Metabolic effects of inhibitors of two enzymes of the branched-chain amino acid pathway in Salmonella typhimurium

Abstract

The metabolic effects of inhibitors of two enzymes in the pathway for biosynthesis of branched-chain amino acids were examined in Salmonella typhimurium mutant strain TV105, expressing a single isozyme of acetohydroxy acid synthase (AHAS), AHAS isozyme II. One inhibitor was the sulfonylurea herbicide sulfometuron methyl (SMM), which inhibits this isozyme and AHAS of other organisms, and the other was N-isopropyl oxalylhydroxamate (IpOHA), which inhibits ketol-acid reductoisomerase (KARI). The effects of the inhibitors on growth, levels of several enzymes of the pathway, and levels of intermediates of the pathway were measured. The intracellular concentration of the AHAS substrate 2-ketobutyrate increased on addition of SMM, but a lack of correlation between increased ketobutyrate and growth inhibition suggests that the former is not the immediate cause of the latter. The levels of the keto acid precursor of valine, but not of the precursor of isoleucine, were drastically decreased by SMM, and valine, but not isoleucine, partially overcame SMM inhibition. This apparent stronger effect of SMM on the flux into the valine arm, as opposed to the isoleucine arm, of the branched-chain amino acid pathway is explained by the kinetics of the AHAS reaction, as well as by the different roles of pyruvate, ketobutyrate, and the valine precursor in metabolism. The organization of the pathway thus potentiates the inhibitory effect of SMM. IpOHA has strong initial effects at lower concentrations than does SMM and leads to increases both in the acetohydroxy acid substrates of KARI and, surprisingly, in ketobutyrate. Valine completely protected strain TV105 from IpOHA at the MIC. A number of explanations for this effect can be ruled out, so that some unknown arrangement of the enzymes involved must be suggested. IpOHA led to initial cessation of growth, with partial recovery after a time whose duration increased with the inhibitor concentration. The recovery is apparently due to induction of new KARI synthesis, as well as disappearance of IpOHA from the medium.Enzymes that participate in biosynthetic pathways of essential amino acids have been recognized as targets for a number of safe and effective herbicides (18). The biosynthetic pathway to the branched-chain amino acids valine, leucine, and isoleucine is of special importance in this respect. At least three classes of very potent and extensively used herbicides, the sulfonylureas, the imidazolinones, and the sulfonanilides (26,30,39), are known to inhibit the first common enzyme in this pathway (Fig. 1), acetohydroxy acid synthase (AHAS; EC 4.1.3.18). The second common enzyme in the pathway (Fig. 1), ketol-acid reductoisomerase (KARI; EC 1.1.1.86), is the target for two additional inhibitors with potential herbicidal activity, 2-dimethylphosphinoyl-2-hydroxyacetate (42) and N-isopropyl oxalylhydroxamate (IpOHA) (3).Sulfometuron methyl (SMM), which belongs to the sulfonylurea class of herbicides and inhibits AHAS activity, is an extremely potent herbicide and bacteriostatic age...

Isoleucine starvation caused by sulfometuron methyl in Salmonella typhimurium measured by translational frameshifting

“…1), and is consistent with the observation that SMM-inhibited S. typhimurium TV105 cells contained reduced amounts of the valine precursor, 2-oxoisovalerate, while the concentration of the parallel isoleucine precursor, 2-oxo-3-methylvalerate, was not reduced (Epelbaum et al, 1996). We suggested (Epelbaum et al, 1996) that the competition between 2-oxobutyrate and pyruvate as AHAS substrates, together with the feedback control of 2-oxobutyrate synthesis by isoleucine, could account for the specific reduction of valine and its precursor, as opposed to isoleucine, when AHAS is inhibited. These findings do not exclude the possibility that accumulation of 2-oxobutyrate could be partly responsible for SMM toxicity through competition with 2-oxoisovalerate, the valine precursor ( Fig.…”

“…This suggests that the inhibition of growth when AHAS activity is decreased is primarily the result of decreased flux in the valine-leucine branch of the branched-chain amino acid path (Fig. 1), and is consistent with the observation that SMM-inhibited S. typhimurium TV105 cells contained reduced amounts of the valine precursor, 2-oxoisovalerate, while the concentration of the parallel isoleucine precursor, 2-oxo-3-methylvalerate, was not reduced (Epelbaum et al, 1996). We suggested (Epelbaum et al, 1996) that the competition between 2-oxobutyrate and pyruvate as AHAS substrates, together with the feedback control of 2-oxobutyrate synthesis by isoleucine, could account for the specific reduction of valine and its precursor, as opposed to isoleucine, when AHAS is inhibited.…”

“…As shown in Fig. 2 and previously described (Epelbaum et al, 1996), this inhibition was almost completely relieved by addition of valine alone. Valine itself does not inhibit this bacterium, as AHAS II, unlike most other AHASs, is valine resistant .…”

“…This enzyme catalyses the decarboxylation of pyruvate and its condensation with a second molecule of pyruvate to yield acetolactate, the precursor of valine (and leucine), or with 2-oxobutyrate to yield acetohydroxybutyrate, the precursor of isoleucine. Because of the branching of the pathway, feedback inhibition of several enzymes by end products, and competition between substrates of the same and different enzymes, the metabolic and physiological consequences of inhibition by sulfonylureas are not simple and are still not completely understood (Epelbaum et al, 1996(Epelbaum et al, , 1998LaRossa et al, 1987 ;Jia et al, 2000). An understanding of the ramifications of AHAS inhibitors is important for the understanding of their mode of action as herbicides and for the development reliable method for direct and quantitative assessment of the degree of starvation for individual amino acids caused by the presence of the inhibitor.…”

Growth Inhibition of Pathogenic Bacteria by Sulfonylurea Herbicides

“…Similar to plants (2,29), a single mutation in the catalytic subunit of AHAS was sufficient to confer resistance to chlorimuron ethyl and other SHs. To counter the rapid emergence of single-step, high-level SH-resistant mutants, AHAS inhibitors could be used in combination with other antibiotics (30) or inhibitors of BCAA biosynthesis (31)(32)(33).…”