Glutathione transferases, also known as Glutathione S-transferases (GSTs), are a diverse group of enzymes that catalyse the conjugation of the tri-peptide glutathione to a wide range of electrophilic substrates. Their biological function in endogenous metabolism in plants is not well characterised, although their role in herbicide metabolism and herbicide selectivity is well documented. Many herbicides used in soybean. Glycine max (L.) Merr., are selective against weeds due to their rapid detoxification in the crop through conjugation with homoglutathione (γ-glu-cys-β-ala), the predominant free thiol in many legumes. However, an in depth characterisation of the GSTs which can potentially catalyse these reactions in soybean has never been performed. This work describes the biochemical and molecular characterisation of GSTs in soybean with emphasis on the identification of specific isoenzymes involved in herbicide metabolism. GST activity toward the chloroacetanilide herbicides acetochlor and metolachlor, the diphenyl ethers acifluorfen and fomesafen and the sulphonyl urea chlorimuron-ethyl were all detected in crude protein extracts from five-day-old suspension cultured soybean cells. GST activity was also determined in five-day-old soybean seedlings, though this activity was significantly lower than that observed with the cell suspension cultures. Treatment of soybean plants with herbicides and herbicide safeners resulted in increased GST activity toward the model substrate l-chloro-2,4-dinitrobenzene (CDNB), but no change in activity toward herbicide substrates. In both plant and cell cultures GST-catalysed conjugation of the diphenyl ethers acifluorfen and fomesafen was over five-fold greater in the presence of homoglutathione as compared with glutathione. The preferential detoxification of these herbicides in the presence of homoglutathione appeared to be an important determinant of their rapid detoxification in soybean and an important factor in herbicide selectivity. GSTs were purified from five-day-old soybean cell cultures using S-hexylglutathione affinity chromatography and anion-exchange chromatography. A combination of reversed-phase HPLC, SDS-PAGE and MALDI-TOF mass spectrometry of the purified fractions indicated the presence of nine putative GST subunits, each with a molecular mass between 25 and 29 kDa. Soybean GST cDNA clones were obtained using a combination of RT-PCR, utilising degenerate oligonucleotides designed to conserved regions within plant GSTs, and screening of cDNA libraries prepared from soybean plants and cell cultures. This process failed to identify any theta-type GSTs, the class associated with herbicide detoxification in maize. In contrast, seven distinct tau-type GSTs were isolated together with a number of clones showing minor variations in individual sequences. Expression of these cDNAs in Escherichia coli showed the purified recombinant GSTs were active toward a diverse range of substrates, and possessed additional glutathione peroxidase activity. GST activities for each recombinant enzyme varied with substrate and thiol type, with a marked preference for homoglutathione with selected substrates. From the work reported in this study it would appear that the tau-type GSTs of soybean are at least as complex as those previously reported in cereals and have an important role in determining herbicide metabolism and selectivity in this major crop.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-09-01T13:05:01Z No. of bitstreams: 2 Dissertação - Wanderson Lucas da Costa - 2017.pdf: 3043089 bytes, checksum: 32c1b9c81dae778ab37d939fdba41eb5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-09-15T13:46:23Z (GMT) No. of bitstreams: 2 Dissertação - Wanderson Lucas da Costa - 2017.pdf: 3043089 bytes, checksum: 32c1b9c81dae778ab37d939fdba41eb5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-09-15T13:46:23Z (GMT). No. of bitstreams: 2 Dissertação - Wanderson Lucas da Costa - 2017.pdf: 3043089 bytes, checksum: 32c1b9c81dae778ab37d939fdba41eb5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Paracoccidioides spp. are thermodymorphic fungi that when inhaled by humans, these conidia find a favorable environment, changing to the yeast phase and becoming pathogenic causing paracoccidioidomycosis (PCM), one of the most prevalent systemic mycoses in Brazil. Some antifungals are used in the treatment of PCM. Treatment depends on the patient's progression and tolerability of each drug, but their treatment may be for long periods and cause various side effects in the patient. The chiquimate pathway is coordinated by 7 enzymes that perform consecutive steps to convert erythrose-4-phosphate and phosphoenol pyruvate (PEP) into chorismate. In microorganisms, this pathway is involved in the production of the amino acids phenylalanine, tyrosine and tryptophan; These amino acids are essential to the maintenance of these organisms. In this work, pGEX4T3 vector cloning and heterologous expression of Pb18 EPSP synthase belonging to the chiquimate pathway were performed. This protein was expressed in E. coli (DE3) strain and purified. Antibodies were produced for expression analysis of the protein in Western blot. The modeling of EPSP synthase was performed aiming to identify the amino acids involved in the active site. The pull down-GST assay with soluble Pb18 proteins allowed the identification of 40 proteins that interact with EPSP synthase. These proteins belong to different functional categories, which are involved with the availability of phosphoenol pyruvate, the substrate necessary for the functioning of the chiquimate pathway. / Paracoccidioides spp. são fungos termodimórficos que ao serem inalados pelo ser humano, esses conídios encontram um ambiente propício, mudando para a fase de levedura e tornando-se patogênico causando a paracoccidioidomicose (PCM), umas das micoses sistêmicas de maior prevalência no Brasil. Alguns antifúngicos são empregados no tratamento da PCM. O tratamento depende do avanço da doença e da capacidade de tolerância do paciente a cada medicamento, mas o seu tratamento pode ser por longos períodos e causando diversos efeitos colaterais no paciente. A via do chiquimato é coordenada pela ação de 7 enzimas que realizam passos consecutivos para transformar a eritrose-4-fosfato e fosfoenol piruvato (PEP) em corismato. Em micro-organismos, esta via está envolvida com a produção dos aminoácidos fenilalanina, tirosina e triptofano; estes aminoácidos são essenciais à manutenção desses organismos. Neste trabalho foi realizado a clonagem em vetor pGEX4T3 e expressão heteróloga da EPSP–sintase de Pb18 pertencente à via do chiquimato. Essa proteína foi expressa em linhagem E. coli (DE3) e purificada. Os anticorpos foram produzidos para análise da expressão da proteína em Western blot. A modelagem da EPSP-sintase foi realizada visando identificar os aminoácidos envolvidos no sítio ativo. O ensaio de pull down-GST com proteínas solúveis de Pb18 possibilitou a identificação de 40 proteínas que interagem com EPSP-sintase. Essas proteínas pertencem a diferentes categorias funcionais, as quais estão envolvidas com a disponibilidade de fosfoenol piruvato, substrato necessário para o funcionamento da via do chiquimato.