' family GH19 chitinase from the moss Bryum coronatum in complex with chitotetraose, (GlcNAc)4, are reported. The crystals were grown using the vapour-diffusion method. They diffracted to 1.58 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 74.5, b = 58.4, c = 48.1 Å, ß = 115.6°. The asymmetric unit of the crystals is expected to contain one protein molecule, with a Matthews coefficient of 2.08 Å(3) Da(-1) and a solvent content of 41%.