Análisis del rol de los residuos E190, Q339 y N340 en la interacción con Mn2+ en la Agmatinase Like Protein (ALP).
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Date
2025
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Publisher
Universidad de Concepción
Abstract
La agmatina es una amina primaria presente en diversos organismos, es metabolizada por las enzimas agmatinasa y/o agmatinase like protein (ALP) generando urea y putrescina. En mamíferos, la agmatina, regula diversos procesos fisiológicos, además de tener un potencial uso para el tratamiento de enfermades a la salud mental. La enzima ALP fue descubierta en tejido cerebral de ratas, esta enzima conserva las características propias de la familia de las metaloureohidrolasas, especialmente la dependencia del cofactor metálico Mn2+ para su actividad catalítica. ALP presenta una región proteica en el extremo carboxilo denominada dominio-LIM, que tiene un efecto auto inhibitorio de la actividad agmatinasa de ALP, la variante trunca en este dominio (ΔLIM-ALP) es mucho más activa y estable, y con esta isoforma se realizaron los estudios de esta tesis. En cuanto a su secuencia aminoacídica, ALP resultó ser diferente de las demás agmatinasas y no presenta los residuos que coordinan con el cofactor Mn2+ altamente conservados en las metalo-ureohidrolasas. Dada la ausencia de información estructural, se construyó un modelo molecular de ALP utilizando como referencia agmatinasas procariotas cuya estructura ha sido resuelta. Pese a la baja identidad de la secuencia, este modelo permitió proponer residuos putativos involucrados en la coordinación de los iones Mn²⁺ en ALP. En estudios anteriores utilizando la isoforma ΔLIM-ALP, se generó una mutante doble (E288A/K290A) y una mutante triple (N213A/Q215A/D217A), donde se reemplazaron los residuos por alanina. Se encontró que estas variantes no presentaron actividad agmatinasa, lo que indica que dichos residuos son necesarios para la actividad catalítica de ALP. A continuación, se generaron 4 mutantes simples de estos residuos reemplazándolos por alanina (N213A, Q215A, D217A, K290A y E288A) y se encontró que cada mutación puntual altera las propiedades cinéticas de ΔLIM-ALP disminuyendo su Km y su Vmáx en todas las variantes, pero no se altera la interacción enzima-Mn2+. En vista de lo anterior, en este proyecto se evaluó la importancia en la actividad catalítica y la coordinación metálica de 3 residuos que se encuentran en la proximidad de los residuos analizados según el modelo estructural, estos son E190, Q339, N340. Se realizaron mutantes simples de cada residuo, reemplazándolos por alanina, luego se expresaron y purificaron parcialmente por cromatografía de intercambio aniónico, se realizaron experimentos cinéticos para determinar los parámetros cinéticos y la interacción enzima metal. Al revisar las secuencias de las mutantes, se encontró una inserción no deseada antes de la mutación N340A. Las tres variantes resultaron activas y sus parámetros cinéticos (Km y Vmáx) sufrieron variaciones respecto al control ΔLIM-ALP, Km aumentó y Vmáx disminuyó a la mitad en la variante que además tiene la inserción. Para analizar la interacción enzima-Mn2+, se realizaron experimentos de activación enzimática, diálisis, hiperactivación y especificidad de la activación con el metal y en todos los resultados las tres mutantes simples no mostraron diferencias con la enzima silvestre. Concluimos que las tres mutaciones simples afectaron el sitio activo de la enzima ya que alteraron sus parámetros cinéticos y dado que las mutaciones no alteraron la interacción enzima-Mn2+concluimos que estos residuos no participan como ligandos coordinantes del metal activador Mn2+.
Agmatine is a primary amine present in various organisms; it is metabolized by the enzymes agmatinase and/or agmatinase-like protein (ALP), generating urea and putrescine. In mammals, agmatine regulates various physiological processes and has potential uses in the treatment of mental health conditions. The ALP enzyme was discovered in rat brain tissue. It retains the characteristics of the metalloureohydrolase family, especially its dependence on the metal cofactor Mn2+ for its catalytic activity. ALP has a protein region at the carboxyl end called the LIM domain, which autoinhibits the agmatinase activity of ALP. The truncated variant in this domain (ΔLIM-ALP) is much more active and stable, and this isoform was used for the studies in this thesis. Regarding its amino acid sequence, ALP was found to be distinct from other agmatinases and lacks the highly conserved Mn2+ cofactor coordinating residues found in metalloureohydrolases. Given the lack of structural information, a molecular model of ALP was constructed using structurally solved prokaryotic agmatinases as a reference. Despite the low sequence identity, this model allowed us to propose putative residues involved in the coordination of Mn²⁺ ions in ALP. In previous studies using the ΔLIM-ALP isoform, a double mutant (E288A/K290A) and a triple mutant (N213A/Q215A/D217A) were generated, in which the residues were replaced by alanine. These variants were found to be inactive, indicating that these residues are required for the catalytic activity of ALP. Next, 4 single mutants of these residues were generated by replacing them with alanine (N213A, Q215A, D217A, K290A and E288A) and it was found that each point mutation alters the kinetic properties of ΔLIM-ALP by decreasing its Km and Vmax in all variants, but the enzyme-Mn2+ interaction is not altered. In view of the above, in this project the importance in the catalytic activity and the metal coordination of 3 residues that are in the proximity of the residues analyzed according to the structural model was evaluated, these are E190, Q339, N340. Single mutants of each residue were made, replacing them with alanine, then they were expressed and partially purified by anion exchange chromatography, kinetic experiments were performed to determine the kinetic parameters and the enzyme metal interaction. When reviewing the mutant sequences, an unwanted insertion was found before the N340A mutation. The three variants were active and their kinetic parameters (Km and Vmax) varied with respect to the ΔLIM-ALP control, Km increased and Vmax decreased by half in the variant that also had the insertion. To analyze the enzyme-Mn2+ interaction, enzyme activation, dialysis, hyperactivation, and metal activation specificity experiments were performed. In all the results, the three single mutants showed no differences with the wild-type enzyme. We conclude that the three single mutations affected the active site of the enzyme since they altered its kinetic parameters. Since the mutations did not alter the enzyme-Mn2+ interaction, we conclude that these residues do not participate as coordinating ligands for the activating metal Mn2+.
Agmatine is a primary amine present in various organisms; it is metabolized by the enzymes agmatinase and/or agmatinase-like protein (ALP), generating urea and putrescine. In mammals, agmatine regulates various physiological processes and has potential uses in the treatment of mental health conditions. The ALP enzyme was discovered in rat brain tissue. It retains the characteristics of the metalloureohydrolase family, especially its dependence on the metal cofactor Mn2+ for its catalytic activity. ALP has a protein region at the carboxyl end called the LIM domain, which autoinhibits the agmatinase activity of ALP. The truncated variant in this domain (ΔLIM-ALP) is much more active and stable, and this isoform was used for the studies in this thesis. Regarding its amino acid sequence, ALP was found to be distinct from other agmatinases and lacks the highly conserved Mn2+ cofactor coordinating residues found in metalloureohydrolases. Given the lack of structural information, a molecular model of ALP was constructed using structurally solved prokaryotic agmatinases as a reference. Despite the low sequence identity, this model allowed us to propose putative residues involved in the coordination of Mn²⁺ ions in ALP. In previous studies using the ΔLIM-ALP isoform, a double mutant (E288A/K290A) and a triple mutant (N213A/Q215A/D217A) were generated, in which the residues were replaced by alanine. These variants were found to be inactive, indicating that these residues are required for the catalytic activity of ALP. Next, 4 single mutants of these residues were generated by replacing them with alanine (N213A, Q215A, D217A, K290A and E288A) and it was found that each point mutation alters the kinetic properties of ΔLIM-ALP by decreasing its Km and Vmax in all variants, but the enzyme-Mn2+ interaction is not altered. In view of the above, in this project the importance in the catalytic activity and the metal coordination of 3 residues that are in the proximity of the residues analyzed according to the structural model was evaluated, these are E190, Q339, N340. Single mutants of each residue were made, replacing them with alanine, then they were expressed and partially purified by anion exchange chromatography, kinetic experiments were performed to determine the kinetic parameters and the enzyme metal interaction. When reviewing the mutant sequences, an unwanted insertion was found before the N340A mutation. The three variants were active and their kinetic parameters (Km and Vmax) varied with respect to the ΔLIM-ALP control, Km increased and Vmax decreased by half in the variant that also had the insertion. To analyze the enzyme-Mn2+ interaction, enzyme activation, dialysis, hyperactivation, and metal activation specificity experiments were performed. In all the results, the three single mutants showed no differences with the wild-type enzyme. We conclude that the three single mutations affected the active site of the enzyme since they altered its kinetic parameters. Since the mutations did not alter the enzyme-Mn2+ interaction, we conclude that these residues do not participate as coordinating ligands for the activating metal Mn2+.
Description
Tesis presentada para optar al título de Biólogo/a.
Keywords
Agmatina, Aminoácidos Análisis, Enzimas Biotecnología