Branched-Chain Amino Acid Biosynthesis in Salmonella typhimurium : a Quantitative Analysis

Abstract

We report here the first quantitative study of the branched-chain amino acid biosynthetic pathway in Salmonella typhimurium LT2. The intracellular levels of the enzymes of the pathway and of the 2-keto acid intermediates were determined under various physiological conditions and used for estimation of several of the fluxes in the cells. The results led to a revision of previous ideas concerning the way in which multiple acetohydroxy acid synthase (AHAS) isozymes contribute to the fitness of enterobacteria. In wild-type LT2, AHAS isozyme I provides most of the flux to valine, leucine, and pantothenate, while isozyme II provides most of the flux to isoleucine. With acetate as a carbon source, a strain expressing AHAS II only is limited in growth because of the low enzyme activity in the presence of elevated levels of the inhibitor glyoxylate. A strain with AHAS I only is limited during growth on glucose by the low tendency of this enzyme to utilize 2-ketobutyrate as a substrate; isoleucine limitation then leads to elevated threonine deaminase activity and an increased 2-ketobutyrate/2-ketoisovalerate ratio, which in turn interferes with the synthesis of coenzyme A and methionine. The regulation of threonine deaminase is also crucial in this regard. It is conceivable that, because of fundamental limitations on the specificity of enzymes, no single AHAS could possibly be adequate for the varied conditions that enterobacteria successfully encounter.

Activation of the Bacillus subtilis global regulator CodY by direct interaction with branched‐chain amino acids

“…Because the accumulation of isoleucine and valine is so closely tied to the concentrations of so many other key metabolites, the cell may monitor its metabolic state by having a regulatory protein (CodY) that responds directly to isoleucine and valine concentrations. The intracellular concentrations of BCAAs in Escherichia coli and Salmonella have been reported to range from 8 mM to 15 mM (Epelbaum et al, 1998). This range is consistent with those used in our experiments for B. subtilis CodY.…”

Many of the functional differences between acetohydroxyacid synthase (AHAS) isozyme I and other AHASs are a result of the rapid formation and breakdown of the covalent acetolactate–thiamin diphosphate adduct in AHAS I

“…Escherichia coli , Salmonella typhimurium and other enterobacteria encode three or more isozymes of AHAS that differ in preference for 2‐KB as the second substrate, inhibition by the pathway end‐product valine, affinity for cofactors and sensitivity to inhibition by the many herbicides that have been developed whose target is AHAS. The expression of each isozyme is regulated in a different way [2–4] and each presumably has a slightly different metabolic role [3–6]. On the other hand, many other prokaryotes have only a single AHAS, with most being similar in sequence and properties to an enterobacterial AHAS isozyme III.…”

Global expression profiling of yeast treated with an inhibitor of amino acid biosynthesis, sulfometuron methyl

“…ALS catalyzes the second step in isoleucine and the first step in valine biosynthesis, the formation of ␣-acetolactate and ␣-aceto-␣-hydroxybutyrate (51). Studies of Salmonella typhimurium and Escherichia coli have suggested that both an imbalance among ␣-ketoacids and end product deficiency contributed to inhibitor efficacy (12,30). Since ␣-ketoacids and their derivative acyl-CoA molecules are quantitatively predominant in biosynthetic metabolism (30a) and since similar imbalances are found in inborn errors of human metabolism Article published online before print.…”