Deciphering The Synergism Of Endogenous Glycoside Hydrolase Families 1 And 9 From Coptotermes Gestroi

Termites can degrade up to 90% of the lignocellulose they ingest using a repertoire of endogenous and symbiotic degrading enzymes. Termites have been shown to secrete two main glycoside hydrolases, which are GH1 (EC 3.2.1.21) and GH9 (EC 3.2.1.4) members. However, the molecular mechanism for lignocellulose degradation by these enzymes remains poorly understood. The present study was conducted to understand the synergistic relationship between GH9 (CgEG1) and GH1 (CgBG1) from Coptotermes gestroi, which is considered the major urban pest of São Paulo State in Brazil. The goal of this work was to decipher the mode of operation of CgEG1 and CgBG1 through a comprehensive biochemical analysis and molecular docking studies. There was outstanding degree of synergy in degrading glucose polymers for the production of glucose as a result of the endo-β-1,4-glucosidase and exo-β-1,4-glucosidase degradation capability of CgEG1 in concert with the high catalytic performance of CgBG1, which rapidly converts the oligomers into glucose. Our data not only provide an increased comprehension regarding the synergistic mechanism of these two enzymes for cellulose saccharification but also give insight about the role of these two enzymes in termite biology, which can provide the foundation for the development of a number of important applied research topics, such as the control of termites as pests as well as the development of technologies for lignocellulose-to-bioproduct applications. © 2013 Elsevier Ltd.4310970981Aguiar-Oliveira, E., Maugeri, F., Thermal stability of the immobilized fructosyltransferase from Rhodotorula sp. (2011) Braz. J. Chem. Eng., 28, pp. 363-372Barsotti, R.C., Costa-Leonardo, A.M., (2005) The Caste System of Coptotermes gestroi (Isoptera:Rhinotermitidae), 46, p. 17Bendtsen, J.D., Nielsen, H., von Heijne, G., Brunak, S., Improved prediction of signal peptides: SignalP 3.0 (2004) J.Mol. Biol., 340, pp. 783-795Benkert, P., Kunzli, M., Schwede, T., QMEAN server for protein model quality estimation (2009) Nucleic Acids Res., 37, pp. W510-W514Bradford, M.M., Arapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding (1976) Anal. Biochem., 72, pp. 248-254Brune, A., Termite guts: the world's smallest bioreactors (1998) Trends Biotechnol, 16, pp. 16-21Brune, A., Ohkuma, M., Role of the termite gut microbiota in symbiotic digestion (2011) Biology of Termites: A Modern Synthesis, pp. 439-475. , Springer, Netherlands, D.E. Bignell, Y. Roisin, N. Lo (Eds.)Buck, M., Bouguet-Bonnet, S., Pastor, R.W., MacKerell, A.D., Importance of the CMAP correction to the CHARMM22 protein force field: dynamics of hen lysozyme (2006) Biophys. J., 90, pp. L36-L38Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard, V., Henrissat, B., The carbohydrate-active EnZymes database (CAZy): an expert resource for Glycogenomics (2009) Nucleic Acids Res., 37, pp. D233-D238Chandrasekharaiah, M., Thulasi, A., Bagath, M., Kumar, D.P., Santosh, S.S., Palanivel, C., Jose, V.L., Sampath, K.T., Molecular cloning, expression and characterization of a novel feruloyl esterase enzyme from the symbionts of termite(Coptotermes formosanus) gut (2011) BMB Rep., 44, pp. 52-57Chen, X.A., Ishida, N., Todaka, N., Nakamura, R., Maruyama, J., Takahashi, H., Kitamoto, K., Promotion of efficient saccharification of crystalline cellulose by Aspergillus fumigatus Swo1 (2010) Appl. Environ. Microbiol., 76, pp. 2556-2561Converse, A.O., Optekar, J.D., Asynergistic kinetics model for enzymatic cellulose hydrolysis compared to degree-of-synergism experimental results (1993) Biotechnol. Bioeng., 42, pp. 145-148Coy, M.R., Salem, T.Z., Denton, J.S., Kovaleva, E.S., Liu, Z., Barber, D.S., Campbell, J.H., Scharf, M.E., Phenol-oxidizing laccases from the termite gut (2010) Insect Biochem. Mol. Biol., 40, pp. 723-732de Vasconcelos, S.M., Santos, A.M.P., Rocha, G.J.M., Souto-Maior, A.M., Diluted phosphoric acid pretreatment for production of fermentable sugars in a sugarcane-based biorefinery (2013) Bioresour. Technol., 135, pp. 46-52Franco Cairo, J.P., Leonardo, F.C., Alvarez, T.M., Ribeiro, D.A., Buchli, F., Costa-Leonardo, A.M., Carazzolle, M.F., Squina, F.M., Functional characterization and target discovery of glycoside hydrolases from lower termite Coptotermes gestroi digestome (2011) Biotechnol. Biofuels, 4, p. 50Fujita, A., Hojo, M., Aoyagi, T., Hayashi, Y., Arakawa, G., Tokuda, G., Watanabe, H., Details of the digestive system in the midgut of Coptotermes formosanus Shiraki (2010) J.Wood Sci., 56, pp. 222-226Gonçalves, T.A., Damasio, A.R., Segato, F., Alvarez, T.M., Bragatto, J., Brenelli, L.B., Citadini, A.P., Squina, F.M., Functional characterization and synergic action of fungal xylanase and arabinofuranosidase for production of xylooligosaccharides (2012) Bioresour. Technol., 119, pp. 293-299Guerin, D.M., Lascombe, M.B., Costabel, M., Souchon, H., Lamzin, V., Beguin, P., Alzari, P.M., Atomic (0.94 A) resolution structure of an inverting glycosidase in complex with substrate (2002) J.Mol. Biol., 316, pp. 1061-1069Hongoh, Y., Toward the functional analysis of uncultivable, symbiotic microorganisms in the termite gut (2011) Cell Mol. Life Sci., 68, pp. 1311-1325Hoogwijk, M., Faaij, A., Eickhout, B., de Vries, B., Turkenburg, W., Potential of biomass energy out to 2100, for four IPCC SRES land-use scenarios (2005) Biomass and Bioenergy, 29, pp. 225-257Jeng, W.Y., Wang, N.C., Lin, M.H., Lin, C.T., Liaw, Y.C., Chang, W.J., Liu, C.I., Wang, A.H., Structural and functional analysis of three beta-glucosidases from bacterium Clostridium cellulovorans, fungus Trichoderma reesei and termite Neotermes koshunensis (2011) J.Struct. Biol., 173, pp. 46-56Jenkins, T.M., Jones, S.C., Lee, C.Y., Forschler, B.T., Chen, Z., Lopez-Martinez, G., Gallagher, N.T., Kleinschmidt, S., Phylogeography illuminates maternal origins of exotic Coptotermes gestroi (Isoptera: Rhinotermitidae) (2007) Mol. Phylogenet. Evol., 42, pp. 612-621Katsumata, K., Jin, Z., Hori, K., Iiyama, K., Structural changes in lignin of tropical woods during digestion by termite, Cryptotermes brevis (2007) J.Wood Sci., 53, pp. 419-426Khademi, S., Guarino, L.A., Watanabe, H., Tokuda, G., Meyer, E.F., Structure of an endoglucanase from termite, Nasutitermes takasagoensis (2002) Acta Crystallogr. D Biol. Crystallogr., 58, pp. 653-659Kiefer, F., Arnold, K., Kunzli, M., Bordoli, L., Schwede, T., The SWISS-MODEL repository and associated resources (2009) Nucleic Acids Res., 37, pp. D387-D392Klose, D.P., Wallace, B.A., Janes, R.W., 2Struc: the secondary structure server (2010) Bioinformatics, 26, pp. 2624-2625Koshland, D.E., Sterrochemistry and the mechanism of enzymatic reactions (1953) Biol. Rev., 28, pp. 416-436Lang, P.T., Brozell, S.R., Mukherjee, S., Pettersen, E.F., Meng, E.C., Thomas, V., Rizzo, R.C., Kuntz, I.D., DOCK 6: combining techniques to model RNA-small molecule complexes (2009) RNA, 15, pp. 1219-1230Leonardo, F.C., da Cunha, A.F., da Silva, M.J., Carazzolle, M.F., Costa-Leonardo, A.M., Costa, F.F., Pereira, G.A., Analysis of the workers head transcriptome of theAsian subterranean termite, Coptotermes gestroi (2011) Bull. Entomol. Res., 101, pp. 383-391Li, Y., Irwin, D.C., Wilson, D.B., Processivity, substrate binding, and mechanism of cellulose hydrolysis by Thermobifida fusca Cel9A (2007) Appl. Environ. Microbiol., 73, pp. 3165-3172Lynd, L.R., Zhang, Y., Quantitative determination of cellulase concentration as distinct from cell concentration in studies of microbial cellulose utilization: analytical framework and methodological approach (2002) Biotechnol. Bioeng., 77, pp. 467-475Miller, G.L., Use of dinitrosalicylic acid reagent for determination of reducing sugar (1959) Anal. Chem., 31, pp. 426-428Murashima, K., Kosugi, A., Doi, R.H., Synergistic effects on crystalline cellulose degradation between cellulosomal cellulases from Clostridium cellulovorans (2002) J.Bacteriol., 184, pp. 5088-5095Naran, R., Pierce, M.L., Mort, A.J., Detection and identification of rhamnogalacturonan lyase activity in intercellular spaces of expanding cotton cotyledons (2007) Plant J., 50, pp. 95-107Ni, J., Takehara, M., Watanabe, H., Heterologous overexpression of a mutant termite cellulase gene in Escherichia coli by DNA shuffling of four orthologous parental cDNAs (2005) Biosci. Biotechnol. Biochem., 69, pp. 1711-1720Ni, J., Tokuda, G., Takehara, M., Watanabe, H., Heterologous expression and enzymatic characterization of β-glucosidase from the drywood-eating termite, Neotermes koshunensis (2007) Appl. Entomol. Zool., 42, pp. 457-463Ohkuma, M., Termite symbiotic systems: efficient bio-recycling of lignocellulose (2003) Appl. Microbiol. Biotechnol., 61, pp. 1-9Ohkuma, M., Hongoh, Y., Noda, S., Symbiotic complex in the termite gut microbial community (2008) Tanpakushitsu. Kakusan. Koso., 53, pp. 1841-1849Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., Ferrin, T.E., UCSF Chimera-a visualization system for exploratory research and analysis (2004) J.Comput. Chem., 25, pp. 1605-1612Pronk, S., Pall, S., Schulz, R., Larsson, P., Bjelkmar, P., Apostolov, R., Shirts, M.R., Lindahl, E., GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit (2013) Bioinformatics, 29 (7), pp. 845-854Ribeiro, D.A., Cota, J., Alvarez, T.M., Bruchli, F., Bragato, J., Pereira, B.M., Pauletti, B.A., Squina, F.M., The Penicillium echinulatum secretome on sugar cane bagasse (2012) PLoS One, 7, pp. e50571Rincones, J., Zeidler, A.F., Grassi, M.C.B., Carazzolle, M.F., Pereira, G.A.G., The golden bridge for nature: the new biology applied to bioplastics (2009) Pol. Rev., 49, pp. 85-106Rudsander, U.J., Sandstrom, C., Piens, K., Master, E.R., Wilson, D.B., Brumer Iii, H., Kenne, L., Teeri, T.T., Comparative NMR analysis of cellooligosaccharide hydrolysis by GH9 bacterial and plant endo-1,4-beta-glucanases (2008) Biochemistry, 47, pp. 5235-5241Sagermann, M., Matthews, B.W., Crystal structures of a T4-lysozyme duplication-extension mutant demonstrate that the highly conserved beta-sheet region has low intrinsic folding propensity (2002) J.Mol. Biol., 316, pp. 931-940Sakon, J., Irwin, D., Wilson, D.B., Karplus, P.A., Structure and mechanism of endo/exocellulase E4 from Thermomonospora fusca (1997) Nat. Struct. Biol., 4, pp. 810-818Scharf, M.E., Karl, Z.J., Sethi, A., Boucias, D.G., Multiple levels of synergistic collaboration in termite lignocellulose digestion (2011) PLoS One, 6, pp. e21709Scharf, M.E., Kovaleva, E.S., Jadhao, S., Campbell, J.H., Buchman, G.W., Boucias, D.G., Functional and translational analyses of a beta-glucosidase gene (glycosyl hydrolase family 1) isolated from the gut of the lower termite Reticulitermes flavipes (2010) Insect Biochem. Mol. Biol., 40, pp. 611-620Schwarz, W.H., The cellulosome and cellulose degradation by anaerobic bacteria (2001) Appl. Microbiol. Biotechnol., 56, pp. 634-649Sethi, A., Slack, J.M., Kovaleva, E.S., Buchman, G.W., Scharf, M.E., Lignin-associated metagene expression in a lignocellulose-digesting termite (2013) Insect Biochem. Mol. Biol., 43, pp. 91-101Soccol, C.R., Vandenberghe, L.P., Medeiros, A.B., Karp, S.G., Buckeridge, M., Ramos, L.P., Pitarelo, A.P., Torres, F.A., Bioethanol from lignocelluloses: status and perspectives in Brazil (2010) Bioresour. Technol., 101, pp. 4820-4825Souza, A., Leite, D., Pattathil, S., Hahn, M., Buckeridge, M., Composition and structure of sugarcane cell wall Polysaccharides: implications for second-generation bioethanol production (2013) Bioenerg. Res., 6, pp. 564-579Squina, F.M., Prade, R.A., Wang, H., Murakami, M.T., Expression, purification, crystallization and preliminary crystallographic analysis of an endo-1,5-alpha-L-arabinanase from hyperthermophilic Thermotoga petrophila (2009) Acta Crystallogr. Sect. F Struct. Biol. Cryst Commun., 65, pp. 902-905Tartar, A., Wheeler, M.M., Zhou, X., Coy, M.R., Boucias, D.G., Scharf, M.E., Parallel metatranscriptome analyses of host and symbiont gene expression in the gut of the termite Reticulitermes flavipes (2009) Biotechnol. Biofuels, 2, p. 25Teeri, T.T., Crystalline cellulose degradation: new insight into the function of cellobiohydrolases (1997) Trends Biotechnol., 15, pp. 160-167Todaka, N., Inoue, T., Saita, K., Ohkuma, M., Nalepa, C.A., Lenz, M., Kudo, T., Moriya, S., Phylogenetic analysis of cellulolytic enzyme genes from representative lineages of termites and a related cockroach (2010) PLoS One, 5, pp. e8636Todaka, N., Moriya, S., Saita, K., Hondo, T., Kiuchi, I., Takasu, H., Ohkuma, M., Kudo, T., Environmental cDNA analysis of the genes involved in lignocellulose digestion in the symbiotic protist community of Reticulitermes speratus (2007) FEMS Microbiol. Ecol., 59, pp. 592-599Tokuda, G., Watanabe, H., Hojo, M., Fujita, A., Makiya, H., Miyagi, M., Arakawa, G., Arioka, M., Cellulolytic environment in the midgut of the wood-feeding higher termite Nasutitermes takasagoensis (2012) J.Insect Physiol., 58, pp. 147-154Uchima, C.A., Tokuda, G., Watanabe, H., Kitamoto, K., Arioka, M., Heterologous expression and characterization of a glucose-stimulated beta-glucosidase from the termite Neotermes koshunensis in Aspergillus oryzae (2011) Appl. Microbiol. Biotechnol., 89, pp. 1761-1771Uchima, C.A., Tokuda, G., Watanabe, H., Kitamoto, K., Arioka, M., Heterologous expression in Pichia pastoris and characterization of an endogenous thermostable and high-glucose-tolerant beta-glucosidase from the termite Nasutitermes takasagoensis (2012) Appl. Environ. Microbiol., 78, pp. 4288-4293Uchima, C.A., Tokuda, G., Watanabe, H., Kitamoto, K., Arioka, M., Anovel glucose-tolerant β-glucosidase from the salivary gland of the termite Nasutitermes takasagoensis (2013) J.Gen. Appl. Microbiol., 59, pp. 141-145van den Brink, J., de Vries, R., Fungal enzyme sets for plant polysaccharide degradation (2011) Appl. Microbiol. Biotechnol., 91, pp. 1477-1492Wallace, A.C., Laskowski, R.A., Thornton, J.M., LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions (1995) Protein Eng., 8, pp. 127-134Wang, H., Squina, F., Segato, F., Mort, A., Lee, D., Pappan, K., Prade, R., High-temperature enzymatic breakdown of cellulose (2011) Appl. Environ. Microbiol., 77, pp. 5199-5206Warnecke, F., Luginbuhl, P., Ivanova, N., Ghassemian, M., Richardson, T.H., Stege, J.T., Cayouette, M., Leadbetter, J.R., Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite (2007) Nature, 450, pp. 560-565Watanabe, H., Tokuda, G., Animal cellulases (2001) Cell Mol. Life Sci., 58, pp. 1167-1178Watanabe, H., Tokuda, G., Cellulolytic systems in insects (2010) Annu. Rev. Entomol., 55, pp. 609-632Wheeler, M.M., Tarver, M.R., Coy, M.R., Scharf, M.E., Characterization of four esterase genes and esterase activity from the gut of the termite Reticulitermes flavipes (2010) Arch. Insect Biochem. Physiol., 73, pp. 30-48Woodward, J., Lima, M., Lee, N.E., The role of cellulase concentration in determining the degree of synergism in the hydrolysis of microcrystalline cellulose (1988) Biochem. J., 255, pp. 895-899Wu, Y., Kondrashkina, E., Kayatekin, C., Matthews, C.R., Bilsel, O., Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein (2008) Proc. Natl. Acad. Sci. U S A, 105, pp. 13367-13372Zhang, D., Allen, A.B., Lax, A.R., Functional analyses of the digestive beta-glucosidase of Formosan subterranean termites (Coptotermes formosanus) (2012) J.Insect Physiol., 58, pp. 205-210Zhang, D., Lax, A.R., Bland, J.M., Allen, A.B., Characterization of a new endogenous endo-beta-1,4-glucanase of Formosan subterranean termite (Coptotermes formosanus) (2011) Insect Biochem. Mol. Biol., 41, pp. 211-218Zhang, D., Lax, A.R., Bland, J.M., Yu, J., Fedorova, N., Nierman, W.C., Hydrolysis of filter-paper cellulose to glucose by two recombinant endogenous glycosyl hydrolases of Coptotermes formosanus (2010) Insect Sci., 17, pp. 245-252Zhang, D., Lax, A.R., Raina, A.K., Bland, J.M., Differential cellulolytic activity of native-form and C-terminal tagged-form cellulase derived from Coptotermes formosanus and expressed in E. coli (2009) Insect Biochem. Mol. Biol., 39, pp. 516-522Zhou, X., Kovaleva, E.S., Wu-Scharf, D., Campbell, J.H., Buchman, G.W., Boucias, D.G., Scharf, M.E., Production and characterization of a recombinant beta-1,4-endoglucanase (glycohydrolase family 9) from the termite Reticulitermes flavipes (2010) Arch. Insect Biochem. Physiol., 74, pp. 147-162Zoete, V., Cuendet, M.A., Grosdidier, A., Michielin, O., SwissParam: a fast force field generation tool for small organic molecules (2011) J.Comput. Chem., 32, pp. 2359-236