Identification and structural analysis of a thermophilic β-1,3-glucanase from compost

Abstract

β-1,3-glucanase can specifically hydrolyze glucans to oligosaccharides and has potential applications in biotechnology. We used the metatranscriptomic technology to discover a thermophilic β-1,3-glucanase from compost. The phylogenetic study shows that it belongs to the family 16 glycoside hydrolase (GH16) and is most homologous with an enzyme from Streptomyces sioyaensis, an actinobacterium. It has the activity of 146.9 U/mg in the optimal reaction condition (75 °C and pH 5.5). Its catalytic domain was crystallized and diffracted to 1.14 Å resolution. The crystal structure shows a sandwich-like β-jelly-roll fold with two disulfide bonds. After analyzing the occurring frequencies of these cysteine residues, we designed two mutants (C160G and C180I) to study the role of these disulfide bonds. Both mutants have decreased their optimal temperature from 75 to 70 °C, which indicate that the disulfide bonds are important to maintain thermostability. Interestingly, the activity of C160G has increased ~ 17% to reach 171.4 U/mg. We speculate that the increased activity of C160G mutant is due to increased dynamics near the active site. Our studies give a good example of balancing the rigidity and flexibility for enzyme activity, which is helpful for protein engineering.

Keywords

β-1,3-glucanase / Crystal structure / Disulfide bond / Mutagenesis / Molecular dynamics

References

[1] Artimo P, Jonnalagedda M, Arnold K, Baratin D, Csardi G, De Castro E, Duvaud S, Flegel V, Fortier A, Gasteiger E, Grosdidier A, Hernandez C, Ioannidis V, Kuznetsov D, Liechti R, Moretti S, Mostaguir K, Redaschi N, Rossier G, Xenarios I, Stockinger H. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res, 2012, 40: W597-W603.

[2] Ashida H, Maskos K, Li SC, Li YT. Characterization of a novel endo-β-galactosidase specific for releasing the disaccharide GlcNAcα1→4Gal from glycoconjugates. Biochemistry, 2002, 41: 2388-2395.

[3] Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr Sect D Biol Crystallogr, 2010, 66: 12-21.

[4] Cheng R, Chen J, Yu X, Wang Y, Wang S, Zhang J. Recombinant production and characterization of full-length and truncated β-1,3-glucanase PglA from Paenibacillus sp. S09. BMC Biotechnol, 2013, 13: 105.

[5] Dehnavi E, Fathi-Roudsari M, Mirzaie S, Arab SS, Ranaei Siadat SO, Khajeh K. Engineering disulfide bonds in Selenomonas ruminantium β-xylosidase by experimental and computational methods. Int J Biol Macromol, 2017, 95: 248-255.

[6] DiMaio F, Echols N, Headd JJ, Terwilliger TC, Adams PD, Baker D. Improved low-resolution crystallographic refinement with Phenix and Rosetta. Nat Methods, 2013, 10: 1102-1104.

[7] Dong W, Huang J, Li Y, Tan Y, Shen Z, Song Y, Dang W, Xiao S, Chen H, Peng G. Crystal structural basis for Rv0315, an immunostimulatory antigen and inactive beta-1,3-glucanase of Mycobacterium tuberculosis. Sci Rep, 2015, 5: 15073.

[8] Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr Sect D Biol Crystallogr, 2010, 66: 486-501.

[9] Fibriansah G, Masuda S, Koizumi N, Nakamura S, Kumasaka T. The 1.3 Å crystal structure of a novel endo-β-1,3-glucanase of glycoside hydrolase family 16 from alkaliphilic Nocardiopsis sp. strain F96. Proteins, 2007, 69: 683-690.

[10] Fields PA, Somero GN. Hot spots in cold adaptation: localized increases in conformational flexibility in lactate dehydrogenase A(4) orthologs of Antarctic notothenioid fishes. Proc Natl Acad Sci USA, 1998, 95: 11476-11481.

[11] Fields PA, Dong Y, Meng X, Somero GN. Adaptations of protein structure and function to temperature: there is more than one way to 'skin a cat'. J Exp Biol, 2015, 218: 1801-1811.

[12] Gouet P, Courcelle E, Stuart D, Metoz F. ESPript: multiple sequence alignments in PostScript. Bioinformatics, 1999, 15: 305-308.

[13] Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Lindblad-Toh K, Friedman N, Regev A. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol, 2011, 297: 644-652.

[14] Gurtler JB, Doyle MP, Erickson MC, Jiang X, Millner P, Sharma M. Composting to inactivate foodborne pathogens for crop soil application: a review. J Food Prot, 2018, 81: 1821-1837.

[15] Hahn M, Olsen O, Politz O, Borriss R, Heinemann U. Crystal structure and site-directed mutagenesis of Bacillus macerans endo-1,3–1,4-glucanase. J Biol Chem, 1995, 270: 3081-3088.

[16] Hong TY, Huang JW, Meng M, Cheng CW. Isolation and biochemical characterization of an endo-1,3-β-glucanase from Streptomyces sioyaensis containing a C-terminal family 6 carbohydrate-binding module that binds to 1,3-β-glucan. Microbiology, 2002, 148: 1151-1159.

[17] Hong TY, Hsiao YY, Meng M, Li TT. The 1.5 Å structure of endo-1,3-β-glucanase from Streptomyces sioyaensis: evolution of the active-site structure for 1,3-β-glucan-binding specificity and hydrolysis. Acta Crystallogr Sect D Biol Crystallogr, 2008, 64: 964-970.

[18] Ismail WM, Ye Y, Tang H. Gene finding in metatranscriptomic sequences. BMC Bioinformatics, 2014, 15: 1-8.

[19] Jaenicke R, Schurig H, Beaucamp N, Ostendorp R. Structure and stability of hyperstable proteins: glycolytic enzymes from hyperthermophilic bacterium Thermotoga maritima. Adv Protein Chem, 1996, 48: 181-269.

[20] Jeng WY, Wang NC, Lin CT, Shyur LF, Wang AH. Crystal structures of the laminarinase catalytic domain from Thermotoga maritima MSB8 in complex with inhibitors: essential residues for β-1,3- and β-1,4-glucan selection. J BIOL CHEM, 2011, 286: 45030-45040.

[21] Jin X, Liao Q, Wei H, Zhang J, Liu B. SMI-BLAST: a novel supervised search framework based on PSI-BLAST for protein remote homology detection. Bioinformatics, 2020, 37: 913-920.

[22] Kobayashi T, Uchimura K, Kubota T, Nunoura T, Deguchi S. Biochemical and genetic characterization of beta-1,3 glucanase from a deep subseafloor Laceyella putida. Appl Microbiol Biotechnol, 2016, 100: 203-214.

[23] Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol, 2016, 33: 1870-1874.

[24] Labourel A, Jam M, Jeudy A, Hehemann JH, Czjzek M, Michel G. The β-glucanase ZgLamA from Zobellia galactanivorans evolved a bent active site adapted for efficient degradation of algal laminarin. J Biol Chem, 2014, 289: 2027-2042.

[25] Laman Trip DS, Youk H. Yeasts collectively extend the limits of habitable temperatures by secreting glutathione. Nat Microbiol, 2020, 5: 943-954.

[26] Laskowski RA, Swindells MB. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model, 2011, 51: 2778-2786.

[27] Masuda S, Endo K, Koizumi N, Hayami T, Fukazawa T, Yatsunami R, Fukui T, Nakamura S. Molecular identification of a novel beta-1,3-glucanase from alkaliphilic Nocardiopsis sp. strain F96. Extremophiles, 2006, 10: 251-255.

[28] McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. J Appl Crystallogr, 2007, 40: 658-674.

[29] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem, 1959, 31: 426-428.

[30] Minor W, Cymborowski M, Otwinowski Z, Chruszcz M. HKL-3000: the integration of data reduction and structure solution–from diffraction images to an initial model in minutes. Acta Crystallogr Sect D Biol Crystallogr, 2006, 62: 859-866.

[31] Murshudov GN, Vagin AA, Dodson EJ. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr Sect D Biol Crystallogr, 1997, 53: 240-255.

[32] Niu C, Zhu L, Xu X, Li Q. Rational design of thermostability in bacterial 1,3–1,4-beta-glucanases through spatial compartmentalization of mutational hotspots. Appl Microbiol Biotechnol, 2017, 101: 1085-1097.

[33] Oda T, Lim K, Tomii K. Simple adjustment of the sequence weight algorithm remarkably enhances PSI-BLAST performance. BMC Bioinf, 2017, 18: 288.

[34] Reyes-Torres M, Oviedo-Ocana ER, Dominguez I, Komilis D, Sanchez A. A systematic review on the composting of green waste: Feedstock quality and optimization strategies. Waste Manage, 2018, 77: 486-499.

[35] Saavedra HG, Wrabl JO, Anderson JA, Li J, Hilser VJ. Dynamic allostery can drive cold adaptation in enzymes. Nature, 2018, 558: 324-328.

[36] Seeliger D, de Groot BL. Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J Comput Aided Mol Des, 2010, 24: 417-422.

[37] Shoseyov O, Shani Z, Levy I. Carbohydrate binding modules: biochemical properties and novel applications. Microbiol Mol Biol R, 2006, 70: 283-295.

[38] Singh N, Mathur AS, Gupta RP, Barrow CJ, Tuli DK, Puri M. Enzyme systems of thermophilic anaerobic bacteria for lignocellulosic biomass conversion. Int J Biol Macromol, 2021, 168: 572-590.

[39] Stahmann KP, Schimz KL, Sahm H. Purification and characterization of four extracellular 1,3-β-glucanases of Botrytis cinerea. J Gen Microbiol, 1993, 139: 2833-2840.

[40] Törnkvist M, Larsson G, Enfors SO. Protein release and foaming in Escherichia coli cultures grown in minimal medium. Bioprocess Eng, 1996, 15: 231-237.

[41] Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem, 2010, 31: 455-461.

[42] Vagin A, Teplyakov A. MOLREP: an automated program for molecular replacement. J Appl Crystallogr, 1997, 30: 1022-1025.

[43] Vuong TV, Wilson DB. Glycoside hydrolases: catalytic base/nucleophile diversity. Biotechnol Bioeng, 2010, 107: 195-205.

[44] Wang X, Kong Z, Wang Y, Wang M, Liu D, Shen Q. Insights into the functionality of fungal community during the large scale aerobic co-composting process of swine manure and rice straw. J Environ Manage, 2020, 270: 110958.

[45] Wang X, Wang M, Zhang J, Kong Z, Wang X, Liu D, Shen Q. Contributions of the biochemical factors and bacterial community to the humification process of in situ large-scale aerobic composting. Bioresour Technol, 2021, 323: 124599.

[46] Webb B, Sali A. Comparative protein structure modeling using MODELLER. Curr Protoc Bioinf, 2016, 54: 5.6.1-5.6.37.

[47] Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AG, McCoy A. Overview of the CCP4 suite and current developments. Acta Crystallogr. Sect d: Biol Crystallogr, 2011, 67: 235-242.

[48] Yennamalli RM, Rader AJ, Wolt JD, Sen TZ. Thermostability in endoglucanases is fold-specific. BMC Struct Biol, 2011, 11: 10.

[49] Zarkar N, Khalili MAN, Khodadadi S, Zeinoddini M, Ahmadpour F. Expression and purification of soluble and functional fusion protein DAB₃₈₉IL-2 into the E.coli strain Rosetta-gami (DE3). Biotechnol Appl Biochem, 2019, 67: 206-212.

[50] Zhong C, Edlund A, Yang Y, McLean JS, Yooseph S. Metagenome and Metatranscriptome Analyses Using Protein Family Profiles. PLoS Comput Biol, 2016, 12: e1004991.

[51] Zhu F, Du B, Bian Z, Xu B. β-glucans from edible and medicinal mushrooms: characteristics, physicochemical and biological activities. J Food Compos Anal, 2015, 41: 165-173.

[52] Zhu N, Jin H, Kong X, Zhu Y, Ye X, Xi Y, Du J, Li B, Lou M, Shah GM. Improving the fermentable sugar yields of wheat straw by high-temperature pre-hydrolysis with thermophilic enzymes of Malbranchea cinnamomea. Microb Cell Fact, 2020, 19: 149.

Funding

National Natural Science Foundation of China(No. 31670790)

Fundamental Research Funds for the Central Universities(No. KYXK202009)