Purificación y caracterización estructural de ΔLIM-ALP.
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Date
2025
Journal Title
Journal ISSN
Volume Title
Publisher
Universidad de Concepción
Abstract
La Agmatinase Like Protein (ALP) es una enzima clonada a partir de una librería de ADNc de cerebro de rata y cataliza la hidrólisis de agmatina en putrescina y urea. La agmatina (1-amino-4-guanidinobutano) es una amina que deriva de la descarboxilación de arginina mediante la arginina descarboxilasa (ADC), cumple funciones críticas en mamíferos como neuromodulador, anticonvulsivante y agente neuroprotector. Hasta la fecha, ALP es la única enzima de mamíferos caracterizada cinéticamente con actividad agmatinasa confirmada. A pesar de su función catalítica, ALP muestra una identidad de secuencia mínima con agmatinasas conocidas, y los residuos involucrados en la catálisis o en la coordinación del cofactor Mn²⁺ (ion metálico divalente crítico para la actividad de ALP y otras ureohidrolasas) permanecen sin identificar. Las herramientas actuales de predicción de plegamiento proteico, como la plataforma AlphaFold 3, no logran modelar la estructura de ALP. En el Laboratorio de Enzimología de la Universidad de Concepción, se han clonado varias isoformas de ALP. La variante de ALP más activa y estable contiene 457 aminoácidos y carece de 66 aminoácidos en su extremo carboxilo, esta secuencia se denomina dominio LIM y ejerce un efecto auto- inhibitorio sobre su actividad agmatinasa (ΔLIM-ALP). En este seminario de título, se estableció un protocolo de purificación para ΔLIM-ALP logrando purificar la enzima a homogeneidad y se realizó un análisis de dicroísmo circular (DC) para determinar el contenido de estructura secundaria. El análisis de DC arrojó que ΔLIM- ALP contiene 2,8% de estructuras helicoidales, 34,4% de hojas ß-plegadas, 14,6% de vueltas y 48,1% de zonas al azar o no definidas. Este trabajo proporciona las primeras aproximaciones respecto de la estructura de ALP.
Agmatinase Like Protein (ALP) is an enzyme cloned from a rat brain cDNA library and catalyzes the hydrolysis of agmatine to putrescine and urea. Agmatine (1-amino- 4-guanidinobutane) is an amine derived from the decarboxylation of arginine by arginine decarboxylase (ADC), fulfilling critical functions in mammals as a neuromodulator, anticonvulsant, and neuroprotective agent. To date, ALP is the only kinetically characterized mammalian enzyme with confirmed agmatinase activity. Despite its catalytic function, ALP shows minimal sequence identity with known agmatinases, and the residues involved in catalysis or in the coordination of the Mn²⁺ cofactor (a divalent metal ion critical for the activity of ALP and other ureohydrolases) remain unidentified. Current protein folding prediction tools, such as the AlphaFold 3 platform, fail to model the structure of ALP. Several ALP isoforms have been cloned at the Enzymology Laboratory of the University of Concepción. The most active and stable ALP variant contains 457 amino acids and lacks 66 amino acids at its carboxyl terminus. This sequence is called the LIM domain and exerts an autoinhibitory effect on its agmatinase activity (ΔLIM-ALP). In this degree seminar, a purification protocol for ΔLIM-ALP was established, achieving homogeneity in the enzyme, and circular dichroism (CD) analysis was performed to determine its secondary structure content. CD analysis showed that ΔLIM-ALP contains 2.8% helical structures, 34.4% β- pleated sheets, 14.6% turns, and 48.1% random or undefined regions. This work provides the first approximations regarding the structure of ALP.
Agmatinase Like Protein (ALP) is an enzyme cloned from a rat brain cDNA library and catalyzes the hydrolysis of agmatine to putrescine and urea. Agmatine (1-amino- 4-guanidinobutane) is an amine derived from the decarboxylation of arginine by arginine decarboxylase (ADC), fulfilling critical functions in mammals as a neuromodulator, anticonvulsant, and neuroprotective agent. To date, ALP is the only kinetically characterized mammalian enzyme with confirmed agmatinase activity. Despite its catalytic function, ALP shows minimal sequence identity with known agmatinases, and the residues involved in catalysis or in the coordination of the Mn²⁺ cofactor (a divalent metal ion critical for the activity of ALP and other ureohydrolases) remain unidentified. Current protein folding prediction tools, such as the AlphaFold 3 platform, fail to model the structure of ALP. Several ALP isoforms have been cloned at the Enzymology Laboratory of the University of Concepción. The most active and stable ALP variant contains 457 amino acids and lacks 66 amino acids at its carboxyl terminus. This sequence is called the LIM domain and exerts an autoinhibitory effect on its agmatinase activity (ΔLIM-ALP). In this degree seminar, a purification protocol for ΔLIM-ALP was established, achieving homogeneity in the enzyme, and circular dichroism (CD) analysis was performed to determine its secondary structure content. CD analysis showed that ΔLIM-ALP contains 2.8% helical structures, 34.4% β- pleated sheets, 14.6% turns, and 48.1% random or undefined regions. This work provides the first approximations regarding the structure of ALP.
Description
Tesis presentada para optar al título de Biólogo/a.
Keywords
Agmatina, Enzimas Análisis, Aminoácidos