Microbial Secondary Metabolite, Phlegmacin B1, as a

Article pubs.acs.org/JAFC

Microbial Secondary Metabolite, Phlegmacin B1, as a Novel Inhibitor of Insect Chitinolytic Enzymes Lei Chen,† Tian Liu,*,†,‡ Yanwei Duan,† Xinhua Lu,§ and Qing Yang*,†,‡ †

State Key Laboratory of Fine Chemical Engineering and School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China ‡ Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China § New Drug Research & Development Center, North China Pharmaceutical Group Corporation, Shijiazhuang 050015, China S Supporting Information *

ABSTRACT: Periodic chitin remodeling during insect growth and development requires a synergistic action of two glycosyl hydrolase (GH) family enzymes, GH18 chitinase and GH20 β-N-acetylhexosaminidase (Hex). Inhibiting either or both of these enzymes is a promising strategy for pest control and management. In this study, Of Chi-h (a GH18 chitinase) and Of Hex1 (a GH20 Hex) from Ostrinia f urnacalis were used to screen a library of microbial secondary metabolites. Phlegmacin B1 was found to be the inhibitor of both Of Chi-h and Of Hex1 with Ki values of 5.5 μM and 26 μM, respectively. Injection and feeding experiments demonstrated that phlegmacin B1 has insecticidal effect on O. furnacalis’s larvae. Phlegmacin B1 was predicted to bind to the active pockets of both Of Chi-h and Of Hex1. Phlegmacin B1 also showed moderate inhibitory activities against other bacterial and insect GH18 enzymes. This work provides an example of exploiting microbial secondary metabolites as potential pest control and management agents. KEYWORDS: chitinase, hexosaminidase, inhibitor, phlegmacin, microbial secondary metabolites

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FO-7314,15 and cyclo (L-Arg-D-Pro) from Pseudomonas sp. IZ208,16 and GH20 Hex inhibitors, such as nagstatin,17 pochonicine,18 and TMG-chitotriomycin,19 which were derived from microbial secondary metabolites, have practical applications. Here, we applied a chitinase, Chi-h, and a Hex, Hex1, derived from the agricultural pest Ostrinia f urnacalis as representatives of insect GH18 and GH20 enzymes, respectively. Our previous work indicates that Of Chi-h and Of Hex1 are indispensable for molting, and their crystal structures have been resolved.9,20 In this work, by screening a library of microbial secondary metabolites, phlegmacin B1 was obtained as a potential pesticide that targets both enzymes. Phlegmacin B1, as well as other dimeric preanthraquinones (Figure 1B), has antiplasmodial activity and could be an antimalarial drug.21−23 This work may provide an additional application for dimeric preanthraquinone compounds and derivatives.

INTRODUCTION Chitin, a polymer of β-1,4-linked N-acetyl-β-D-glucosamine (GlcNAc), is an essential structural component of insects.1 To meet their growth and developmental demands, insects periodically shed their old cuticles through a process known as chitin degradation.2 Two glycoside hydrolase family members are required for chitin degradation, the glycosyl hydrolase family 18 (GH18) chitinase (EC 3.2.1.14)3 and the glycosyl hydrolase family 20 (GH20) β-N-acetylhexosaminidase (Hex; EC 3.2.1.52).4 By using structural and chemical biology, GH18 and GH20 were shown to have the same substrateassisted mechanism.5,6 The 2-acetamido group of the sugar at the reducing end acts as the nucleophile to attack its anomeric carbon atom to form an oxazolinium intermediate (Figure 1A). A typical GH18 chitinase possesses a long substrate-binding cleft (or groove) composed of multiple subsites.7 Catalysis takes place between subsite +1 and subsite −1. However, a GH20 Hex usually contains one subsite, termed as subsite −1, for binding substrates and catalysis.8 An exception is the insect Group I Hex, which contains two subsites, −1 and +1.9 Because chitin degradation is essential for insect development, small molecules that have inhibitory activities toward chitinase and Hex are promising pest control reagents. Microbial secondary metabolites represent a large family of diverse chemical entities that have been developed as commercial products for human medicine, animal health, and plant crop protection.10−12 The inherent advantages of microbial secondary metabolites are their taxonomic, functional, and chemical diversities.10 GH18 chitinase inhibitors, such as allosamidin from Streptomyces sp.,13 argifin from Gliocladium sp. FTD-0668,14 argadin from Clonostachys sp. © 2017 American Chemical Society

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MATERIALS AND METHODS

Materials. The chromatographic columns for protein purification were purchased from GE Life Sciences (Beijing, China). The BCA protein assay kit was purchased from TaKaRa (Dalian, China). HexB from Homo sapiens, plant Hex from Canavalia ensiformis, 4methylumbelliferyl-β-D-N,N′-diacetylchitobiose [MU-(GlcNAc)2] and 4-methylumbelliferyl-β-D-N-acetylglucosamine (MU-GlcNAc) were purchased from Sigma-Aldrich (Shanghai, China). All other chemicals of the highest purity were purchased from commercial sources. Received: Revised: Accepted: Published: 3851

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2017 2017 2017 2017 DOI: 10.1021/acs.jafc.7b01710 J. Agric. Food Chem. 2017, 65, 3851−3857

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Journal of Agricultural and Food Chemistry

Figure 1. (A) Catalytic mechanisms of GH18 chitinases and GH20 Hexs; (B) structure of phlegmacin B1. Enzyme Preparation. For GH18 chitinases, Chi-h and ChtI from O. f urnacalis and Cht from H. sapiens were recombinantly expressed in Pichia pastoris GS115 and purified by immobilized metal affinity chromatography (IMAC).20,24,25 ChiA, ChiB, and ChiC from Serratia marcescens and ChiB1 from Aspergillus f unigatus were expressed in Escherichia coli BL21 (DE3) and purified by IMAC as described previously.26,27 For GH20 β-N-acetylhexosaminidases (Hexs), Of Hex1 and Of Hex2 were expressed in P. pastoris GS115 and purified by IMAC as described previously.28,29 Chb from S. marcescens was recombinantly expressed and purified using previously reported methods.30 All of the purified proteins were desalted using a 5 mL HiTrap desalting column with 20 mM sodium phosphate, pH 6.0, and quantitated using a commercial BCA protein assay kit. The purities of the target proteins were analyzed by SDS-PAGE followed by Coomassie Brilliant Blue R-250 staining. Inhibitory Activity Assay. Of Chi-h and Of Hex1 were screened against a small molecule library of microbial secondary metabolites from NCPC New Drug Research and Development Co. Ltd., China. Briefly, in a final assay volume of 100 μL, 20 nM enzyme was incubated with 50 μM substrate [MU-(GlcNAc)2 for Of Chi-h and MU-GlcNAc for Of Hex1] in 20 mM sodium phosphate buffer (pH 6.0) containing 13 ppm inhibitor at 30 °C. The reaction in the absence of inhibitors was used as a positive control. After reacting for an appropriate time, an equal volume of 0.5 M Na2CO3 was added to the reaction mixture to terminate the reaction, and the fluorescence of the liberated MU was quantitated using a Varioskan Flash microplate reader (Thermo Fisher Scientific, Waltham, MA, USA), with excitation and emission wavelengths of 360 and 450 nm, respectively. Phlegmacin B1 was reassayed at 20 μM and 100 μM to determine its inhibitory activity against enzymes from other species. The GH18 chitinases included Of ChtI from the insect O. f urnacalis; SmChiA, SmChiB, and SmChiC from the bacterium S. marcescens; Af ChiB1 from the fungus A. f umigatus; and HsCht from H. sapiens. These chitinases were assayed using 50 μM of MU-(GlcNAc)2 in 20 mM sodium phosphate buffer (pH 6.0). The GH20 Hexs included Of Hex2 from the insect O. f urnacalis, SmChb from the bacterium S. marcescens, CeHex from the plant C. ensiformis, and HsHexB from H. sapiens. These Hexs were assayed using 50 μM of MU-GlcNAc in buffers with different pH levels, 20 mM sodium phosphate buffer (pH 6.0) for Of Hex1, Of Hex2, and SmChb, and 50 mM sodium acetate (pH 4.0) for HsHexB and CeHex. For the determination of the mode of inhibition and the Ki value, the reaction mixtures contained four substrate concentrations (15, 30, 40, and 50 μM) in the presence of increasing concentrations of the putative inhibitors. For Of ChtI and SmChiB, the substrate concentrations were changed to 5, 10, 15, and 20 μM to prevent substrate inhibition. The production of MU was linear for the incubation period used, and less than 10% of the available substrate was hydrolyzed. After quantifying the fluorescence of the liberated MU, the Ki values and types of inhibition were determined using Lineweaver−Burk plots. Isolation of Phlegmacin B1. The fungus was isolated from a soil sample collected on Mount Qingcheng, Sichuan Province, China. The strain was identified as Talaromyces on the basis of morphology and

sequence analysis of the ITS region of the genomic DNA and assigned the accession number F08Z-0631 in the culture collection center of NCPC New Drug Research and Development Co., Ltd. The fungal strain was cultured on slants of potato dextrose agar (PDA) at 25 °C for 10 d. The agar plugs were inoculated in 500 mL Erlenmeyer flasks containing 80 mL of seed media (2.0% starch, 1.0% glucose, 0.6% malt extract, 0.3% yeast extract, 0.2% hot-pressed soybean cake meal, 0.2% NaCl, 0.1% MgSO4·7H2O, and 0.2% CaCO3, final pH 7.0 before sterilization), and the flask cultures were incubated at 26 °C on a rotary shaker at 220 rpm for 3 d. Thirty 1 L Fernbach flasks, each containing 100 g of solid media (97.5% rice, 2.5% hotpressed soybean cake meal) were individually inoculated with 1.0 mL of the seed culture and fermented at 26 °C under static conditions for 14 d. The fermented rice substrate (3.0 kg) was extracted with ethyl acetate (4.5 L), and the organic solvent was evaporated to dryness under vacuum to afford a crude extract (10.5 g). The extract was subjected to a 3.5 cm × 50 cm silica gel column and eluted with a stepwise gradient of n-hexane/ethyl acetate from 100:0 to 0:100 to give fractions F1−F5. Then fraction F2 (300 mg) was purified using a 21.2 mm × 250 mm Unisil 10 C18 HPLC column with 55% acetonitrile as the movable phase at a flow rate of 18 mL/min, and 23.0 mg of phlegmacin B1 was obtained. The structure of phlegmacin B1 was confirmed using ESI−MS, CD, and 1H NMR spectra.23 Molecular Docking. The program database (PDB) files of phlegmacin B1 were prepared using PRODRG.31 The ligand-free PDB files of Of Chi-h and Of Hex1 were prepared by PyMOL (DeLano Scientific LLC, San Carlos, CA, USA) from the TMGchitotriomycin-complexed structure of Of Hex1 (PDB code: 3NSN) and chitohepatose-complexed structure of Of Chi-h (PDB code: 5GQB). MGLTools32 was used to generate the PDBQT files of the proteins and compounds. Affinity grids of 50 × 50 × 50 Å3 and 80 × 60 × 80 Å3 for Of Hex1 and Of Chi-h, respectively, were calculated using AutoGrid4. 32 Molecular dockings were performed by AutoDock432 using the Lamarckian genetic algorithm with a population size of 100 individuals, 25 000 000 energy evaluations, and 27 000 generations. Plausible docking models were selected from the abundant clusters [root−mean−square deviation (RMSD) = 2 Å] that had lower binding energies. Weak intermolecular interactions such as hydrogen bonding and hydrophobic interactions were analyzed by LigPlus+33 and visualized by PyMOL (DeLano Scientific LLC, San Carlos, CA). Molecular Dynamics (MD) Simulations. To generate a convincing conformation, the MD simulation was carried out after docking. The initial conformations of Of Hex1−phlegmacin B1 and Of Chi-h−phlegmacin B1 were obtained using X-ray crystal structures (PDB ID: 3NSN for Of Hex1 and PDB ID: 5GQB for Of Chi-h) and docking studies, respectively. The topologies and force field parameters of phlegmacin B1 were automatically generated by SwissParam based on the Merck molecular force field.34 The protein−phlegmacin B1 complexes were then immersed in a rectangular simulation box that was 16 Å thick. In addition, a 0.10 M NaCl aqueous solution with excess counterions was dissolved into the simulation box to keep the system electrically neutral. The final system comprised 97 269 atoms for Of Chi-h−phlegmacin B1 and 3852

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Figure 2. Inhibitory kinetics of phlegmacin B1 against (A) Of Chi-h, (B) Of Hex1, (C) SmChiB, and (D) Of ChtI.

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96 546 atoms for Of Hex1−phlegmacin B1. Production MD simulations were performed using NAMD35 with the CHARMM27 force field36 and the TIP3P water model37 for 10 ns at constant temperature (303 K) and pressure (1 atm) employing the Langevin barostat and thermostat35 after the initial 1000 steps of energy minimization. Periodic boundary conditions were employed using particle-mesh Ewald38 to manage long-range Coulomb interactions with a cutoff value of 12 Å for truncating short-range potentials. Harmonic constraints were treated to restrain the protein backbones of Of Chih and Of Hex1. Bonds involving hydrogen atoms were constrained at their equilibrium lengths, and a time step of 2 fs was used to integrate the equations of motion. RMSD and the hydrogen bond interactions between ligand and protein were obtained through VMD.39 In Vivo Activity of Phlegmacin B1. O. f urnacalis larvae were reared using an artificial diet with a photoperiod of 16 h of light and 8 h of darkness and a relative humidity of 70−90% at 26−28 °C. Day 4 fifth-instar larvae were selected for the microinjection experiment. In the experimental group, 1 μg of phlegmacin B1 (dissolved in DMSO) was injected into the penultimate abdominal segment of the larvae. In the control group, 5% DMSO was injected. Each group contained 30 individual larvae. After injection, all of the treated larvae were reared under the same conditions, as described above. Mortality and developmental defects were recorded every day until eclosion. Day 1 third-instar larvae were selected for the feeding experiment. In the experimental group, artificial diet containing 0.5 mM of phlegmacin B1 (dissolved in ethanol) was used. In the control group, normal artificial diet was used. Each group contained 30 individual larvae. Mortality and developmental defects were recorded every day until all of the larvae in the experimental group died.

RESULTS AND DISCUSSION Phlegmacin B 1 Activities against Of Chi-h and Of Hex1. A small library of microbial secondary metabolites was screened using their inhibitory activities toward Of Chi-h and Of Hex1. At a concentration of 13 ppm, phlegmacin B1 had inhibitory rates of 90.4% and 72.4% toward Of Chi-h and Of Hex1, respectively. The phlegmacin B1 tested in this work was purified from the culture of Talaromyces sp., a strain isolated from a soil sample taken at Mount Qingcheng, Sichuan Province, China. The structure of phlegmacin B1 was confirmed using ESI−MS, CD, and 1H NMR spectra.23 To characterizeits inhibitory mode, a steady-state kinetic analysis of phlegmacin B1 was then performed. By using Lineweaver−Burk plots, phlegmacin B1 was determined to be a competitive inhibitor of both Of Chi-h and Of Hex1, with Ki values of 5.5 μM and 26 μM, respectively (Figure 2A,B). The inhibitory activities of allosamidin13 (a GH18 chitinase inhibitor) for Of Chi-h and NAG-thiazoline40 (a GH20 Hex inhibitor) for Of Hex1 was also studied for comparison. The Ki value of allosamidin for Of Chi-h was determined to be 0.84 μM, while the Ki value of NAG-thiazoline for Of Hex1 was determined in our previous work as 75 μM.41 Inhibitory Mechanisms of Phlegmacin B1. Because the crystal structures of both Of Hex1 and Of Chi-h were resolved in our laboratory, the inhibitory mechanism of phlegmacin B1 was studied using structure-based molecular docking and MD. The intermolecular interactions were analyzed using LigPlus+. Phlegmacin B1 binds to the subsites −3 to +1 in the substrate-binding cleft of Of Chi-h through hydrogen bonds and 3853

DOI: 10.1021/acs.jafc.7b01710 J. Agric. Food Chem. 2017, 65, 3851−3857

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Figure 3. Binding mechanism of phlegmacin B1 revealed by molecular docking and MD. The binding modes of phlegmacin B1 with (A) Of Chi-h and (B) Of Hex1. Phlegmacin B1 is shown in green. The key residues involved in compound binding are shown in blue, and the hydrogen bonds are represented by black dashes. (C) RMSDs of phlegmacin B1 during MD simulations. (D) RMSDs of binding-site residues during MD simulations.

binding pocket, and it stacked with Trp490 (Figure 3B). The other preanthraquinone moiety is positioned in a shadow pocket outside the substrate-binding pocket consisting of Trp322, Lys323, Pro329, Ala482, Trp483, Val484, Asn488, and Asn489. Two hydrogen bonds are formed between phlegmacin B1 and two residues, Val484 and Lys323. MD simulation indicated that the binding of phlegmacin B1 to Of Hex1 was stable, with the RMSD values of phlegmacin B1 and binding-site residues being 0.65 and 0.93, respectively (Figure 3C,D). However, the hydrogen bond occupancy rates were very low (Table 1). Unlike other GH20 Hexs, Of Hex1 contains an extra subsite +1, which is characterized by residues Val327, Glu328, and Trp490.7 The interaction between phlegmacin B1 and Trp490 may be further used in developing species-specific inhibitors. Phlegmacin B1 Impairment of Insect Molting. To test its in vivo activity, phlegmacin B1 was injected into fifth-instar day 4 O. f urnacalis larvae prior to pupation. The development of these larvae was severely affected when compared with the 5% DMSO-injected group (Figure 4A,B). In the DMSOinjected group, 80% of larvae molted into normal pupae 4 days after injection. However, in the phlegmacin B1-injected group, only 40% molted to normal pupae, 30% died, and 30% molted to abnormal pupae. The dead larvae had the phenotype of shrunken thoraxes and abdomens (Figure 4C). The abnormal pupae had the phenotype of prepupae with undetached head capsules (Figure 4C). By 10 days after injection, most of the abnormal pupae were dead. The phenotypes of phlegmacin B1injected insects were very similar to those receiving injections

hydrophobic interactions (Figure 3A). The naming of the sugar-binding subsites was according to our previous work.18 One of the preanthraquinones binds to a small pocket characterized by Trp160, Ile200, Thr269, and Leu270 and stacks well with Trp160. Additionally, the other preanthraquinone moiety is positioned between Trp268 and Trp532. The O9 and O2 hydroxyl groups of phlegmacin B1 form hydrogen bonds with the catalytic residues Glu308 and Trp160, respectively. The positioning of phlegmacin B1 was validated by MD. By analyzing the trajectory from the 10 ns MD simulation, the binding of phlegmacin B1 to Of Chi-h was determined to be stable. The average RMSD values of phlegmacin B1 and the binding-site residues were 0.54 and 1.02, respectively (Figure 3C,D). Notably, the two hydrogen bonds between phlegmacin B1 and Of Chi-h showed occupancy rates of greater than 50% (Table 1). Unlike the binding mode of phlegmacin B1 in Of Chi-h, only one preanthraquinone moiety is inserted into the substrateTable 1. Hydrogen Bonds between Proteins and Ligands, and Their Occupancy Rates (%)

Of Chi-h Of Hex1

hydrogen bond donor

hydrogen bond acceptor

occupancy (%)

OH9 (phlegmacin B1) NE1 (W160) NH (V484) NZ (K323)

OE1 (E308) OH2 (phlegmacin B1) OH8 (phlegmacin B1) OH5 (phlegmacin B1)

58.9 54.6 1.0 0.5 3854

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Figure 4. Bioactivity of phlegmacin B1 for O. f urnacalis. (A) 5% DMSO-injected group; (B) phlegmacin B1-injected group (1 μg per insect); (C) phenotypes of phlegmacin B1-injected insects; (D) normal diet-fed group; (E) phlegmacin B1 containing diet-fed group; (F) phenotypes of phlegmacin B1-fed insects.

of Chi-h dsRNA from Spodoptera exigua (the ortholog of Of Chi-h) and Of Hex1,9,41 which suggested that in vivo targets of phlegmacin B1 are Of Chi-h and Of Hex1. To further test its bioactivity, a phlegmacin B1-containing artificial diet was used to feed third-instar day 1 O. f urnacalis larvae. Compared to the control group, the development of these larvae was also severely affected (Figure 4D,E). In the control group, 100% of larvae molted into fourth-instar larvae in 5 days, and 78% of larvae molted into fifth-instar larvae in 10 days. However, in the phlegmacin B1-fed group, only 3% of larvae survived to fourth-instar and were finally dead in 10 days without molting into fifth-instar. Some of the dead larvae had the phenotype of shrunken thoraxes and abdomens, the other larvae dead from molting failure (Figure 4F). The dead larvae were still fully or partially covered by the old cuticle. Phlegmacin B1 Activities against Both chitinases and Hexs. To evaluate the specificity of phlegmacin B1, its inhibitory activities against GH18 chitinases and GH20 Hexs from different species were determined at 20 μM and 100 μM concentrations. The representative GH18 chitinases were bacterial SmChiA, SmChiB, and SmChiC; fungal Af ChiB1; insect Of Chi-h and Of ChtI; and human HsCht, and the representative GH20 Hexs were bacterial SmChb, plant CeHex, insect Of Hex1 and Of Hex2, and human HsHexB. At the concentration of 100 μM, phlegmacin B1 showed 94%, 61%, and 51% inhibitory rates for Of Chi-h, SmChiB, and Of ChtI, but showed very weak inhibitory activities against SmChiC, Af ChiB, and HsCht (Table 2). The Ki values of phlegmacin B1 against SmChiB and Of ChtI were 40 μM and 79 μM, respectively (Figure 2). Phlegmacin B1 showed a greater than 50% inhibitory rate only for Of Hex1 at the concentration of 100 μM (Table 2). Thus, phlegmacin B1 is a potent and specific inhibitor of the insect enzymes Of Chi-h and Of Hex1, but a weak inhibitor of other GH18 chitinases and GH20 Hexs. In conclusion, the microbial secondary metabolite, phlegmacin B1, was obtained as an inhibitor of both GH18 chitinase and GH20 Hex. Because phlegmacin B1 can induce abnormal

Table 2. Inhibition of GH18 and GH20 Enzymes by Phlegmacin B1 inhibition rate (%) (mean ± SD) 100 μM GH18

GH20

a

Of Chi-h Of ChtI HsCht Af ChiB1 SmChiA SmChiB SmChiC Of Hex1 Of Hex2 HsHexB CeHex SmChb

93.8 50.5 35.3 36.4 32.8 60.7 12.2 88.8 39.5 21.8 46.3 37.4

± ± ± ± ± ± ± ± ± ± ± ±

0.4 2.0 2.1 2.7 1.4 1.1 3.0 1.4 0.7 2.9 2.4 0.4

20 μM

Ki (μM)

± ± ± ± ± ±

0.3 0.7 3.2 1.3 1.6 0.9

± ± ± ± ±

0.2 2.0 0.5 0.2 2.3

5.5 79.3 NDa ND ND 40 ND 26 ND ND ND ND

80.9 30.6 12.6 9.0 16.8 37.0 0 60.2 20.2 4.9 22.9 16.1

ND, not determined.

pupation and lethality in an insect pest, it could be developed into a pest control agent.

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ASSOCIATED CONTENT

S Supporting Information *

Additional data (Table S1−S4, Figure S1−S4). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b01710. Chemical identification of phlegmacin B1; inhibitory kinetic parameters; CD spectra; Inhibitory kinetics of allosamidin against Of Chi-h; ligplot analyses of phlegmacin B1 binding in Of Chi-h and Of Hex1 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: 86-411-84707245. Fax: 86-411-84707245. 3855

DOI: 10.1021/acs.jafc.7b01710 J. Agric. Food Chem. 2017, 65, 3851−3857

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Journal of Agricultural and Food Chemistry *E-mail: [email protected]. Phone: 86-411-84707245. Fax: 86-411-84707245.

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ORCID

Tian Liu: 0000-0001-9768-5496 Funding

This work was supported by the Program for National Natural Science Funds for Distinguished Young Scholar (31425021), the Program for Liaoning Excellent Talents in University (LJQ2014006), the Open Research Fund of the State Key Laboratory for Biology of Plant Diseases and Insect Pests (SKLOF201706), and the Fundamental Research Funds for the Central Universities (DUT16TD22). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Jianjun Zhang (China Agricultural University) for providing MU-GlcNAc and MU-(GlcNAc)2 (synthesized in the NKT R&D Program of China, 2015BAK45B01, CAU). We thank Dr. Yuesheng Dong (Dalian University of Technology) for the assistance in CD experiments. We also thank Thomas Malott (Dalian University of Technology) for the contribution in the language editing of the manuscript.

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ABBREVIATIONS USED Af ChiB1, chitinase B1 from Aspergillus f unigatus; CeHex, β-Nacetylhexosaminidase from Canavalia ensiformis; GH, glycosyl hydrolase; GlcNAc, N-acetyl-β-D-glucosamine; Hex, β-Nacetylhexosaminidase; HsCht, chitotriosidase from Homo sapiens; HsHexB, β-N-acetylhexosaminidase B from H. sapiens; IMAC, immobilized metal affinity chromatography; MD, molecular dynamics; MU-GlcNAc, 4-methylumbelliferyl-β-DN-acetylglucosamine; MU-(GlcNAc)2, 4-methylumbelliferyl-βD-N,N′-diacetylchitobiose; Of Chi-h and Of ChtI, chitinase-h and group I chitinase from Ostrinia f urnacalis; Of Hex1, group I β-N-acetylhexosaminidase from O. furnacalis; RMSD, root− mean−square deviation; SeChi-h, chitinase-h from Spodoptera exigua; SmChiA, SmChiB and SmChiC, chitinase A, chitinase B, and chitinase C from Serratia marcescens

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