O-acetilserina(tiol) liase: estudos in silico, in vitro e in vivo
| Ano de defesa: | 2021 |
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| Autor(a) principal: | |
| Orientador(a): | |
| Banca de defesa: | |
| Tipo de documento: | Tese |
| Tipo de acesso: | Acesso aberto |
| Idioma: | por |
| Instituição de defesa: |
Universidade Estadual de Maringá
Dois Vizinhos Brasil Programa de Pós-Graduação em Ciências Biológicas UEM |
| Programa de Pós-Graduação: |
Não Informado pela instituição
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| Departamento: |
Não Informado pela instituição
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| País: |
Não Informado pela instituição
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| Palavras-chave em Português: | |
| Link de acesso: | http://repositorio.utfpr.edu.br/jspui/handle/1/32139 |
Resumo: | Sulfur is a vital macronutrient for plants and animals and can be found in nature in many different oxidation states. Animals and humans can only incorporate organic forms of sulfur presents in amino acids such as L-methionine and L-cysteine. Plants, in the other hand, can uptake and reduce inorganic forms of sulfur and assimilate it into several important compounds for their survival, such as biotin, coenzyme A, thiamine pyrophosphate, lipoic acid, iron-sulfur centers and glucosinolates. Plants can incorporate sulfate through a reaction catalyzed by the enzyme O-acetylserine(thiol) lyase (OAS-TL) (E.C. 2.5.1.47). The product of this reaction, L-cysteine is an amino acid singularly important for the maintenance of three-dimensional structures of proteins and precursor of several sulfated organic compounds. The OAS-TL is a potential metabolic target for the development of new herbicides, due to the importance of its activity for the survival of plants. Therefore, the objective of this work was to model the structure of the OAS-TL of maize (Zea mays L.), complexed with the O-acetylserine substrate (OAS) and the 5'-phosphate pyridoxal cofactor, to be used as a target in studies of virtual screening. S-benzyl-L-cysteine (SBC), one of the selected inhibitors, had its herbicidal potential evaluated on maize and morning-glory (Ipomoea grandifolia L.). For this, the amino acid sequence of maize OAS-TL was obtained from Uniprot (P80608) and used to search for templates using BlastP against a Protein Data bank database. Subsequently, the library for virtual screening was built from the search for compounds structurally similar to OAS in the database ZINC 12 and 15. The programs used in docking simulations were AutoDock 4.2.3 and Vina, implemented in the graphical interface of PyRx 0.9. Different ligand orientations were researched and classified based on their energy scores and optimum positioning of the ligand with the enzyme. The protocols were considered validated when the position of the exogenous OAS substrate overlapped the position of the OAS substrate modeled with the enzyme, which provided a root mean square deviation (RMSD) less than 2.0 Å. After validation using the Lipinski rules and toxicity (ADMETox), maize and morning-glory seedlings were grown in nutritive solution (pH 6.0) with and without S-benzyl-L-cysteine (SBC) (125-1000 ?M) for incubation periods between 24 to 96 h and the effects on growth parameters were estimated. Enzymatic kinetic experiments were carried out to verify the inhibitory activity of SBC on the OAS-TL of Arabidopsis thaliana (AtOAS-TL) in vitro. The impacts of SBC on the production of L-cysteine in vivo were determined by quantifying the levels of L-cysteine in the roots and by growing experiments in a medium supplemented with L-cysteine. In addition, experiments with maize plants in more mature phenological tilling stages were carried out to assess the impacts of SBC on the growth, photosynthesis, content of reactive oxygen species (ROS) and proteome of these plants. Maize plants were grown in pots containing substrate and vermiculite supplemented with 1 and 5 mM SBC for 14 days. Biometric parameters were determined at the end of the cultivation period. The effects on photosynthesis were monitored by performing, on the 14th day of cultivation, curves of response to light and CO2 (A/ci), as well as by determining the chlorophyll a fluorescence. To investigate the responses of maize plants exposed to SBC at the proteome level, we performed a comparative quantitative proteomic analysis of leaves. The modeling and virtual screening experiments made it possible to identify two candidate compounds for inhibitors of the maize OAS-TL. The SBC showed the best energy score (-7.0 kcal mol-1) than the substrate OAS (- 6.6 kcal mol-1), indicating its better interaction with the active site of OAS-TL. In addition, SBC significantly reduced the growth of maize and morning-gloryseedlings, besides to causing a significant reduction in L-cysteine content in vivo. Enzymatic kinetic experiments revealed that SBC is a moderate inhibitor of AtOAS-TL in vitro. SBC also significantly reduced the growth of 14-days-oldmaize plants, the rate of CO2 assimilation (due to stomatal and non-stomatal limitations) and the expression of important components of the photosynthetic electron transport chain (photosystems I and II, cytochrome b6f and ATP synthase) and Calvin cycle enzymes. Photosynthetic electron transport was inhibited, with a consequent increase in ROS content of leaves. In conclusion, our data show that SBC can inhibit OAS-TL activity in vitro and in vivo. The inhibitory effect of SBC on the growth of maize and morning-glory seedlings seem to be related primarily to the reduction of L-cysteine synthesis. The negative impacts on the photosynthesis of maize plants were shown to be associated with reduced expression of components of photosystems I and II, cytochrome b6f and ATP synthase. Such results suggest that the sulphate assimilation pathway was affected by exposure to SBC, thus interfering in the synthesis of sulfur-countaining compounds essential for the development and survival of plants. Future studies that aim to improve the inhibitory activity of SBC on OAS-TL through chemical modifications may be important for the development of a new potential herbicide. |