Purification and characterization of embryo-specific soy urease (Glycine max) and its antifungal potential against Paracoccidioides brasiliensis
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Abstract
Ureases are amidohydrolases that catalyze the hydrolysis of urea to ammonia and carbamate. In addition to the enzymatic function, ureases have fungitoxic and insecticidal function, which are independent of their catalytic activity. Soy (Glycine max) has two main urease isoforms: ubiquitous and embryo-specific, the latter is present in beans. In view of the potential applications of ureases, this work aimed to extract, purify, characterize the structure, activity and fungitoxic activity of soy urease against Paracoccidioides brasiliensis. The biochemical characterization was performed, in terms of optimal pH and temperature, as well as the determination of the Michaelis–Menten constant (KM) and maximum velocity (Vmax). The protein sequence was identified by mass spectrometry and used in computational modeling of the biological structure. The optimum pH and temperature of the enzyme were 6.5 and 65 °C, respectively, KM 526 mmol L-1 and Vmax 7.4 mmol L-1 NH3×µgurease-1×s-1 and biological unity as a trimer. The antifungal activity assays (in vitro) were promising, showing a fungicidal profile of the urease, with a minimum inhibitory concentration of 10 µg×mL-1. This work demonstrated, for the first time, the fungitoxic activity of embryo-specific soy urease against the Pb18 strain of P. brasiliensis.
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References
Balasubramanian, A., Durairajpandian, V., Elumalai, S., Mathivanan, N., Munirajan, A. K., Ponnuraj, K., Structural and functional studies on urease from pigeon pea (Cajanus cajan), International Journal of Biological Macromolecules 58 (2013) 301-309. https://doi.org/10.1016/j.ijbiomac.2013.04.055.
Carlini, C. R., Ligabue-Braun, R., Ureases as multifunctional toxic proteins: A review, Toxicon 110 (2016) 90-109. https://doi.org/10.1016/j.toxicon.2015.11.020.
Kappaun, K., Piovesan, A. R., Carlini, C. R., Ligabue-Braun, R., Ureases: Historical aspects, catalytic, and non-catalytic properties – A review, Journal of Advanced Research 13 (2018) 3–17. https://doi.org/10.1016/j.jare.2018.05.010.
Fisher, K. A., Yarwood, S.A., James, B. R., Soil urease activity and bacterial ureC gene copy numbers: Effect of pH, Geoderma 285 (2017) 1–8. https://doi.org/10.1016/j.geoderma.2016.09.012.
Wessén, E., Nyberg, K., Jansson, J. K., Hallin, S., Responses of bacterial and archaeal ammonia oxidizers to soil organic and fertilizer amendments under long-term management, Applied Soil Ecology 45 (2010) 193–200. https://doi.org/10.1016/j.apsoil.2010.04.003.
Krajewska, B., Ureases I. Functional, catalytic and kinetic properties: A review, Journal of Molecular Catalysis B: Enzymatic 59 (2009) 9–21. https://doi.org/10.1016/j.molcatb.2009.01.003.
Wiebke-Strohm, B., Ligabue-Braun, R., Rechenmacher, C., De Oliveira-Busatto, L. A., Carlini, C. R., Bodanese-Zanettini, M. H., Structural and transcriptional characterization of a novel member of the soybean urease gene family, Plant Physiology and Biochemistry 101 (2016) 96–104. https://doi.org/10.1016/j.plaphy.2016.01.023.
Polacco, J. C., Mazzafera, P., Tezotto, T., Opinion – Nickel and urease in plants: Still many knowledge gaps, Plant Science 199–200 (2013) 79–90. https://doi.org/10.1016/j.plantsci.2012.10.010.
Follmer, C., Barcellos, G. B. S., Zingali, R. B., Machado, O. L. T., Alves, E. W., Barja-Fidalgo, C., Guimarães, J. A., Carlini, C. R., Canatoxin, a toxic protein from jack beans (Canavalia ensiformis), is a variant form of urease (EC 3.5.1.5): biological effects of urease independent of its ureolytic activity, Biochemical Journal 360 (2001) 217–224. https://doi.org/10.1042/0264-6021:3600217.
Postal, M., Martinelli, A. H. S., Becker-Ritt, A. B., Ligabue-Braun, R., Demartini, D. R., Ribeiro, S. F. F., Pasquali, G., Gomes, V. M., Carlini, C. R., Antifungal properties of Canavalia ensiformis urease and derived peptides, Peptides 38 (2012) 22–32. https://doi.org/10.1016/j.peptides.2012.08.010.
Becker-Ritt, A. B., Martinelli, A. H. S., Mitidieri, S., Feder, V., Wassermann, G.E., Santi, L., Vainstein, M. H., Oliveira, J. T. A., Fiuza, L. M., Pasquali, G., Carlini, C. R., Antifungal activity of plant and bacterial ureases, Toxicon 50 (2007) 971-983. https://doi.org/10.1016/j.toxicon.2007.07.008.
Martinelli, A. H. S., Kappaun, K., Ligabue-Braun, R., Defferrari, M. S., Piovesan, A. R., Stanisçuaski, F., Demartini, D. R., Dal Belo, C. A., Almeida, C. G. M., Follmer, C., Verli, H., Carlini, C. R., Pasquali, G., Structure–function studies on jaburetox, a recombinant insecticidal peptide derived from jack bean (Canavalia ensiformis) urease, Biochimica et Biophysica Acta (BBA) - General Subjects 1840 (2014) 935-944. https://doi.org/10.1016/j.bbagen.2013.11.010.
Martinelli, A. H. S., Lopes, F. C., Broll, V., Defferrari, M. S., Ligabue-Braun, R., Kappaun, K., Tichota, D. M., Fruttero, L. L., Moyetta, N. R., Demartini, D. R., Postal, M., Medeiros-Silva, M., Becker-Ritt, A. B., Pasquali, G., Carlini, C. R., Soybean ubiquitous urease with purification facilitator: An addition to the moonlighting studies toolbox, Process Biochemistry 53 (2017) 245-258. https://doi.org/10.1016/j.procbio.2016.12.003.
Wanke, B., Aidê, M.A., Capítulo 6 - Paracoccidioidomicose, Jornal Brasileiro de Pneumologia 35 (2009) 1245–1249. https://doi.org/10.1590/S1806-37132009001200013.
Ferreira, M. S., Paracoccidioidomycosis, Paediatric Respiratory Reviews 10 (2009) 161–165. https://doi.org/10.1016/j.prrv.2009.08.001.
Ricci, C.D., Evangelista, C., Tomaz, B.C.A., da Silva, M.V., Barbo, M.L.P., Paracoccidioidomicose: forma crônica cutânea, Revista da Faculade Ciências Médicas Sorocaba 20 (2018) 51. https://doi.org/10.23925/1984-4840.2018v20i1a12.
Queiroz-Telles, F., Fahal, A. H., Falci, D. R., Caceres, D. H., Chiller, T., Pasqualotto, A. C., Neglected endemic mycoses, The Lancet Infectious Disease 17 (2017) e367–e377. https://doi.org/10.1016/S1473-3099(17)30306-7.
Shikanai-Yasuda, M. A., Mendes, R. P., Colombo, A. L., Telles, F. de Q., Kono, A., Paniago, A. M. M., Nathan, A., do Valle, A. C. F., Bagagli, E., Benard, G., Ferreira, M.S ., Teixeira, M. de M., Vergara, M. L. S., Pereira, R. M., Cavalcante, R de S., Hahn, R., Durlacher, R.R., Khoury, Z., de Camargo, Z.P., Moretti, M. L., Martinez, R., II Consenso Brasileiro em Paracoccidioidomicose - 2017, Epidemiologia e Serviços Saúde. 27 (2018). https://doi.org/10.5123/S1679-49742018000500001.
Borba, J. V. V. B., Tauhata, S. B. F., de Oliveira, C. M. A., Marques, M. F., Bailão, A. M., Soares, C. M. de A., Pereira, M., Chemoproteomic identification of molecular targets of antifungal prototypes, thiosemicarbazide and a camphene derivative of thiosemicarbazide, in Paracoccidioides brasiliensis, PLoS One. 13 (2018) e0201948. https://doi.org/10.1371/journal.pone.0201948.
Nicola, A. M., Albuquerque, P., Paes, H. C., Fernandes, L., Costa, F. F., Kioshima, E. S., Abadio, A. K. R., Bocca, A. L., Felipe, M. S., Antifungal drugs: New insights in research & development, Pharmacology & Therapeutics 195 (2019) 21–38. https://doi.org/10.1016/j.pharmthera.2018.10.008.
Bracco, L. F., Levin, G. J., Urtasun, N., del Cañizo, A. A. N., Wolman, F. J., Miranda, M. V., Cascone, O., Covalent immobilization of soybean seed hull urease on chitosan mini-spheres and the impact on their properties, Biocatalysis and Agricultural Biotechnology 18 (2019) 101093. https://doi.org/10.1016/j.bcab.2019.101093.
Laemmli, U. K., Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4, Nature 227 (1970) 680–685. https://doi.org/10.1038/227680a0.
Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Analytical Biochemistry 72 (1976) 248–254. https://doi.org/10.1006/abio.1976.9999.
Bzura, J., Koncki, R., A mechanized urease activity assay, Enzyme and Microbial Technology 123 (2019) 1–7. https://doi.org/10.1016/j.enzmictec.2019.01.001.
El-Hefnawy M. E., Sakran, M., Ismail, A. I., Aboelfetoh, E., Extraction, purification, kinetic and thermodynamic properties of urease from germinating Pisum Sativum L. seeds, BMC Biochemistry 15 (2014) 15. https://doi.org/10.1186/1471-2091-15-15.
Shevchenko, A., Jensen, O. N., Podtelejnikov, A. V., Sagliocco, F., Wilm, M., Vorm, O., Mortensen, P., Shevchenko, A., Boucherie, H., Mann, M., Linking genome and proteome by mass spectrometry: Large-scale identification of yeast proteins from two dimensional gels, Proceedings of the National Academy of Sciences of the United States of America 93 (1996) 14440–14445. https://doi.org/10.1073/pnas.93.25.14440.
Shevchenko, A., Wilm, M., Vorm, O., Mann, M., Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels, Analytical Chemistry 68 (1996) 850–858. https://doi.org/10.1021/ac950914h.
Balasubramanian, A., Ponnuraj, K., Crystal Structure of the First Plant Urease from Jack Bean: 83 Years of Journey from Its First Crystal to Molecular Structure, Journal of Molecular Biology 400 (2010) 274–283. https://doi.org/10.1016/j.jmb.2010.05.009.
Webb, B., Sali, A., Comparative Protein Structure Modeling Using MODELLER, Current Protocols in Bioinformatics 54 (2016). https://doi.org/10.1002/cpbi.3.
Collaborative Computational Project Number 4, The CCP4 suite: programs for protein crystallography, Acta Crystallographica Section D Structural Biology 50 (1994) 760–763. https://doi.org/10.1107/S0907444994003112.
Britton, H. T. S., Robinson, R. A., CXCVIII—Universal buffer solutions and the dissociation constant of veronal, Journal of the Chemical Society (1931) 1456–1462. https://doi.org/10.1039/JR9310001456.
CLSI, Método de Referência para Testes de Diluição em Caldo para a Determinação da Sensibilidade a Terapia Antifúngica das Leveduras, 22 (2002) 1–50.
Wayne, P. A., CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-second Informational supplement. CLSI document M100-S22. Clinical and Laboratory Standards Institute, 2015.
Rodrigues-Vendramini, F. A. V., Faria, D. R., Arita, G. S., Capoci, I. R. G., Sakita, K. M., Caparroz-Assef, S. M., Becker, T. C. A., Bonfim-Mendonça, P. S., Felipe, M. S., Svidzinski, T. I. E., Maigret, B., Kioshima, É. S., Antifungal activity of two oxadiazole compounds for the paracoccidioidomycosis treatment, PLoS Neglected Tropical Diseases 13 (2019) e0007441. https://doi.org/10.1371/journal.pntd.0007441.
Rodrigues-Vendramini, F. A. V., Marschalk, C., Toplak, M., Macheroux, P., Bonfim-Mendonça, P. S., Svidzinski, P. T. I. E., Seixas, F. A. V., Kioshima, E. S., Promising New Antifungal Treatment Targeting Chorismate Synthase from Paracoccidioides brasiliensis, Antimicrobial Agents and Chemotherapy 63 (2018) e01097-18. https://doi.org/10.1128/AAC.01097-18.
Polacco, J. C., Havir, E. A., Comparisons of soybean urease isolated from seed and tissue culture., Journal of Biological Chemistry, 254 (1979) 1707-1715.
Polacco, J. C., Thomas, A. L., Bledsoe, P. J., A Soybean Seed Urease-Null Produces Urease in Cell Culture, Plant Physiology 69 (1982) 1233-1240. https://doi.org/10.1104/pp.69.5.1233.
Torisky, R. S., Polacco, J. C., Soybean Roots Retain the Seed Urease Isozyme Synthesized during Embryo Development, Plant Physiology 94 (1990) 681-689. https://doi.org/10.1104/pp.94.2.681.
Polacco, J. C., Winkler, R. G., Soybean Leaf Urease: A Seed Enzyme?, Plant Physiology 74 (1984) 800-803. https://doi.org/10.1104/pp.74.4.800.
Yoo, Y. J., Feng, Y., Kim, Y.-H., Yagonia, C. F. J., Fundamentals of Enzyme Engineering, New York, Springer, 2017. https://doi.org/10.1007/978-94-024-1026-6.
Desjardins, C. A., Champion, M. D., Holder, J. W., Muszewska, A., Goldberg, J., Bailão, A. M., Brigido M. M., Ferreira, M. E. S., Garcia, A. M., Grynberg, M., Gujja, S., Heiman, D. I., Henn, M. R., Kodira, C. D., León-Narváez, H., Longo, L. V. G., Ma, L.-J., Malavazi, I., Matsuo, A. L., Morais, F. V., Pereira, M., Rodriguéz-Brito, S., Sakthikumar, S., Salem-Izacc, S. M., Sykes, S. M., Teixeira, M. M., Vallejo, M. C., Walter, M. E. M. T., Yandava, C., Young, S., Zeng, Q., Zucker, J., Felipe, M. S., Goldman, G. H., Haas, B. J., McEwan, J. G., Nino-Vega, G., Puccia, R., San-Blas, G., Soares, C. M. A., Birres, B. W., Cuomo, C. A., Comparative Genomic Analysis of Human Fungal Pathogens Causing Paracoccidioidomycosis, PLoS Genetics 7 (2011) e1002345. https://doi.org/10.1371/journal.pgen.1002345.