Alternative relay regulates the adenosine triphosphatase activity of Locusta migratoria striated muscle myosin.

Locust (Locusta migratoria) has a single striated muscle myosin heavy chain (Mhc) gene, which contains 5 clusters of alternative exclusive exons and 1 differently included penultimate exon. The alternative exons of Mhc gene encode 4 distinct regions in the myosin motor domain, that is, the N-terminal SH3-like domain, one lip of the nucleotide-binding pocket, the relay, and the converter. Here, we investigated the role of the alternative regions on the motor function of locust muscle myosin. Using Sf9-baculovirus protein expression system, we expressed and purified 5 isoforms of the locust muscle myosin heavy meromyosin (HMM), including the major isoform in the thorax dorsal longitudinal flight muscle (FL1) and 4 isoforms expressed in the abdominal intersegmental muscle (AB1 to AB4). Among these 5 HMMs, FL1-HMM displayed the highest level of actin-activated adenosine triphosphatase (ATPase) activity (hereafter referred as ATPase activity). To identify the alternative region(s) responsible for the elevated ATPase activity of FL1-HMM, we produced a number of chimeras of FL1-HMM and AB4-HMM. Substitution with the relay of AB4-HMM (encoded by exon-14c) substantially decreased the ATPase activity of FL1-HMM, and conversely, the relay of FL1-HMM (encoded by exon-14a) enhanced the ATPase activity of AB4-HMM. Mutagenesis showed that the exon-14a-encoded residues Gly474 and Asn509 are responsible for the elevated ATPase activity of FL1-HMM. Those results indicate that the alternative relay encoded by exon-14a/c play a key role in regulating the ATPase activity of FL1-HMM and AB4-HMM.

Group h Chitinase: A Molecular Target for the Development of Lepidopteran-Specific Insecticides.

Sustainable agriculture requires insecticides that are selective between insects and mammals and even between harmful and beneficial insects. Lepidoptera includes the largest number of insect pests that threaten crops, and Hymenoptera contains the natural enemies for these pests. Discovery of lepidopteran-specific molecular targets is one route to develop such selective pesticides. Group h chitinase (Chi-h) is an ideal target for lepidopteran-specific insecticides because it is only distributed in Lepidoptera and is critical to their molting processes. This minireview focuses on the latest progress in developing Chi-h as a lepidopteran-specific insecticide target. We describe the biological function, crystal structure, and small-molecule inhibitors of the enzyme. Notably, two unique pockets were discovered in the crystal structure of Chi-h for the binding of the selective inhibitors, phlegmacin B1 and lynamicin B. Moreover, lynamicin B was found to exhibit significant insecticidal activity toward lepidopteran pests but is harmless toward their natural enemies. These findings are advancing the development of selective insecticides to meet the needs of sustainable agriculture.

AA15 lytic polysaccharide monooxygenase is required for efficient chitinous cuticle turnover during insect molting.

Microbial lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of crystalline polysaccharides including chitin and cellulose. The discovery of a large assortment of LPMO-like proteins widely distributed in insect genomes suggests that they could be involved in assisting chitin degradation in the exoskeleton, tracheae and peritrophic matrix during development. However, the physiological functions of insect LPMO-like proteins are still undetermined. To investigate the functions of insect LPMO15 subgroup I-like proteins (LPMO15-1s), two evolutionarily distant species, Tribolium castaneum and Locusta migratoria, were chosen. Depletion by RNAi of T. castaneum TcLPMO15-1 caused molting arrest at all developmental stages, whereas depletion of the L. migratoria LmLPMO15-1, prevented only adult eclosion. In both species, LPMO15-1-deficient animals were unable to shed their exuviae and died. TEM analysis revealed failure of turnover of the chitinous cuticle, which is critical for completion of molting. Purified recombinant LPMO15-1-like protein from Ostrinia furnacalis (rOfLPMO15-1) exhibited oxidative cleavage activity and substrate preference for chitin. These results reveal the physiological importance of catalytically active LPMO15-1-like proteins from distant insect species and provide new insight into the enzymatic mechanism of cuticular chitin turnover during molting.

A midgut-specific lytic polysaccharide monooxygenase of Locusta migratoria is indispensable for the deconstruction of the peritrophic matrix.

Lytic polysaccharide monooxygenases (LPMOs) are important enzymes that boost the hydrolysis of recalcitrant polysaccharides, such as chitin. They are found extensively in different insect species and are classified as auxiliary activities family 15 (AA15) LPMOs (LPMO15). Some of them were identified from the insect midgut and proven to act on chitin. However, knowledge about their physiological roles during insect growth and development remains limited. Here, we found that midgut-specific LPMO15s are widely distributed in different insect orders, such as the orthopteran Locusta migratoria and the lepidopteran Bombyx mori. Using L. migratoria as a model insect, the function of midgut-specific LmLPMO15-3 during development was investigated. Double-stranded RNA-mediated downregulation of LmLPMO15-3 expression at the 4th or 5th instar nymph stage severely decreased the survival rate and resulted in lethal phenotypes. Hematoxylin and eosin staining results indicated that the deficient individuals exhibited incompletely digested peritrophic matrix (PM), which suggested that LmLPMO15-3 is essential for the deconstruction of the PM during molting. This study provides direct evidence of the physiological importance of a midgut-specific LPMO15 during insect development. As L. migratoria is one of the most destructive agricultural pests, LmLPMO15-3 is a potential target for pest management.

Insect group II chitinase OfChtII promotes chitin degradation during larva-pupa molting.

The insect group II chitinase (ChtII, also known as Cht10) is a unique chitinase with multiple catalytic and chitin-binding domains. It has been proven genetically to be an essential chitinase for molting. However, ChtII's role in chitin degradation during insect development remains poorly understood. Obtaining this knowledge is the key to fully understanding the chitin degradation system in insects. Here, we investigated the role of OfChtII during the molting of Ostrinia furnacalis, a model lepidopteran pest insect. OfChtII was expressed earlier than OfChtI (OfCht5) and OfChi-h, at both the gene and protein levels during larva-pupa molting as evidenced by quantitative polymerase chain reaction and western blot analyses. A truncated OfChtII, OfChtII-B4C1, was recombinantly expressed in Pichia pastoris cells and purified to homogeneity. The recombinant OfChtII-B4C1 loosened compacted chitin particles and produced holes in the cuticle surface as evidenced by scanning electron microscopy. It synergized with OfChtI and OfChi-h when hydrolyzing insoluble α-chitin. These findings suggested an important role for ChtII during insect molting and also provided a strategy for the coordinated degradation of cuticular chitin during insect molting by ChtII, ChtI and Chi-h.

Phylogenetic relationships and biological features reveal that male Ostrinia furnacalis (Lepidoptera: Crambidae) in Northeast China can be categorized into postmedial line-based clades.

Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae), often called the Asian corn borer, is a complicated pest because of its complex biological features, such as its adult dynamics, host choice, and life span. This complexity has been causing difficulties in both pest forecasting and control for more than 60 years. One likely explanation for this complexity is that O. furnacalis has several varieties that vary based on some specific features. During 2015-2017, postmedial line-based varieties of male O. furnacalis were identified as distinct clades (I, II, and III), which were then compared based on COI gene sequences, male sacculus construction, life span, male dynamics, and host preference. The results showed that: (1) clades II and III were more closely related to each other than Clade I, because they both completed two generations per year, more were captured in 2016 or fewer were captured in 2015, and they were more closely related according to phylogenetic inference; (2) all three clades shared some features, such as life spans under various rearing conditions, similar dynamic trends, and three teeth on the male sacculus; and (3) all three clades were significantly different from O. nubilalis based on genetic sequences, postmedial line pattern of the forewing, and sacculus construction. Overall, if O. furnacalis is categorized into clades, the species' features are likely to be a combination or mixture of the features of each individual clade. Our findings help explain the biological complexity of O. furnacalis. Future investigations on each individual clade are essential for improving forecasting and control of this pest.

Structural and biochemical insights into the catalytic mechanisms of two insect chitin deacetylases of the carbohydrate esterase 4 family.

Insect chitin deacetylases (CDAs) catalyze the removal of acetyl groups from chitin and modify this polymer during its synthesis and reorganization. CDAs are essential for insect survival and therefore represent promising targets for insecticide development. However, the structural and biochemical characteristics of insect CDAs have remained elusive. Here, we report the crystal structures of two insect CDAs from the silk moth Bombyx mori: BmCDA1, which may function in cuticle modification, and BmCDA8, which may act in modifying peritrophic membranes in the midgut. Both enzymes belong to the carbohydrate esterase 4 (CE4) family. Comparing their overall structures at 1.98-2.4 Å resolution with those from well-studied microbial CDAs, we found that two unique loop regions in BmCDA1 and BmCDA8 contribute to the distinct architecture of their substrate-binding clefts. These comparisons revealed that both BmCDA1 and BmCDA8 possess a much longer and wider substrate-binding cleft with a very open active site in the center than the microbial CDAs, including VcCDA from Vibrio cholerae and ArCE4A from Arthrobacter species AW19M34-1. Biochemical analyses indicated that BmCDA8 is an active enzyme that requires its substrates to occupy subsites 0, +1, and +2 for catalysis. In contrast, BmCDA1 also required accessory proteins for catalysis. To the best of our knowledge, our work is the first to unveil the structural and biochemical features of insect proteins belonging to the CE4 family.

Biochemical characterization of three midgut chitin deacetylases of the Lepidopteran insect Bombyx mori.

Peritrophic membrane (PM) is a chitin and protein-containing extracellular matrix that lines the midgut in most insect species, functioning as a barrier to exogenous toxins and pathogens. Midgut chitin deacetylases (CDAs) are chitin-modifying enzymes known to alter the mechanical property and permeability of PM. However, biochemical properties and specific roles of these enzymes remain elusive. In this study, the midgut-expressed CDAs (BmCDA6, BmCDA7 and BmCDA8) from Bombyx mori were cloned, recombinantly expressed and purified and their enzymatic activities toward PM chitin were determined. Of the three enzymes, BmCDA7 exhibited the highest activity (0.284 µmol/min/µmol), while BmCDA8 showed lower activity of 0.061 µmol/min/µmol. BmCDA6 was inactive towards PM chitin. Gene expression patterns indicated that although all three CDA genes were specifically expressed in the anterior midgut, they differed in their temporal expression patterns. BmCDA6 was expressed almost exclusively at the mid-molt stage, the stage when the PM was thick and with multiple chitin layers. Unlike BmCDA6, high expression levels of BmCDA7 and BmCDA8 were observed only at the feeding stage, the stage when the PM is thin and with fewer chitin layers. The different gene expression patterns and biochemical characteristics provide new information about the functional specialization among BmCDA6, BmCDA7 and BmCDA8 proteins.

Biochemical characteristics of a nitroreductase with diverse substrate specificity from Streptomyces mirabilis DUT001.

A nitroreductase-encoded gene from an efficient nitro-reducing bacterium Streptomyces mirabilis DUT001, named snr, was cloned and heterogeneously expressed in Escherichia coli. The purified Streptomyces nitroreductase SNR was a homodimer with an apparent subunit molecular weight of 24 kDa and preferred NADH to NADPH as a cofactor. By enzyme incubation and isothermal calorimetry experiments, flavin mononucleotide (FMN) was found to be the preferred flavin cofactor; the binding process was exothermic and primarily enthalpy driven. The enzyme can reduce multiple nitro compounds and flavins, including antibacterial drug nitrofurazone, priority pollutants 2,4-dinitrotoluene and 2,4,6-trinitrotoluene, as well as key chemical intermediates 3-nitrophthalimide, 4-nitrophthalimide, and 4-nitro-1,8-naphthalic anhydride. Among the substrates tested, the highest activity of kcat(app) /Km(app) (0.234 µM-1  Sec-1 ) was observed for the reduction of FMN. Multiple sequence alignment revealed that the high FMN reduction activity of SNR may be due to the absence of a helix, constituting the entrance to the substrate pocket in other nitroreductases.

Structural analysis of group II chitinase (ChtII) catalysis completes the puzzle of chitin hydrolysis in insects.

Chitin is a linear homopolymer of N-acetyl-ß-d-glucosamines and a major structural component of insect cuticles. Chitin hydrolysis involves glycoside hydrolase family 18 (GH18) chitinases. In insects, chitin hydrolysis is essential for periodic shedding of the old cuticle ecdysis and proceeds via a pathway different from that in the well studied bacterial chitinolytic system. Group II chitinase (ChtII) is a widespread chitinolytic enzyme in insects and contains the greatest number of catalytic domains and chitin-binding domains among chitinases. In Lepidopterans, ChtII and two other chitinases, ChtI and Chi-h, are essential for chitin hydrolysis. Although ChtI and Chi-h have been well studied, the role of ChtII remains elusive. Here, we investigated the structure and enzymology of OfChtII, a ChtII derived from the insect pest Ostrinia furnacalis We present the crystal structures of two catalytically active domains of OfChtII, OfChtII-C1 and OfChtII-C2, both in unliganded form and complexed with chitooligosaccharide substrates. We found that OfChtII-C1 and OfChtII-C2 both possess long, deep substrate-binding clefts with endochitinase activities. OfChtII exhibited structural characteristics within the substrate-binding cleft similar to those in OfChi-h and OfChtI. However, OfChtII lacked structural elements favoring substrate binding beyond the active sites, including an extra wall structure present in OfChi-h. Nevertheless, the numerous domains in OfChtII may compensate for this difference; a truncation containing one catalytic domain and three chitin-binding modules (OfChtII-B4C1) displayed activity toward insoluble polymeric substrates that was higher than those of OfChi-h and OfChtI. Our observations provide the last piece of the puzzle of chitin hydrolysis in insects.

The deduced role of a chitinase containing two nonsynergistic catalytic domains.

The glycoside hydrolase family 18 chitinases degrade or alter chitin. Multiple catalytic domains in a glycoside hydrolase family 18 chitinase function synergistically during chitin degradation. Here, an insect group III chitinase from the agricultural pest Ostrinia furnacalis (OfChtIII) is revealed to be an arthropod-conserved chitinase that contains two nonsynergistic GH18 domains according to its catalytic properties. Both GH18 domains are active towards single-chained chitin substrates, but are inactive towards insoluble chitin substrates. The crystal structures of each unbound GH18 domain, as well as of GH18 domains complexed with hexa-N-acetyl-chitohexaose or penta-N-acetyl-chitopentaose, suggest that the two GH18 domains possess endo-specific activities. Physiological data indicated that the developmental stage-dependent gene-expression pattern of OfChtIII was the same as that of the chitin synthase OfChsA but significantly different from that of the chitinase OfChtI, which is indispensable for cuticular chitin degradation. Additionally, immunological staining indicated that OfChtIII was co-localized with OfChsA. Thus, OfChtIII is most likely to be involved in the chitin-synthesis pathway.

Fully deacetylated chitooligosaccharides act as efficient glycoside hydrolase family 18 chitinase inhibitors.

Small molecule inhibitors against chitinases have potential applications as pesticides, fungicides, and antiasthmatics. Here, we report that a series of fully deacetylated chitooligosaccharides (GlcN)2-7 can act as inhibitors against the insect chitinase OfChtI, the human chitinase HsCht, and the bacterial chitinases SmChiA and SmChiB with IC50 values at micromolar to millimolar levels. The injection of mixed (GlcN)2-7 into the fifth instar larvae of the insect Ostrinia furnacalis resulted in 85% of the larvae being arrested at the larval stage and death after 10 days, also suggesting that (GlcN)2-7 might inhibit OfChtI in vivo. Crystal structures of the catalytic domain of OfChtI (OfChtI-CAD) complexed with (GlcN)5,6 were obtained at resolutions of 2.0 Å. These structures, together with mutagenesis and thermodynamic analysis, suggested that the inhibition was strongly related to the interaction between the -1 GlcN residue of the inhibitor and the catalytic Glu(148) of the enzyme. Structure-based comparison showed that the fully deacetylated chitooligosaccharides mimic the substrate chitooligosaccharides by binding to the active cleft. This work first reports the inhibitory activity and proposed inhibitory mechanism of fully deacetylated chitooligosaccharides. Because the fully deacetylated chitooligosaccharides can be easily derived from chitin, one of the most abundant materials in nature, this work also provides a platform for developing eco-friendly inhibitors against chitinases.

Proteomic analysis of insect molting fluid with a focus on enzymes involved in chitin degradation.

Cuticular chitin degradation is extremely important for insect growth and development, which has not been fully understood thus far. One obstacle to understanding this mechanism is the lack of a systematic analysis of the chitinolytic enzymes involved in cuticular chitin degradation. In this study, we used the silkmoth Bombyx mori as a model organism and compared proteomic analyses for larval-pupal (L-P) and pupal-adult (P-A) molting fluids using tandem mass tag quantitative mass spectrometry. There were 195 proteins identified from both L-P and P-A molting fluids. A total of 170 out of 195 proteins were deduced to be secretory and were enriched for GO terms associated with chitin metabolism and proteolysis by using AgriGO. Although the chitinolytic enzymes are encoded by many insect genes, the proteomics analysis unexpectedly showed that only four chitinolytic enzymes with the combination "211" were abundant in both molting fluids, namely, two insect GH18 Chitinase family members (ChtI and ChtII), one bacterial-type GH18 Chitinase (Chi-h), and one insect GH20 hexosaminidase (Hex1). A tissue-specific and stage-specific gene expression pattern verified that the "211" enzymes are involved in cuticular chitin degradation. This work first demonstrates that specific enzymes ChtI, ChtII, Chi-h, and Hex1 can be assigned to cuticular chitin degradation.

A sperm-plasma ß-N-acetyl-D-hexosaminidase interacting with a Chitinolytic ß-N-Acetyl-D-hexosaminidase in insect molting fluid.

Insects require molting fluids to shed the old cuticle during molting. ß-N-acetyl-D-hexosaminidase, known as Hex1, together with various chitinases, is responsible for degrading the chitin component of the old cuticle. This study showed that another ß-N-acetyl-D-hexosaminidase, termed OfHex3, interacted with Hex1 and functioned in the molting fluid, although the homolog of OfHex3 was known as a sperm-plasma enzyme functioning in egg-sperm recognition. OfHex3 is an enzyme cloned from the insect Asian corn borer, Ostrinia furnacalis, which is one of the most destructive pests of maize. The enzymatic activity analysis indicated that OfHex3 was able to degrade chitooligosaccharides, but at a lower rate than that of OfHex1. Because OfHex3 did not have substrate inhibition, we deduced that the presence of OfHex3 might help OfHex1 relieve substrate inhibition during chitin degradation during molting. The expression patterns of OfHex3 during O. furnacalis development were studied by real-time PCR as well as western blot. The results showed that both gene transcription and protein translation levels of OfHex3 were up-regulated during larval-larval molting. The tissue-specific expression pattern analysis indicated that OfHex3 was mostly localized in the fat body and testis. All these data further supported that Hex3 was involved in molting as well as in fertilization. This study may help to understand the complexity of cuticle degradation during insect molting, and may provide a possible target for pest control.

Biochemical characterization of a novel ß-N-acetylhexosaminidase from the insect Ostrinia furnacalis.

The ß-N-acetylhexosaminidase FDL specifically removes the ß-1,2-GlcNAc residue conjugated to the α-1,3-mannose residue of the core structure of insect N-glycans, playing significant physiological roles in post-translational modification in the Golgi apparatus. Little is known about its enzymatic properties. We obtained the OfFDL gene from the insect Ostrinia furnacalis by RT-PCR. The full length cDNA of FDL is 2241 bp carrying an opening reading frame of 1923 bp encoding 640 amino acids. The recombinant protein OfFDL in a soluble and active form was obtained with high purity through a two-step purification strategy. The recombinant OfFDL exclusively hydrolyzes the terminal ß-1,2-GlcNAc residue from the α-1,3 branch instead of the α-1,6 branch of the substrate GnGn-PA. Several kinetic parameters including kcat/Km values toward four artificial substrates and Ki values of three representative hexosaminidase inhibitors were obtained.

Molecular and biochemical characterization of a novel ß-N-acetyl-D-hexosaminidase with broad substrate-spectrum from the Aisan corn borer, Ostrinia furnacalis.

Insect ß-N-acetyl-D-hexosaminidases with broad substrate-spectrum (IBS-Hex) are the homologues of human ß-N-acetyl-D-hexosaminidase A/B (HsHex A/ B). These enzymes are distributed in most insect species and vary in physiological roles. In this study, the gene encoding an IBS-Hex, OfHEX2, was cloned from the Asian corn borer, Ostrinia furnacalis. Recombinant OfHex2 was expressed in Pichia pastoris and purified to homogeneity. By structure-based sequence alignment, three sequence segments with high diversity among IBS-Hexs were firstly concluded. Furthermore, the residue pair N423-R424/ D452-L453 important for the specificity of human ß-N-acetyl-D-hexosaminidase subunits α/ß toward charged/ non-charged substrates was not conserved in OfHex2 and other IBS-Hexs. Unlike HsHex A, OfHex2 could not degrade charged substrates such as 4-methylumbelliferyl-6-sulfo-N-acetyl-ß-D-glucosaminide, ganglioside GM2 and peptidoglycan. OfHex2 showed a broad substrate-spectrum by hydrolyzing ß1-2 linked N-acetyl-D-glucosamines from both α3 and α6 branches of biantennary N-glycan and ß1-4 linked GlcNAc from chitooligosaccharides as well as ß1-3 linked or ß1-4 linked N-acetyl-D-galactosamine from oligosaccharides of glycolipids. Real-time PCR analysis demonstrated that the expression of OfHEX2 was up-regulated in the intermolt stages (both larva and pupa), and mainly occurred in the carcass rather than in the midgut during the feeding stage of fifth (final) instar larva. This study reported a novel IBS-Hex with specific biochemical properties, suggesting biodiversity of this class of enzymes.

A novel alternative splicing site of class A chitin synthase from the insect Ostrinia furnacalis - gene organization, expression pattern and physiological significance.

Insect chitin synthase A (CHSA) catalyzes chitin biosynthesis in tissues that develop from ectoderm. Since only one gene copy encodes CHSA, we hypothesized that CHSA is very likely to exist as isoforms through alternative splicing, and the functions of these isoforms may be tissue-specific. Besides the known alternative splicing exons in the mid-ORF region, we report here the alternative exons (OfCHSA-2a and OfCHSA-2b) of OfCHSA, the chitin synthase A from the lepidopteran pest Ostrinia furnacalis. Sequence analysis of the 5' upstream region of the transcription start site indicated that presences of two independent promoters for controlling the expression of OfCHSA-2a/b. Both OfCHSA-2a and OfCHSA-2b transcripts were preferentially expressed in the epidermis. During growth and development of O. furnacalis, OfCHSA-2a was mainly expressed during larval-larval molting and larval-pupal transformation, as well as in newly-laid eggs, while OfCHSA-2b was expressed only during the larval-larval molting. Gene silencing of OfCHSA-2a caused incomplete molting, while silencing of OfCHSA-2b exclusively influenced the head cuticle formation of the 3rd instar larval. Since O. furnacalis is phylogenetically close to the model insect Bombyx mori, the same undiscovered alternative splicing exon was also identified in BmCHSA by gDNA sequence alignment. This work may lead to greater understanding of the mechanism by which a single copy of the CHSA gene could fulfill various functions with tissue specificity.

The gene, expression pattern and subcellular localization of chitin synthase B from the insect Ostrinia furnacalis.

Insect midgut peritrophic membrane (PM) is a functional structure that protects insects against chemical damage and microorganism infection. The essential component in PM is chitin and its synthesis is catalyzed by Class B chitin synthase (CHSB), which plays a unique role in chitin-containing organisms and thus represents a potential target for eco-friendly pesticides. cDNA and gDNA of CHSB from a widely spread pest Ostrinia furnacalis (OfCHSB) were obtained and their sequences and transcription patterns were characterized. Results indicated that OfCHSB may be indirectly stimulated by ecdysone because the binding sites of only early ecdysone-inducible elements (BR-C and E74A) rather than ecdysone response elements (EcR and USP) were found within the core promoter of OfCHSB. In addition, the transcripts of OfCHSB increased in vivo at the feeding stage of the 4th and 5th instar larvae. The subcellular localization of OfCHSB was studied using an insect midgut cell line. Puncta structures of the recombinant OfCHSB were observed co-localized with Golgi marker Man II-GFP, suggesting a possible localization of chitin synthases under physiological conditions.

A novel beta-N-acetyl-D-hexosaminidase from the insect Ostrinia furnacalis (Guenée).

Exploiting specific targets is of specific interest in developing eco-friendly pesticides. We isolated, purified and characterized a novel beta-N-acetyl-D-hexosaminidase (OfHex1) from the fifth instar larva integument of the Asian corn borer, Ostrinia furnacalis (Guenée). OfHex1 was purified 1468-fold to homogeneity with an activity yield of 20% by four column chromatography steps. Under denaturing conditions, the molecular mass of OfHex1 was determined to be 67.0 kDa by MS and SDS/PAGE, but 128 kDa by gel filtration chromatography, suggesting that it was in the form of a homodimer. Its pI was 4.7 as determined by IEF electrophoresis. OfHex1 was shown to be an exo-splitting enzyme, acting by cutting one beta-GlcNAc unit once from the nonreducing ends of substrates, with a preference for shorter substrates. OfHex1 could hydrolyze p-nitrophenyl beta-GlcNAc, p-nitrophenyl beta-GalNAc and chito-oligosaccharides (degree of polymerization from 2 to 6), but it could not hydrolyze the complex N-glycan substrate (GlcNAc beta-1,2Man alpha-1,6)(GlcNAc beta-1,2Man alpha-1,3)Man beta-1,4GlcNAc beta-1,4GlcNAc-PA as well as the long polymer chitin. Certain structural elements of substrates, the 2-acetamido group and the beta-glycoside bond linkage, were determined to be essential for its activity. The 2.6 kb cDNA encoding OfHex1 was obtained by RT-PCR. Sequence analysis indicated that it was different from reported beta-N-acetyl-D-hexosaminidases with N-glycan hydrolytic activities. Real-time PCR determined that its transcriptional level increased dramatically before the molting stage. According to its hydrolytic mode, substrate spectrum, cDNA sequence and mRNA transcriptional level, we deduced that OfHex1 could be mostly involved in insect chitin catabolism and might be a specific target for pesticide development.