Caracterización preliminar de la enzima Dihidroorotato Deshidrogenasa de la especie Solanum Tuberosum como estrategia para el control del patógeno Phytophthora Infestans
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The potato-solanum tuberosum-is a species belonging to the family of solanaceous that is cultivated in more than 100 countries, which makes it one of the most important crops worldwide. According to the food and Agriculture Organization of the United Nations (FAO), the potato is located in the fourth place of crops most importantly as a foodstuff after rice, wheat and corn. Potato crops, as well as other crops, are subject to attacks by microorganisms which are causing serious diseases that cause economic and production losses. The Oomycete Phytophthora infestans (P. Infestans), is the microorganism that causes late blight, which is one of the diseases of greater prevalence and severity in solanaceous crops, within which are plants of agricultural and economic importance like the potato and the tomato. By the summer of 1845, late blight disease attacked potato crops, spreading throughout Europe, with Ireland being one of the hardest hit countries. The disease spread uncontrollably in the following years, generating the well-known "Irish Famine" that resulted in the deaths of one and a half million Irish peasants and the exodus of a large part of this population to countries like the United States and Canada. Recently, studies with parasites of phylum apicomplexa have shown that the metabolic pathway of pyrimidines, specifically the synthesis of novo, may become a potential target for the inhibition of different pathogens, because the nucleotides of pyrimidines are involved in several critical cellular processes and perform structural, metabolic and regulatory functions. Currently some enzymes of this route of synthesis are important in the study of the inhibition of these pathogens, as is the case of toxoplasma gondii, causing toxoplasmosis, which has been evidence that parasites in the absence of the enzyme CPSII (An enzyme that catalyzes the first reaction of the route) loses its virulence; hence the importance of this research in the inhibition of pathogens as P. Infestans in hosts as S. Tuberosum. Therefore, studies highlight the importance of the enzyme DHOD because it catalyzes the only redox stage of the pathway, so it would be of great importance the kinetic and functional characterization of the enzymes of both the pathogen (PiDHOD) and the host (StDHOD). Thanks to previous work, carried out by the research group BBMP (Biochemistry and Molecular Biology of parasites) of the University of the Andes, already has the kinetic characterization of the PiDHOD, however, it has not been characterized StDHOD because the complete recombinant protein is highly hydrophobic and therefore insoluble after being induced under osmotic stress. That is why, this research aimed to characterize in preliminary form the enzyme DHOD of the species S. tuberosum, by cloning and expression of three truncated versions of this in the vector of expression pET-19b, in order to obtain a version and preserving its biological activity for its subsequent enzymatic characterization. For which it was carried out through a bioinformatic analysis the determination of the region N '-terminal to be suppressed, from this were carried out three independent truncations of the protein, Delecionando the first 69.73 and 76 amino acids called AST, DEA and TFC respectively; Which were cloned in the vector PET-19b, expressed in cells of BL21- odonPlus (DE3) RP of E. coli, then were purified by affinity chromatography using cobalt resins. Finally, the respective kinetic characterization was performed, by means of the Spectrophotometer DU 800 (Coulter Beckmann) Evaluating the reduction reaction of the 2.6 diclorofenolindofenol (DCIP), of blue color, in a reaction coupled to the dihidroorotato substrate (L-DHO) through the oxidation of the substrate of quinone (QD) that changes to colorless, which allowed to obtain the saturation curves of each of the substrates of the StDHOD (L- HO and QD) and to calculate by means of the Michaelis-Menten equation The most relevant kinetic constants for the two versions that were more likely to preserve enzymatic activity (AST and DEA): Km and Vmax (Recombinant enzyme AST KmL-DHO: 23.65 µ m, KmQd: 22.85 µ m, and VmaxL-DHO: 21.40 Μmol/mg/min VmaxQd: 20.60 Μmol/mg/ Min, whereas for the recombinant enzyme DEA, only the kinetic variables were established for the substrate L-DHO: KmL-DHO: 22.59 Μ m and VmaxL-DHO: 17.67 Μmol/mg/min and the concentration of Qd remained constant, which allowed to show that for both versions of the truncated enzymes, the Km are very close. The difference in the kinetic constants between the enzyme StDHOD and PiDHOD (KmL-DHO: 16.9 Μ m, KmQd: 6 Μ m and Vmax: 5.8 Μmol/mg/min), could be explored in the search for compounds with inhibitory capacity that had a great effect on the enzyme of the pathogen without affect the activity of the plant enzyme. In conclusion, this study opens the way for research related to the control of the pathogen P. Infestans in potato crops, strengthening the enzyme DHOD as a promising target for the inhibition of this oomycete. It is expected that this study will facilitate future research in the field of pharmacology, as well as consolidate the path of synthesis de novo de pyrimidines as a target for the control of various pathogens, such as P. Infestans, have become A harmful organism for crops of great nutritional importance for humanity.