RESUMEN
As glutamate and ammonia play a pivotal role in nitrogen homeostasis, their production is mediated by various enzymes that are widespread in living organisms. Here, we report on an effective electrophoretic method to monitor these enzymes. The in gel activity visualization is based on the interaction of the products, glutamate and ammonia, with glutamate dehydrogenase (GDH, EC: 1.4.1.2) in the presence of either phenazine methosulfate (PMS) or 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium (INT). The intensity of the activity bands was dependent on the amount of proteins loaded, the incubation time and the concentration of the respective substrates. The following enzymes were readily identified: glutaminase (EC: 3.5.1.2), alanine transaminase (EC: 2.6.1.2), aspartate transaminase (EC: 2.6.1.1), glycine transaminase (EC: 2.6.1.4), ornithine oxoacid aminotransferase (EC: 2.6.1.13), and carbamoyl phosphate synthase I (EC: 6.3.4.16). The specificity of the activity band was confirmed by high pressure liquid chromatography (HPLC) following incubation of the excised band with the corresponding substrates. These bands are amenable to further molecular characterization by a variety of analytical methods. This electrophoretic technology provides a powerful tool to screen these enzymes that contribute to nitrogen homeostasis in Pseudomonas fluorescens and possibly in other microbial systems.
Asunto(s)
Proteínas Bacterianas/química , Electroforesis en Gel de Poliacrilamida/métodos , Homeostasis , Nitrógeno/metabolismo , Pseudomonas fluorescens/metabolismo , 2,6-Dicloroindofenol/química , Alanina Transaminasa/química , Alanina Transaminasa/aislamiento & purificación , Alanina Transaminasa/metabolismo , Amoníaco/química , Aspartato Aminotransferasas/química , Aspartato Aminotransferasas/aislamiento & purificación , Aspartato Aminotransferasas/metabolismo , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Carbamoil-Fosfato Sintasa (Amoniaco)/química , Carbamoil-Fosfato Sintasa (Amoniaco)/aislamiento & purificación , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Pruebas de Enzimas , Glutamato Deshidrogenasa/química , Ácido Glutámico/química , Glutaminasa/química , Glutaminasa/aislamiento & purificación , Glutaminasa/metabolismo , Glicina-Transaminasa/química , Glicina-Transaminasa/aislamiento & purificación , Glicina-Transaminasa/metabolismo , Metosulfato de Metilfenazonio/química , Ornitina-Oxo-Ácido Transaminasa/química , Ornitina-Oxo-Ácido Transaminasa/aislamiento & purificación , Ornitina-Oxo-Ácido Transaminasa/metabolismo , Proteómica , Pseudomonas fluorescens/enzimología , Sales de Tetrazolio/químicaRESUMEN
Many plants, fungi, and bacteria catabolize allantoin as a mechanism for nitrogen assimilation. Recent reports have shown that in plants and some bacteria the product of hydrolysis of allantoin by allantoinase is the unstable intermediate ureidoglycine. While this molecule can spontaneously decay, genetic analysis of some bacterial genomes indicates that an aminotransferase may be present in the pathway. Here we present evidence that Klebsiella pneumoniae HpxJ is an aminotransferase that preferentially converts ureidoglycine and an alpha-keto acid into oxalurate and the corresponding amino acid. We determined the crystal structure of HpxJ, allowing us to present an explanation for substrate specificity.