A three-step "ping-pong" mechanism of a GH20 ß-N-acetylglucosaminidase from Vibrio campbellii belonging to a major Clade A-I of the phylogenetic tree of the enzyme superfamily.

ß-N-acetylglucosaminidase (GlcNAcase) is an essential biocatalyst in chitin assimilation by marine Vibrio species, which rely on chitin as their main carbon source. Structure-based phylogenetic analysis of the GlcNAcase superfamily revealed that a GlcNAcase from Vibrio campbellii, formerly named V. harveyi, (VhGlcNAcase) belongs to a major clade, Clade A-I, of the phylogenetic tree. Pre-steady-state and steady-state kinetic analysis of the reaction catalysed by VhGlcNAcase with the fluorogenic substrate 4-methylumbelliferyl N-acetyl-ß-D-glucosaminide suggested the following mechanism: (1) the Michaelis-Menten complex is formed in a rapid enzyme-substrate equilibrium with a Kd of 99.1 ± 1 µM. (2) The glycosidic bond is cleaved by the action of the catalytic residue Glu438, followed by the rapid release of the aglycone product with a rate constant (k2) of 53.3 ± 1 s-1. (3) After the formation of an oxazolinium ion intermediate with the assistance of Asp437, the anomeric carbon of the transition state is attacked by a catalytic water, followed by release of the glycone product with a rate constant (k3) of 14.6 s-1, which is rate-limiting. The result clearly indicated a three-step "ping-pong" mechanism for VhGlcNAcase.

Periplasmic chitooligosaccharide-binding protein requires a three-domain organization for substrate translocation.

Periplasmic solute-binding proteins (SBPs) specific for chitooligosaccharides, (GlcNAc)n (n = 2, 3, 4, 5 and 6), are involved in the uptake of chitinous nutrients and the negative control of chitin signal transduction in Vibrios. Most translocation processes by SBPs across the inner membrane have been explained thus far by two-domain open/closed mechanism. Here we propose three-domain mechanism of the (GlcNAc)n translocation based on experiments using a recombinant VcCBP, SBP specific for (GlcNAc)n from Vibrio cholerae. X-ray crystal structures of unliganded or (GlcNAc)3-liganded VcCBP solved at 1.2-1.6 Å revealed three distinct domains, the Upper1, Upper2 and Lower domains for this protein. Molecular dynamics simulation indicated that the motions of the three domains are independent and that in the (GlcNAc)3-liganded state the Upper2/Lower interface fluctuated more intensively, compared to the Upper1/Lower interface. The Upper1/Lower interface bound two GlcNAc residues tightly, while the Upper2/Lower interface appeared to loosen and release the bound sugar molecule. The three-domain mechanism proposed here was fully supported by binding data obtained by thermal unfolding experiments and ITC, and may be applicable to other translocation systems involving SBPs belonging to the same cluster.

Green-Chemical Strategies for Production of Tailor-Made Chitooligosaccharides with Enhanced Biological Activities.

Chitooligosaccharides (COSs) are b-1,4-linked homo-oligosaccharides of N-acetylglucosamine (GlcNAc) or glucosamine (GlcN), and also include hetero-oligosaccharides composed of GlcNAc and GlcN. These sugars are of practical importance because of their various biological activities, such as antimicrobial, anti-inflammatory, antioxidant and antitumor activities, as well as triggering the innate immunity in plants. The reported data on bioactivities of COSs used to contain some uncertainties or contradictions, because the experiments were conducted with poorly characterized COS mixtures. Recently, COSs have been satisfactorily characterized with respect to their structures, especially the degree of polymerization (DP) and degree of N-acetylation (DA); thus, the structure-bioactivity relationship of COSs has become more unambiguous. To date, various green-chemical strategies involving enzymatic synthesis of COSs with designed sequences and desired biological activities have been developed. The enzymatic strategies could involve transglycosylation or glycosynthase reactions using reducing end-activated sugars as the donor substrates and chitinase/chitosanase and their mutants as the biocatalysts. Site-specific chitin deacetylases were also proposed to be applicable for this purpose. Furthermore, to improve the yields of the COS products, metabolic engineering techniques could be applied. The above-mentioned approaches will provide the opportunity to produce tailor-made COSs, leading to the enhanced utilization of chitin biomass.

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.

Plant Chitinase Mutants as the Catalysts for Chitooligosaccharide Synthesis Using the Sugar Oxazoline Derivatives.

Sugar oxazolines, (GlcNAc)n-oxa (n = 2, 3, 4, and 5), were synthesized from a mixture of chitooligosaccharides, (GlcNAc)n (n = 2, 3, 4, and 5), and utilized for synthesis of (GlcNAc)7 with higher elicitor activity using plant chitinase mutants as the catalysts. From isothermal titration calorimetry, the binding affinity of (GlcNAc)2-oxa toward an inactive mutant obtained from Arabidopsis thaliana GH18 chitinase was found to be higher than those of the other (GlcNAc)n-oxa (n = 3, 4, and 5). To synthesize (GlcNAc)7, the donor/acceptor substrates with different size combinations, (GlcNAc)2-oxa/(GlcNAc)5 (1), (GlcNAc)3-oxa/(GlcNAc)4 (2), (GlcNAc)4-oxa/(GlcNAc)3 (3), and (GlcNAc)5-oxa/(GlcNAc)2 (4), were incubated with hypertransglycosylating mutants of GH18 chitinases from A. thaliana and Cycas revoluta. The synthetic activities of these plant chitinase mutants were lower than that of a mutant of Bacillus circulans chitinase A1. Nevertheless, in the plant chitinase mutants, the synthetic efficiency of combination (1) was higher than those of the other combinations (2), (3), and (4), suggesting that the synthetic reaction is mostly dominated by the binding affinities of (GlcNAc)n-oxa. In contrast, the Bacillus enzyme mutant with a different subsite arrangement synthesized (GlcNAc)7 from combination (1) in the lowest efficiency. Donor/acceptor-size dependency of the enzymatic synthesis appeared to be strongly related to the subsite arrangement of the enzyme used as the catalyst. The A. thaliana chitinase mutant was found to be useful when combination (1) is employed for the substrates.

Structure, mechanism, and phylogeny of LysM-chitinase conjugates specifically found in fern plants.

A unique GH18 chitinase containing two N-terminal lysin motifs (PrLysM1 and PrLysM2) was first found in fern, Pteris ryukyuensis (Onaga and Taira, Glycobiology, 18, 414-423, 2008). This type of LysM-chitinase conjugates is not usually found in plants but in fungi. Here, we produced a similar GH18 chitinase with one N-terminal LysM module (EaLysM) from the fern, Equisetum arvense (EaChiA, Inamine et al., Biosci. Biotechnol. Biochem., 79, 1296-1304, 2015), using an Escherichia coli expression system and characterized for its structure and mechanism of action. The crystal structure of EaLysM exhibited an almost identical fold (ßααß) to that of PrLysM2. From isothermal titration calorimetry and nuclear magnetic resonance, the binding mode and affinities of EaLysM for chitooligosaccharides (GlcNAc)n (3, 4, 5, and 6) were found to be comparable to those of PrLysM2. The LysM module in EaChiA is likely to bind (GlcNAc)n almost independently through CH-π stacking of a Tyr residue with the pyranose ring. The (GlcNAc)n-binding mode of LysMs in the LysM-chitinase conjugates from fern plants appears to differ from that of plant LysMs acting in chitin- or Nod-signal perception, in which multiple LysMs cooperatively act on (GlcNAc)n. Phylogenetic analysis suggested that LysM-GH18 conjugates of fern plants formed a monophyletic group and had been separated earlier than forming the clade of fungal chitinases with LysMs.

A conserved loop structure of GH19 chitinases assists the enzyme function from behind the core-functional region.

Plant GH19 chitinases have several loop structures, which may define their enzymatic properties. Among these loops, the longest loop, Loop-III, is most frequently conserved in GH19 enzymes. A GH19 chitinase from the moss Bryum coronatum (BcChi-A) has only one loop structure, Loop-III, which is connected to the catalytically important ß-sheet region. Here, we produced and characterized a Loop-III-deleted mutant of BcChi-A (BcChi-A-ΔIII) and found that its stability and chitinase activity were strongly reduced. The deletion of Loop-III also moderately affected the chitooligosaccharide binding ability as well as the binding mode to the substrate-binding groove. The crystal structure of an inactive mutant of BcChi-A-ΔIII was successfully solved, revealing that the remaining polypeptide chain has an almost identical fold to that of the original protein. Loop-III is not necessarily essential for the folding of the enzyme protein. However, closer examination of the crystal structure revealed that the deletion of Loop-III altered the arrangement of the catalytic triad, Glu61, Glu70 and Ser102, and the orientation of the Trp103 side chain, which is important for sugar residue binding. We concluded that Loop-III is not directly involved in the enzymatic activity but assists the enzyme function by stabilizing the conformation of the ß-sheet region and the adjacent substrate-binding platform from behind the core-functional regions.

Bioeconomic production of high-quality chitobiose from chitin food wastes using an in-house chitinase from Vibrio campbellii.

Marine Vibrio species are natural degraders of chitin and usually secrete high levels of chitinolytic enzymes to digest recalcitrant chitin to chitooligosaccharides. This study used an endochitinase (VhChiA) from Vibrio campbellii to produce high-quality chitobiose from crustacean chitins. The enzyme was shown to be fully active and stable over 24 h when BSA was used as an additive. When different chitin sources were tested, VhChiA preferentially digested shrimp and squid (α) chitins compared to crab (ß) chitin and did not utilize non-chitin substrates. The overall yields of chitobiose obtained from small-scale production using a single-step reaction was 96% from shrimp, and 91% from squid pen and crab-shell chitins. Larger-scale production yielded 200 mg of chitobiose, with > 99% purity after a desalting and purification step using preparative HPLC. In conclusion, we report the employment of an in-house produced chitinase as an effective biocatalyst to rapidly convert chitin food wastes to chitobiose, in a quantity and quality suitable for use in research and commercial purposes. Chitobiose production by this economical and eco-friendly approach can be easily scaled up to obtain multi-gram quantities of chitobiose for chemo-enzymic synthesis of rare chitooligosaccharide derivatives and long chain chitooligosaccharides, as well as preparation of sugar-based functionalized nanomaterials.

A structural model for (GlcNAc)2 translocation via a periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria.

VhCBP is a periplasmic chitooligosaccharide-binding protein mainly responsible for translocation of the chitooligosaccharide (GlcNAc)2 across the double membranes of marine bacteria. However, structural and thermodynamic understanding of the sugar-binding/-release processes of VhCBP is relatively less. VhCBP displayed the greatest affinity toward (GlcNAc)2, with lower affinity for longer-chain chitooligosaccharides [(GlcNAc)3-4]. (GlcNAc)4 partially occupied the closed sugar-binding groove, with two reducing-end GlcNAc units extending beyond the sugar-binding groove and barely characterized by weak electron density. Mutation of three conserved residues (Trp363, Asp365, and Trp513) to Ala resulted in drastic decreases in the binding affinity toward the preferred substrate (GlcNAc)2, indicating their significant contributions to sugar binding. The structure of the W513A-(GlcNAc)2 complex in a 'half-open' conformation unveiled the intermediary step of the (GlcNAc)2 translocation from the soluble CBP in the periplasm to the inner membrane-transporting components. Isothermal calorimetry data suggested that VhCBP adopts the high-affinity conformation to bind (GlcNAc)2, while its low-affinity conformation facilitated sugar release. Thus, chitooligosaccharide translocation, conferred by periplasmic VhCBP, is a crucial step in the chitin catabolic pathway, allowing Vibrio bacteria to thrive in oceans where chitin is their major source of nutrients.

Thermodynamic Analysis for Binding of 4-O-ß-tri-N-acetylchitotriosyl Moranoline, a Transition State Analogue Inhibitor for Hen Egg White Lysozyme.

4-O-ß-tri-N-acetylchitotriosyl moranoline (GN3M) is a transition-state analogue for hen egg white lysozyme (HEWL) and identified as the most potent inhibitor till date. Isothermal titration calorimetry experiments provided the thermodynamic parameters for binding of GN3M to HEWL and revealed that the binding is driven by a favorable enthalpy change (ΔH° = -11.0 kcal/mol) with an entropic penalty (-TΔS° = 2.6 kcal/mol), resulting in a free energy change (ΔG°) of -8.4 kcal/mol [Ogata et al. (2013) 288, 6,072-6,082]. Dissection of the entropic term showed that a favorable solvation entropy change (-TΔS solv° = -9.2 kcal/mol) is its sole contributor. The change in heat capacity (ΔC p°) for the binding of GN3M was determined to be -120.2 cal/K·mol. These results indicate that the bound water molecules play a crucial role in the tight interaction between GN3M and HEWL.

An electrochemical method for detecting the biomarker 4-HPA by allosteric activation of Acinetobacterbaumannii reductase C1 subunit.

The C1 (reductase) subunit of 4-hydroxy-phenylacetate (4-HPA) 3-hydroxylase (HPAH) from the soil-based bacterium Acinetobacterbaumannii catalyzes NADH oxidation by molecular oxygen, with hydrogen peroxide as a by-product. 4-HPA is a potent allosteric modulator of C1, but also a known urinary biomarker for intestinal bacterial imbalance and for some cancers and brain defects. We thus envisioned that C1 could be used to facilitate 4-HPA detection. The proposed test protocol is simple and in situ and involves addition of NADH to C1 in solution, with or without 4-HPA, and direct acquisition of the H2O2 current with an immersed Prussian Blue-coated screen-printed electrode (PB-SPE) assembly. We confirmed that cathodic H2O2 amperometry at PB-SPEs is a reliable electrochemical assay for intrinsic and allosterically modulated redox enzyme activity. We further validated this approach for quantitative NADH electroanalysis and used it to evaluate the activation of NADH oxidation of C1 by 4-HPA and four other phenols. Using 4-HPA, the most potent effector, allosteric activation of C1 was related to effector concentration by a simple saturation function. The use of C1 for cathodic biosensor analysis of 4-HPA is the basis of the development of a simple and affordable clinical routine for assaying 4-HPA in the urine of patients with a related disease risk. Extension of this principle to work with other allosteric redox enzymes and their effectors is feasible.

Multi-functionality of a tryptophan residue conserved in substrate-binding groove of GH19 chitinases.

GH19 and GH22 glycoside hydrolases belonging to the lysozyme superfamily have a related structure/function. A highly conserved tryptophan residue, Trp103, located in the binding groove of a GH19 chitinase from moss Bryum coronatum (BcChi-A) appears to have a function similar to that of well-known Trp62 in GH22 lysozymes. Here, we found that mutation of Trp103 to phenylalanine (W103F) or alanine (W103A) strongly reduced the enzymatic activity of BcChi-A. NMR experiments and the X-ray crystal structure suggested a hydrogen bond between the Trp103 side chain and the -2 sugar. Chitooligosaccharide binding experiments using NMR indicated that the W103F mutation reduced the sugar-binding abilities of nearby amino acid residues (Tyr105/Asn106) in addition to Trp103. This appeared to be derived from enhanced aromatic stacking of Phe103 with Tyr105 induced by disruption of the Trp103 hydrogen bond with the -2 sugar. Since the stacking with Tyr105 was unlikely in W103A, Tyr105/Asn106 of W103A was not so affected as in W103F. However, the W103A mutation appeared to reduce the catalytic potency, resulting in the lowest enzymatic activity in W103A. We concluded that Trp103 does not only interact with the sugar, but also controls other amino acids responsible for substrate binding and catalysis. Trp103 (GH19) and Trp62 (GH22) with such a multi-functionality may be advantageous for enzyme action and conserved in the divergent evolution in the lysozyme superfamily.

Kinetic and thermodynamic insights into the inhibitory mechanism of TMG-chitotriomycin on Vibrio campbellii GH20 exo-ß-N-acetylglucosaminidase.

We investigated the inhibition kinetics of VhGlcNAcase, a GH20 exo-ß-N-acetylglucosaminidase (GlcNAcase) from the marine bacterium Vibrio campbellii (formerly V. harveyi) ATCC BAA-1116, using TMG-chitotriomycin, a natural enzyme inhibitor specific for GH20 GlcNAcases from chitin-processing organisms, with p-nitrophenyl N-acetyl-ß-d-glucosaminide (pNP-GlcNAc) as the substrate. TMG-chitotriomycin inhibited VhGlcNAcase with an IC50 of 3.0 ± 0.7 µM. Using Dixon plots, the inhibition kinetics indicated that TMG-chitotriomycin is a competitive inhibitor, with an inhibition constant Ki of 2.2 ± 0.3 µM. Isothermal titration calorimetry experiments provided the thermodynamic parameters for the binding of TMG-chitotriomycin to VhGlcNAcase and revealed that binding was driven by both favorable enthalpy and entropy changes (ΔH° = -2.5 ± 0.1 kcal/mol and -TΔS° = -5.8 ± 0.3 kcal/mol), resulting in a free energy change, ΔG°, of -8.2 ± 0.2 kcal/mol. Dissection of the entropic term showed that a favorable solvation entropy change (-TΔSsolv° = -16 ± 2 kcal/mol) is the main contributor to the entropic term.

Potent Fungal Chitinase for the Bioconversion of Mycelial Waste.

Aspergillus niger mycelial waste is a good raw material for production of N-acetyl-d-glucosamine (GlcNAc). In this study, AnChiB, an A. niger chitinase which is upregulated during autolysis, was found to degrade A. niger mycelial waste with high efficiency. It could produce 1.45 mM (GlcNAc)2 in 8 h from raw mycelial waste, outperforming other chitinases, including bacterial SmChiA, human HsCht, and insect OfChtI and OfChi-h. The crystal structure of AnChiB was determined, and residues Trp106 and Trp118 were found to be important for the activity of AnChiB toward mycelial waste; mutation of either Trp106 or Trp118 into phenylalanine or alanine resulted in dramatically decreased activity. A recombinant strain of Bacillus subtilis was constructed to extracellularly produce AnChiB, and the culture supernatant was used to treat mycelial waste. This eco-friendly strategy could produce 3.7 mM of GlcNAc from 10 g of mycelial waste in 94 h with a yield of 71.3%.

Crystal structure and biochemical characterization of CJP38, a ß-1,3-glucanase and allergen of Cryptomeria japonica pollen.

A 38 kDa ß-1,3-glucanase allergen from Cryptomeria japonica pollen (CJP38) was recombinantly produced in E. coli and purified to homogeneity with the use of Ni-affinity resin. CJP38 hydrolyzed ß-1,3-glucans such as CM-curdlan and laminarioligosaccharides in an endo-splitting manner. The optimum pH and temperature for ß-1,3-glucanase activity were approximately 4.5 and 50 °C, respectively. The enzyme was stable at 30-60 °C and pH 4.0-10.5. Furthermore, CJP38 catalyzed a transglycosylation reaction to yield reaction products with a molecular weight higher than those of the starting laminarioligosaccharide substrates. The three-dimensional structure of CJP38 was determined using X-ray crystallography at 1.5 Å resolution. CJP38 exhibited the typical (ß/α)8 TIM-barrel motif, similar to allergenic ß-1,3-glucanases from banana (Mus a 5) and rubber tree latex (Hev b 2). Amino acid sequence alignment of these proteins indicated that the two-consensus IgE epitopes identified on the molecular surfaces of Mus a 5 and Hev b 2 were highly conserved in CJP38. Their conformations and surface locations were quite similar for these proteins. Sequence and structural conservation of these regions suggest that CJP38 is a candidate allergen responsible for the pollen-latex-fruit syndrome relating to Japanese cedar pollinosis.

An Introduction to the Book.

Chitin is a linear biopolymer composed of ß-1,4-linked N-acetylglucosamine (GlcNAc), and an essential component in exoskeleton of insects and crustaceans, the egg shells of parasitic nematodes, and the fungal cell wall. Since the chitin-containing organisms often threaten human health, food safety, and agricultural production, it has been highly desirable to control the hazardous chitin-containing organisms. This book will provide researchers and students with information on the recent research progress about the biology of chitin-containing organisms and their cross-talk with other organisms. This book also contains essential knowledge of drug design for controlling chitin-containing organisms. The authors deeply hope this book brings more attention to the fascinating yet unexploited world of chitin. We would like to thank all contributors for their expertise and generous support.

Chitin/Chitosan-Active Enzymes Involved in Plant-Microbe Interactions.

Plant chitinase hydrolyzing ß-1,4-glycosidic linkages of chitin are major enzymes acting in plant-microbe interactions and are involved in self-defense against fungal pathogens. Chitosanases from soil bacteria are also involved in plant defense by hydrolyzing chitosan components of the fungal cell wall. The crystal structures of these enzymes in complex with their substrates have been solved, and the mechanisms of substrate binding were elucidated at the atomic level. These findings enabled us to speculate on the enzyme targets under physiological conditions, leading us to define the physiological roles of the enzymes. The structures and functions of chitin/chitosan-binding modules appended to modular chitinases/chitosanases were analyzed by NMR and isothermal titration calorimetry (ITC), and the enzymes were found to form an appropriate modular organization to fulfill their roles in plant-microbe interactions.

Structures and chitin-binding properties of two N-terminal lysin motifs (LysMs) found in a chitinase from Volvox carteri.

Two N-terminal lysin motifs (LysMs) found in a chitinase from the green alga Volvox carteri (VcLysM1 and VcLysM2) were produced, and their structures and chitin-binding properties were characterized. The binding affinities of VcLysM1 toward chitin oligomers determined by isothermal titration calorimetry (ITC) were higher than those of VcLysM2 by 0.8-1.1 kcal/mol of ΔG°. Based on the NMR solution structures of the two LysMs, the differences in binding affinities were found to result from amino acid substitutions at the binding site. The NMR spectrum of a two-domain protein (VcLysM1+2), in which VcLysM1 and VcLysM2 are linked in tandem through a flexible linker, suggested that the individual domains of VcLysM1+2 independently fold and do not interact with each other. ITC analysis of chitin-oligomer binding revealed two different binding sites in VcLysM1+2, showing no cooperativity. The binding affinities of the VcLysM1 domain in VcLysM1+2 were lower than those of VcLysM1 alone, probably due to the flexible linker destabilizing the interaction between the chito-oligosaccahrides and VcLysM1 domain. Overall, two LysMs attached to the chitinase from the primitive plant species, V. carteri, were found to resemble bacterial LysMs reported thus far.

Periplasmic solute-binding proteins: Structure classification and chitooligosaccharide recognition.

Periplasmic solute-binding proteins (SBPs) serve as molecular shuttles that assist the transport of small solutes from the outer membrane to the inner membrane of all Gram-negative bacteria. Based on the available crystal structures, SBPs are classified into seven clusters, A-G, and are further divided into subclusters, IV. This minireview is focused on the classification, structure and substrate specificity of a distinct class of SBPs specific for chitooligosaccharides (CBPs). To date, only two structures of CBP homologues, VhCBP and VcCBP, have been reported in the marine Vibrio species, with exposition of their limited function. The Vibrio CBPs are structurally classified as cluster C/subcluster IV SBPs that exclusively recognize ß-1,4- or ß-1,3-linked linear oligosaccharides. The overall structural feature of the Vibrios CBPs is most similar to the cellobiose-binding orthologue from the hyperthermophilic bacterium Thermotoga maritima. This similarity provides an opportunity to engineer the substrate specificity of the proteins and to control the uptake of chitinous and cellulosic nutrients in marine bacteria.