N-acetylglucosamine Units Research Articles

Insights into the N-Sulfation Mechanism: Molecular Dynamics Simulations of the N-Sulfotransferase Domain of Ndst1 and Mutants

Sulfation patterns along glycosaminoglycan (GAG) chains dictate their functional role. The N-deacetylase N-sulfotransferase family (NDST) catalyzes the initial downstream modification of heparan sulfate and heparin chains by removing acetyl groups from subsets of N-acetylglucosamine units and, subsequently, sulfating the residual free amino groups. These enzymes transfer the sulfuryl group from 3′-phosphoadenosine-5′-phosphosulfate (PAPS), yielding sulfated sugar chains and 3′-phosphoadenosine-5′-phosphate (PAP). For the N-sulfotransferase domain of NDST1, Lys833 has been implicated to play a role in holding the substrate glycan moiety close to the PAPS cofactor. Additionally, Lys833 together with His716 interact with the sulfonate group, stabilizing the transition state. Such a role seems to be shared by Lys614 through donation of a proton to the bridging oxygen of the cofactor, thereby acting as a catalytic acid. However, the relevance of these boundary residues at the hydrophobic cleft is still unclear. Moreover, whether Lys833, His716 and Lys614 play a role in both glycan recognition and glycan sulfation remains elusive. In this study we evaluate the contribution of NDST mutants (Lys833, His716 and Lys614) to dynamical effects during sulfate transfer using comprehensive combined docking and essential dynamics. In addition, the binding location of the glycan moiety, PAPS and PAP within the active site of NDST1 throughout the sulfate transfer were determined by intermediate state analysis. Furthermore, NDST1 mutants unveiled Lys833 as vital for both the glycan binding and subsequent N-sulfotransferase activity of NDST1.

Linear and cyclic oligo-β-(1→6)-D-glucosamines: Synthesis, conformations, and applications for design of a vaccine and oligodentate glycoconjugates

Poly-β-(1→6)-N-acetyl-D-glucosamine is an exopolysaccharide secreted by numerous pathogenic bacteria, including Staphylococcus aureus, Escherichia coli, Yersinia pestis, Bordetella pertussis, Acinetobacter baumannii, Burkholderia spp., and others. A convergent approach was developed for the synthesis of oligosaccharide fragments consisting of 5, 7, 9, and 11 glucosamine or N-acetylglucosamine units and for the preparation of five nona-β-(1→6)-D-glucosamines with various N-acetylation patterns. Penta- and nona-β‑(1→6)-D-glucosamines conjugated to protein carriers through a specially developed sulfhydryl linker proved to be highly immunogenic in mice and rabbits and elicited antibodies that mediated opsonic killing of multiple strains of S. aureus (including methicillin-resistant S. aureus, MRSA) and E. coli, and protected against S. aureus skin abscesses and lethal E. coli and B. cenocepacia peritonitis. These findings provide a basis for the construction of a unique semisynthetic vaccine against multiple bacterial targets. Conformational studies by means of special NMR experiments and computer modeling revealed that the oligo-β-(1→6)-D-glucosamine chain exists mostly in a helix-like conformation, where the terminal monosaccharides are arranged close to each other. Owing to this feature, oligoglucosamines consisting of 2 to 7 residues easily form products of cycloglycosylation. Cyclooligo-β-(1→6)-D-glucosamines represent a new family of functionalized cyclic oligosaccharides. Owing to their molecular architectonics, these compounds are convenient scaffolds for the design of conjugates with defined valency, symmetry, flexibility, and function.

Characterization of Chitosan and fabrication of Chitosan hydrogels matrices for biomedical applications

The development of injectable hydrogels for drug delivery is a major challenge. Chitosan is a copolymer of N-acetylglucosamine and glucosamine units and is represented as a copolymer. Chitosan occurs in nature in the cell walls of some fungi, exoskeletons of insects and marine animals such as crabs and prawns. Chitosan and its derivatives possess a wide range of useful properties. They are biodegradable, and biocompatible with antibacterial and antioxidant activities. They are useful in drug delivery formulations and tissue engineering. The objective of the present study was to characterize chitosan, prepare chitosan hydrogels and study the gelation of this hydrogels over time. Chitosan with DDA% ∼ 80%–90% were characterized by infrared spectroscopy, conductimetry and pH-metry. In addition, chitosan hydrogels were prepared using an ionic gelation method making it suitable for biomedical applications.

High Resolution Crystal Structure of the Endo-N-Acetyl-β-D-Glucosaminidase Responsible for the Deglycosylation of Hypocrea jecorina Cellulases

Endo-N-acetyl-β-D-glucosaminidases (ENGases) hydrolyze the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. The endo-N-acetyl-β-D-glucosaminidases classified into glycoside hydrolase family 18 are small, bacterial proteins with different substrate specificities. Recently two eukaryotic family 18 deglycosylating enzymes have been identified. Here, the expression, purification and the 1.3Å resolution structure of the ENGase (Endo T) from the mesophilic fungus Hypocrea jecorina (anamorph Trichoderma reesei) are reported. Although the mature protein is C-terminally processed with removal of a 46 amino acid peptide, the protein has a complete (β/α)8 TIM-barrel topology. In the active site, the proton donor (E131) and the residue stabilizing the transition state (D129) in the substrate assisted catalysis mechanism are found in almost identical positions as in the bacterial GH18 ENGases: Endo H, Endo F1, Endo F3, and Endo BT. However, the loops defining the substrate-binding cleft vary greatly from the previously known ENGase structures, and the structures also differ in some of the α-helices forming the barrel. This could reflect the variation in substrate specificity between the five enzymes. This is the first three-dimensional structure of a eukaryotic endo-N-acetyl-β-D-glucosaminidase from glycoside hydrolase family 18. A glycosylation analysis of the cellulases secreted by a Hypocrea jecorina Endo T knock-out strain shows the in vivo function of the protein. A homology search and phylogenetic analysis show that the two known enzymes and their homologues form a large but separate cluster in subgroup B of the fungal chitinases. Therefore the future use of a uniform nomenclature is proposed.

Structural Studies on Molecular Interactions between Camel Peptidoglycan Recognition Protein, CPGRP-S, and Peptidoglycan Moieties N-Acetylglucosamine and N-Acetylmuramic Acid

Peptidoglycan (PGN) consists of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked by short peptides. It is well known that PGN forms a major cell wall component of bacteria making it an important ligand for the recognition by peptidoglycan recognition proteins (PGRPs) of the host. The binding studies showed that PGN, GlcNAc, and MurNAc bind to camel PGRP-S (CPGRP-S) with affinities corresponding to dissociation constants of 1.3 × 10(-9), 2.6 × 10(-7), and 1.8 × 10(-7) M, respectively. The crystal structure determinations of the complexes of CPGRP-S with GlcNAc and MurNAc showed that the structures consist of four crystallographically independent molecules, A, B, C, and D, in the asymmetric unit that exists as A-B and C-D units of two neighboring linear polymers. The structure determinations showed that compounds GlcNAc and MurNAc bound to CPGRP-S at the same subsite in molecule C. Both GlcNAc and MurNAc form several hydrogen bonds and extensive hydrophobic interactions with protein atoms, indicating the specific nature of their bindings. Flow cytometric studies showed that PGN enhanced the secretions of TNF-α and IL-6 from human peripheral blood mononuclear cells. The introduction of CPGRP-S to the PGN-challenged cultured peripheral blood mononuclear cells reduced the expressions of proinflammatory cytokines, TNF-α and IL-6. This showed that CPGRP-S inhibited PGN-induced production of proinflammatory cytokines and down-regulated macrophage-mediated inflammation, indicating its potential applications as an antibacterial agent.

Effects of Organic Solvents on Hyaluronic Acid Nanoparticles Obtained by Precipitation and Chemical Crosslinking

Hyaluronic acid is a hydrophilic mucopolysaccharide composed of alternating units of D-glucuronic acid and N-acetylglucosamine. It is used in many medical, pharmaceutical, and cosmetic applications, as sponges, films, or particle formulations. Hyaluronic acid nanoparticles can be synthesized free of oil and surfactants by nanoprecipitation in organic solvents, followed by chemical crosslinking. The organic solvent plays an important role in particles size and structure. Therefore, this study aimed to investigate the influence of acetone, ethanol, and isopropyl alcohol on the synthesis and physico-chemical properties of hyaluronic acid nanoparticles. Particles were crosslinked with adipic hydrazide and chloride carbodiimide under controlled conditions. The nanoparticles obtained with all three studied solvents were moderately electrostatically stable. Experiments with acetone produced the smallest particle size (120.44 nm) and polydispersity (0.27). The size and polydispersity of hyaluronic acid nanoparticles correlated with the surface tension between water and the organic solvents, not with the thermodynamic affinity of water for the organic solvents.

Association of fucosyltransferase 8 (FUT8) polymorphism Thr267Lys with pulmonary emphysema

The fucosyltransferase 8 gene (FUT8) encodes an enzyme that transfers fucose to the innermost N-acetylglucosamine unit of N-glycan chains. Recent study showed that fut8-deficient mice develop pathological and physiological phenotypes resembling pulmonary emphysema (PE). The role of FUT8 in human PE is not known. A non-synonymous single-nucleotide polymorphism at the amino-acid position of 267 in FUT8 (rs35949016; C/A, C allele=Thr, A allele=Lys) was genotyped in a total of 1149 consecutive autopsies of elderly Japanese. A following study included 182 outpatients with chronic obstructive pulmonary disease, whose emphysematous changes were assessed quantitatively as the percentage of low attenuation area (%LAA) by high-resolution computed tomography. PE was detected in 163 of 1149 autopsy subjects (14.2%). Comparison of patient with vs without PE indicated that the FUT8 A allele was associated with PE (AA+AC vs CC; odds ratio=1.74, 95% confidence intervals=1.19-2.56, P=0.005). In the clinical study, presence of the FUT8 A allele significantly correlated with %LAA after adjustment (AA+AC vs CC=37.5±14.7 vs 32.7±13.9, P=0.02). The FUT8 gene Thr267Lys polymorphism is associated with human PE, and the Lys allele is the risk. The core fucosylation might be involved in the molecular pathogenesis of human PE.

Cell Surface Heparan Sulfate Chains Regulate Local Reception of FGF Signaling in the Mouse Embryo

Heparan sulfate (HS) proteoglycans modulate the activity of multiple growth factors on the cell surface and extracellular matrix. However, it remains unclear how the HS chains control the movement and reception of growth factors into targeted receiving cells during mammalian morphogenetic processes. Here, we found that HS-deficient Ext2 null mutant mouse embryos fail to respond to fibroblast growth factor (FGF) signaling. Marker expression analyses revealed that cell surface-tethered HS chains are crucial for local retention of FGF4 and FGF8 ligands in the extraembryonic ectoderm. Fine chimeric studies with single-cell resolution and expression studies with specific inhibitors for HS movement demonstrated that proteolytic cleavage of HS chains can spread FGF signaling to adjacent cells within a short distance. Together, the results show that spatiotemporal expression of cell surface-tethered HS chains regulate the local reception of FGF-signaling activity during mammalian embryogenesis.

A striking common O‐linked N‐acetylglucosaminyl moiety between cruzipain and myosin

Single units of O-linked N-acetylglucosamine (GlcNAc), usually components of nuclear and cytoplasmatic proteins, are present at the C-terminal domain of cruzipain (Cz), a lysosomal major antigen from Trypanosoma cruzi. On the other hand, antibodies directed against some self-antigens like myosin are associated with Chagas heart disease. The participation of O-GlcNAc moieties in the molecular antigenicity of Cz was determined using GlcNAc linked to aprotinin by ELISA. The immune cross-reactivity between Cz and myosin is mainly focused in the C-T domain. ELISA inhibition assays using rabbit sera specific for Cz and C-T in conjunction with immune-gold electron microscopy analysis of heart tissues from mice immunized with C-T confronted with polyclonal rabbit sera specific for Cz and C-T prior and after myosin adsorption provided evidence which indicates that O-GlcNAc moieties constitute a common epitope between Cz and either myosin or other cardiac O-GlcNAc-containing proteins, showing a new insight into the molecular immune pathogenesis of Chagas heart disease.

Inhibitory Peptides of the Sulfotransferase Domain of the Heparan Sulfate Enzyme, N-Deacetylase-N-sulfotransferase-1

N-Deacetylase-N-sulfotransferase 1 (Ndst1) catalyzes the initial modification of heparan sulfate and heparin during their biosynthesis by removal of acetyl groups from subsets of N-acetylglucosamine units and subsequent sulfation of the resulting free amino groups. In this study, we used a phage display library to select peptides that interact with Ndst1, with the aim of finding inhibitors of the enzyme. The phage library consisted of cyclic random 10-mer peptides expressed in the phage capsid protein pIII. Selection was based on the ability of engineered phage to bind to recombinant murine Ndst1 (mNdst1) and displacement with heparin. Peptides that were enriched through multiple cycles of binding and disassociation displayed two specific sequences, CRGWRGEKIGNC and CNMQALSMPVTC. Both peptides inhibited mNdst1 activity in vitro, however, by distinct mechanisms. The peptide CRGWRGEKIGNC presents a chemokine-like repeat motif (BXX, where B represents a basic amino acid and X is a noncharged amino acid) and binds to heparan sulfate, thus blocking the binding of substrate to the enzyme. The peptide NMQALSMPVT inhibits mNdst1 activity by direct interaction with the enzyme near the active site. The discovery of inhibitory peptides in this way suggests a method for developing peptide inhibitors of heparan sulfate biosynthesis.

Water-Soluble Substrates of the Peptidoglycan-Modifying Enzyme O-Acetylpeptidoglycan Esterase (Ape1) from Neisseria gonorrheae

Peptidoglycan is the component of the bacterial cell wall that is essential for maintaining the shape and rigidity of the cell. As such, its polymeric structure, consisting of alternating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), is also a target for the action of host defense enzymes, such as lysozymes. Many bacteria have developed methods of masking their cell wall from these environmental dangers through the addition of aglycon moieties that prevent recognition or sterically hinder the degradative action of exogenous enzymes that would otherwise prove detrimental to the cell. Peptidoglycan acetyl-transferases (Pat's) and O-acetylpeptidoglycan esterases (Ape's) are the enzymes responsible for the controlled addition and removal of acetate onto the C-6 hydroxyl group of MurNAc residues in peptidoglycan. Studies on Ape1, an O-acetylpeptidoglycan esterase found in Neisseria gonorrheae, have suggested that this enzyme is essential for bacterial viability and thus presents an attractive target for antibacterial design. Previous studies on Ape1 have been hindered by the fact that Ape1's natural substrate is an insoluble polymer. In this paper we outline the design, synthesis, and testing of the water-soluble di- and monosaccharide substrate analogues 1 and 2. Both 1 and 2 serve as substrates of Ape1 with k(cat)/K(M) values of (5.1 ± 1.7) × 10(3) M(-1) s(-1) and (3.1 ± 0.8) × 10(3) M(-1) s(-1), respectively. It was determined that the substitution of the GlcNAc residue in compound 1 with an O-benzyl group in compound 2 did not significantly decrease the enzyme's affinity for the monosaccharide. These findings are important as they demonstrate that the catalytic prowess of Ape1 is not dependent on its binding to a polymeric substrate. This ensures that small molecule transition state/intermediate analogues can also capture the transition state binding energy of Ape1 and potentially serve as potent inhibitors. The synthetic route to compounds 1 and 2 could readily be modified to allow for the installation of a wide variety of functional groups at the MurNAc C-6 position in both the mono- and disaccharide scaffolds. This will serve as a general method for the construction of Ape1 substrates and inhibitors.

A Direct, Heavy Metal Free Synthesis of the ?-1,6-Linked GlcNAc Disaccharide

The major component of the matrix of many bacterial biofilms is a linear polymer of β-1,6-linked units of N-acetylglucosamine (polysaccharide intercellular adhesin or poly-N-acetyl-β-1,6-d-glucosamine). In order to facilitate synthetic vaccine construction we have developed a direct, inexpensive, and biologically compatible synthesis of the minimal building block of this polymer, a β-1,6-linked GlcNAc disaccharide, namely 6-O-[2-acetamido-2-deoxy-β-d-glucopyranosyl]-2-acetamido-2-deoxy-d-glucopyranose.

Crystallization and preliminary X-ray diffraction analysis of the lytic transglycosylase MltE from Escherichia coli.

MltE from Escherichia coli (193 amino acids, 21,380 Da) is a lytic transglycosylase that initiates the first step of cell-wall recycling. This enzyme is responsible for the cleavage of the cell-wall peptidoglycan at the β-1,4-glycosidic bond between the N-acetylglucosamine and N-acetylmuramic acid units. At the end this reaction generates a disaccharide that is internalized and initiates the recycling process. To obtain insights into the biological functions of MltE, crystallization trials were performed and crystals of MltE protein that were suitable for X-ray diffraction analysis were obtained. The MltE protein of E. coli was crystallized using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture consisting of 28% polyethylene glycol 4000, 0.1 M Tris pH 8.4 and 0.2 M magnesium chloride. Further optimization was performed using the microbatch technique. Single crystals were obtained that belonged to the orthorhombic space group C222(1), with unit-cell parameters a=123.32, b=183.93, c=35.29 Å, and diffracted to a resolution of 2.1 Å.

Chitin deacetylase product inhibition

Chitin deacetylase is the only known enzyme catalyzing the hydrolysis of the acetamino linkage in the N-acetylglucosamine units of chitin and chitosan. This reaction can play an important role in enzymatic production of chitosan from chitin, or in enzymatic modification of chitosan, which has applications in medicine, pharmacy or plant protection. It was previously shown that acetic acid, a product of the deacetylation process, may act as an inhibitor of chitin deacetylase. Here we show the mechanism of inhibition of chitin deacetylase isolated from Absidia orchidis vel coerulea by acetic acid released during the deacetylation process. The process follows competitive inhibition with respect to acetic acid with an inhibition constant of K(i) = 0.286 mmol/L. These results will help to find the optimal system to carry out the enzymatic deacetylation process for industrial applications.

Conformational Flexibility of Chitosan: A Molecular Modeling Study

Chitin and chitosan are naturally occurring polysaccharides composed of β-(1,4) linked N-acetylglucosamine units (GlcNAc) and, for chitosan, also glucosamine units (GlcN). In recent years, chitosan has attracted much interest because of its special physical and chemical properties related to drug delivery, wound healing, and tissue engineering. However, limited structural knowledge is available for chitosan because of its composition of the randomly mixed building blocks, GlcNAc and GlcN. In this study, we present exhaustive combined molecular dynamics and Monte Carlo simulations that unravel the conformational flexibility of the β-(1,4)-linkage in di-, tri-, and tetrasaccharide models of chitin and chitosan. The most flexible disaccharide unit was found to be GlcN-GlcNAc, populating four conformations. Furthermore, it is found that the conformational freedom of a glycosidic bond is independent of the flexibility of the neighboring linkages along the oligomer. The results are interpreted with respect to hydrogen bond formation and implications for polymer properties.

Synthesis of glycosaminoglycan oligosaccharides. Part 5: Synthesis of a putative heparan sulfate tetrasaccharide antigen involved in prion diseases

The synthesis of the putative prion-associated heparan sulfate tetrasaccharide containing two d-glucuronic acid units is reported. Key to the synthesis were the differentiation of the N-acetylated and N-unsubstituted glucosamine units, the high-yielding glucosylation at O-4 of an N-acetyl-d-glucosamine derivative and the α-selective glycosylation of the 4′-OH group of a β-d-GlcpA-(1→4)-d-GlcpNAc disaccharide building block with a disaccharide thioglycoside donor.

Echinocystic acid 3,16- O-bisglycosides from Albizia procera

Three triterpene glycosides and two known ones were isolated from the bark of Albizia procera by using chromatographic techniques. The structures of the compounds were determined to be 3- O-β- d-xylopyranosyl-(1 → 2)-β- d-galactopyranosyl-(1 → 6)-2-acetamido-2-deoxy-β- d-glucopyranosyl echinocystic acid 16- O-β- d-glucopyranoside, 3- O-β- d-xylopyranosyl-(1 → 2)-α- l-arabinopyranosyl-(1 → 6)-2-acetamido-2-deoxy-β- d-glucopyranosyl echinocystic acid 16- O-β- d-glucopyranoside and 3- O-α- l-arabinopyranosyl-(1 → 2)-α- l-arabinopyranosyl-(1 → 6)-2-acetamido-2-deoxy-β- d-glucopyranosyl echinocystic acid 16- O-β- d-glucopyranoside. Their structures were determined by NMR techniques including HOHAHA, 1H- 1H COSY, ROE, HMQC and HMBC experiments together with FABMS as well as acid hydrolysis. To the best of our knowledge, the new compounds are considered the first examples of echinocystic acid 3,16- O-bisglycosides. In contrast to other cytotoxic echinocystic acid glycosides with N-acetyl glucosamine unit, the new glycosides were found inactive when assayed by MTT method for their cytotoxicities against the human tumor cell lines HEPG2, A549, HT29 and MCF7. The results showed the importance of the free hydroxyl group at the aglycone C-16 for exhibiting cytotoxic properties.

ChemInform Abstract: Synthesis of a Novel Sialyl Lewis X Analogue Containing a Pyrrolidine in Place of N-Acetyl Glucosamine.

ABSTRACT The synthesis of the new sialyl Lewis X analogue, 4-O-(α-L-fucopyranosyl)-3-O-(3-O-sodium sulfonato-β-D-galactopyranosyl)-(2S,3R, 4R)-2-ethyl-3,4-dihydroxypyrrolidine 2 has been achieved. The N-acetyl glucosamine unit of natural Lewis X has been replaced by a rigid 3R/4R-dihydroxylated pyrrolidine 12. This one has been synthezised from the methyl 4-O-benzoyl-2,3-di-O-benzyl-6-deoxy-6-iodo-α-D-altropyranoside sugar precursor 10 using the Ganem/Bernotas one-pot elimination-reductive amination ring contraction reaction. The (2S, 3R, 4R)-1-benzyloxycarbonyl-3,4-dihydroxy-2-ethylpyrrolidine 12 obtained was subsequently regioselectively glycosylated, using 2,3,4-tri-O-benzyl-α-L-fucopyranosyl fluoride and 2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl bromide as glycosyl donors. Disaccharide containing pyrrolidine 21 was finally transformed into the target O-sulfated analog 2, after regioselective sulfation and usual deprotection.

Hyaluronic acid degradation during initial steps of downstream processing

Hyaluronic acid (HA) is a linear glycosaminoglycan composed of disaccharide units of glucuronic acid and N-acetylglucosamine. It has interesting and distinctive viscoelastic properties that are functions of chain length, concentration and environmental conditions, like pH and ionic strength. These characteristics, coupled with its lack of immunogenicity or toxicity, have led to a wide range of applications in the pharmaceutical and cosmetic industries where molecular mass is of primary importance. Biotechnological production of HA was established many years ago, but HA obtained from bacterial fermentation results in shorter chains compared with HA extracted from animal sources. In the present research the issue of HA degradation during the initial phases of downstream processing is addressed. In particular, the effects of clarification and protein separation on molecular weight have been studied using a triple-detector chromatographic system. Environmental factors affecting degradation (pH, shear stresses, etc.) were uncoupled in order to identify the main causes of molecular weight reduction.

The first crystal structures of a family 19 class IV chitinase: the enzyme from Norway spruce

Chitinases help plants defend themselves against fungal attack, and play roles in other processes, including development. The catalytic modules of most plant chitinases belong to glycoside hydrolase family 19. We report here x-ray structures of such a module from a Norway spruce enzyme, the first for any family 19 class IV chitinase. The bi-lobed structure has a wide cleft lined by conserved residues; the most interesting for catalysis are Glu113, the proton donor, and Glu122, believed to be a general base that activate a catalytic water molecule. Comparisons to class I and II enzymes show that loop deletions in the class IV proteins make the catalytic cleft shorter and wider; from modeling studies, it is predicted that only three N-acetylglucosamine-binding subsites exist in class IV. Further, the structural comparisons suggest that the family 19 enzymes become more closed on substrate binding. Attempts to solve the structure of the complete protein including the associated chitin-binding module failed, however, modeling studies based on close relatives indicate that the binding module recognizes at most three N-acetylglucosamine units. The combined results suggest that the class IV enzymes are optimized for shorter substrates than the class I and II enzymes, or alternatively, that they are better suited for action on substrates where only small regions of chitin chain are accessible. Intact spruce chitinase is shown to possess antifungal activity, which requires the binding module; removing this module had no effect on measured chitinase activity.