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.

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.

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.

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.

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.

The Arabidopsis CERK1-associated kinase PBL27 connects chitin perception to MAPK activation.

Perception of microbe-associated molecular patterns by host cell surface pattern recognition receptors (PRRs) triggers the intracellular activation of mitogen-activated protein kinase (MAPK) cascades. However, it is not known how PRRs transmit immune signals to MAPK cascades in plants. Here, we identify a complete phospho-signaling transduction pathway from PRR-mediated pathogen recognition to MAPK activation in plants. We found that the receptor-like cytoplasmic kinase PBL27 connects the chitin receptor complex CERK1-LYK5 and a MAPK cascade. PBL27 interacts with both CERK1 and the MAPK kinase kinase MAPKKK5 at the plasma membrane. Knockout mutants of MAPKKK5 compromise chitin-induced MAPK activation and disease resistance to Alternaria brassicicola PBL27 phosphorylates MAPKKK5 in vitro, which is enhanced by phosphorylation of PBL27 by CERK1. The chitin perception induces disassociation between PBL27 and MAPKKK5 in vivo Furthermore, genetic evidence suggests that phosphorylation of MAPKKK5 by PBL27 is essential for chitin-induced MAPK activation in plants. These data indicate that PBL27 is the MAPKKK kinase that provides the missing link between the cell surface chitin receptor and the intracellular MAPK cascade in plants.

Structure and function of a novel periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria.

Periplasmic solute-binding proteins in bacteria are involved in the active transport of nutrients into the cytoplasm. In marine bacteria of the genus Vibrio, a chitooligosaccharide-binding protein (CBP) is thought to be the major solute-binding protein controlling the rate of chitin uptake in these bacteria. However, the molecular mechanism of the CBP involvement in chitin metabolism has not been elucidated. Here, we report the structure and function of a recombinant chitooligosaccharide-binding protein from Vibrio harveyi, namely VhCBP, expressed in Escherichia coli Isothermal titration calorimetry revealed that VhCBP strongly binds shorter chitooligosaccharides ((GlcNAc) n , where n = 2, 3, and 4) with affinities that are considerably greater than those for glycoside hydrolase family 18 and 19 chitinases but does not bind longer ones, including insoluble chitin polysaccharides. We also found that VhCBP comprises two domains with flexible linkers and that the domain-domain interface forms the sugar-binding cleft, which is not long extended but forms a small cavity. (GlcNAc)2 bound to this cavity, apparently triggering a closed conformation of VhCBP. Trp-363 and Trp-513, which stack against the two individual GlcNAc rings, likely make a major contribution to the high affinity of VhCBP for (GlcNAc)2 The strong chitobiose binding, followed by the conformational change of VhCBP, may facilitate its interaction with an active-transport system in the inner membrane of Vibrio species.

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.

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.

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.

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.

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.

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.

NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase.

Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases.

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.

Mechanism of chitosan recognition by CBM32 carbohydrate-binding modules from a Paenibacillus sp. IK-5 chitosanase/glucanase.

An antifungal chitosanase/glucanase isolated from the soil bacterium Paenibacillus sp. IK-5 has two CBM32 chitosan-binding modules (DD1 and DD2) linked in tandem at the C-terminus. In order to obtain insights into the mechanism of chitosan recognition, the structures of DD1 and DD2 were solved by NMR spectroscopy and crystallography. DD1 and DD2 both adopted a ß-sandwich fold with several loops in solution as well as in crystals. On the basis of chemical shift perturbations in(1)H-(15)N-HSQC resonances, the chitosan tetramer (GlcN)4 was found to bind to the loop region extruded from the core ß-sandwich of DD1 and DD2. The binding site defined by NMR in solution was consistent with the crystal structure of DD2 in complex with (GlcN)3, in which the bound (GlcN)3 stood upright on its non-reducing end at the binding site. Glu(14)of DD2 appeared to make an electrostatic interaction with the amino group of the non-reducing end GlcN, and Arg(31), Tyr(36)and Glu(61)formed several hydrogen bonds predominantly with the non-reducing end GlcN. No interaction was detected with the reducing end GlcN. Since Tyr(36)of DD2 is replaced by glutamic acid in DD1, the mutation of Tyr(36)to glutamic acid was conducted in DD2 (DD2-Y36E), and the reverse mutation was conducted in DD1 (DD1-E36Y). Ligand-binding experiments using the mutant proteins revealed that this substitution of the 36th amino acid differentiates the binding properties of DD1 and DD2, probably enhancing total affinity of the chitosanase/glucanase toward the fungal cell wall.

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.

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.