Browsing by Subject "Aspartate"

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Metabolic rewiring compensates for the loss of amino acid biosynthesis in Bacillus subtilis
(2024) Yousef Mardoukhi, Mohammad Saba; Commichau, Fabian M.
Amino acids are considered as some of the earliest organic molecules to form on Earth. Serving as the building blocks of proteins, they are intricately connected to nearly every life process. Therefore, amino acid metabolism needs to be precisely regulated in any living organism. Amino acid metabolism includes the biochemical pathways responsible for the synthesis, degradation, and utilization of amino acids. Most of the bacteria, particularly the Gram-positive model bacterium Bacillus subtilis, have the capability to synthesize all proteinogenic amino acids or, if available, import them from the environment. Throughout evolution, different metabolic pathways have emerged to maintain metabolites level inside the cells. Some biosynthetic pathways are unknown as they are not primary routes or are typically inactive under normal conditions. However, they may become active under specific circumstances. Two very important pathways, previously not known to be substituted by alternative routes, involve de novo biosynthesis of glutamate, which is an essential amino group donor in every cell. Many bacteria can synthesize glutamate using a NADPH + H+-dependent glutamate dehydrogenase (GDH). Alternatively, glutamate can be produced by the combined action of the ATP-dependent glutamine synthetase (GS) and the NADPH + H+-dependent glutamate synthase (GOGAT). B. subtilis only employs the GS-GOGAT pathway for de novo synthesis of glutamate. In the context of this work, it was shown that a B. subtilis deficient for the GS-GOGAT pathway may employ the aspartase AnsB and aspartate transaminase AspB for the synthesis of glutamate in biologically significant amounts. Genetic analyses revealed that the aspartase AnsB converts ammonium and the tricarboxylic acid cycle intermediate fumarate to aspartate. Subsequently, the aspartate transaminase AspB transfers the amino group from aspartate to α-ketoglutarate, resulting in the production of L-glutamate and oxaloacetate. This observation challenges the well-established point of view of whether the GS-GOGAT-dependent pathway is indeed the only route for de novo synthesis of glutamate in nature. It was also set out to explore which amino acids could serve as the sole sources of carbon and nitrogen in the background of a B. subtilis strain that is a genetically stable glutamate auxotroph. The aim was to understand the conversion of the amino acids into glutamate and further to α-ketoglutarate, a reaction that is facilitated by the enzymatic activity of the GDHs RocG/GudB. It turned out that some of the amino acids are toxic for B. subtilis. However, B. subtilis can quickly develop resistance by the acquisition of mutations that result in reduced and enhanced amino acid uptake and export, respectively. Moreover, the toxicity of some amino acids may be reduced by increased degradation of glutamate. Furthermore, with focus on the toxicity of asparagine, it could be demonstrated that AimA, which has been characterized as a general amino acid importer, serves as a low affinity asparagine transporter in B. subtilis. Finally, AzlCD, which was previously described as an exporter for histidine and branched-chain amino acids, also exports asparagine. Thus, B. subtilis can adapt to amino acid toxicity in various ways.