ABSTRACT
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.
Subject(s)
Bacterial Proteins/chemistry , Electrophoresis, Polyacrylamide Gel/methods , Homeostasis , Nitrogen/metabolism , Pseudomonas fluorescens/metabolism , 2,6-Dichloroindophenol/chemistry , Alanine Transaminase/chemistry , Alanine Transaminase/isolation & purification , Alanine Transaminase/metabolism , Ammonia/chemistry , Aspartate Aminotransferases/chemistry , Aspartate Aminotransferases/isolation & purification , Aspartate Aminotransferases/metabolism , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Carbamoyl-Phosphate Synthase (Ammonia)/chemistry , Carbamoyl-Phosphate Synthase (Ammonia)/isolation & purification , Carbamoyl-Phosphate Synthase (Ammonia)/metabolism , Enzyme Assays , Glutamate Dehydrogenase/chemistry , Glutamic Acid/chemistry , Glutaminase/chemistry , Glutaminase/isolation & purification , Glutaminase/metabolism , Glycine Transaminase/chemistry , Glycine Transaminase/isolation & purification , Glycine Transaminase/metabolism , Methylphenazonium Methosulfate/chemistry , Ornithine-Oxo-Acid Transaminase/chemistry , Ornithine-Oxo-Acid Transaminase/isolation & purification , Ornithine-Oxo-Acid Transaminase/metabolism , Proteomics , Pseudomonas fluorescens/enzymology , Tetrazolium Salts/chemistryABSTRACT
Aminotransferases catalyse synthetic and degradative reactions of amino acids, and serve as a key linkage between central carbon and nitrogen metabolism in most organisms. In this study, three aminotransferases (AT1, AT2 and AT3) were purified and characterized from Hydrogenobacter thermophilus, a hydrogen-oxidizing chemolithoautotrophic bacterium, which has been reported to possess unique features in its carbon and nitrogen anabolism. AT1, AT2 and AT3 exhibited glutamate:oxaloacetate aminotransferase, glutamate:pyruvate aminotransferase and alanine:glyoxylate aminotransferase activities, respectively. In addition, both AT1 and AT2 catalysed a glutamate:glyoxylate aminotransferase reaction. Interestingly, phylogenetic analysis showed that AT2 belongs to aminotransferase family IV, whereas known glutamate:pyruvate aminotransferases and glutamate:glyoxylate aminotransferases are members of family Igamma. In contrast, AT3 was classified into family I, distant from eukaryotic alanine:glyoxylate aminotransferases which belong to family IV. Although Thermococcus litoralis alanine:glyoxylate aminotransferase is the sole known example of family I alanine:glyoxylate aminotransferases, it is indicated that this alanine:glyoxylate aminotransferase and AT3 are derived from distinct lineages within family I, because neither high sequence similarity nor putative substrate-binding residues are shared by these two enzymes. To our knowledge, this study is the first report of the primary structure of bacterial glutamate:glyoxylate aminotransferase and alanine:glyoxylate aminotransferase, and demonstrates the presence of novel types of aminotransferase phylogenetically distinct from known eukaryotic and archaeal isozymes.
