Mechanism and Inhibitor Exploration with Binuclear Mg Ketol-Acid Reductoisomerase: Targeting the Biosynthetic Pathway of Branched-Chain Amino Acids.

Binuclear Mg ketol-acid reductoisomerase (KARI), which converts (S)-2-acetolactate into (R)-2,3-dihydroxyisovalerate, is responsible for the second step of the biosynthesis of branched-chain amino acids in plants and microorganisms and thus serves as a key inhibition target potentially without effects on mammals. Here, through the use of density functional calculations and a chemical model, the KARI-catalyzed reaction has been demonstrated to include the initial deprotonation of the substrate C2 hydroxy group, bridged by the two Mg ions, alkyl migration from the C2-alkoxide carbon atom to the C3-carbonyl carbon atom, and hydride transfer from a nicotinamide adenine dinucleotide phosphate [NAD(P)H] cofactor to C2. A dead-end mechanism with a hydride transferred to the C3 carbonyl group has been ruled out. The nucleophilicity (migratory aptitude) of the migrating carbon atom and the provision of additional negative charge to the di-Mg coordination sphere have significant effects on the steps of alkyl migration and hydride transfer, respectively. Other important mechanistic characteristics are also revealed. Inspired by the mechanism, an inhibitor (2-carboxylate-lactic acid) was designed and predicted by barrier analysis to be effective in inactivating KARI, hence probably enriching the antifungal and antibacterial library. Two types of slow substrate analogues (2-trihalomethyl acetolactic acids and 2-glutaryl lactic acid) were also found.

Metformin Promotes Anxiolytic and Antidepressant-Like Responses in Insulin-Resistant Mice by Decreasing Circulating Branched-Chain Amino Acids.

Epidemiological studies indicate that insulin resistance (IR), a hallmark of type 2 diabetes, is associated with an increased risk of major depression. Here, we demonstrated that male mice fed a high-fat diet (HFD) exhibited peripheral metabolic impairments reminiscent of IR accompanied by elevated circulating levels of branched-chain amino acids (BCAAs), whereas both parameters were normalized by chronic treatment with metformin (Met). Given the role of BCAAs in the regulation of tryptophan influx into the brain, we then explored the activity of the serotonin (5-HT) system. Our results indicated that HFD-fed mice displayed impairment in the electrical activity of dorsal raphe 5-HT neurons, attenuated hippocampal extracellular 5-HT concentrations and anxiety, one of the most visible and early symptoms of depression. On the contrary, Met stimulated 5-HT neurons excitability and 5-HT neurotransmission while hindering HFD-induced anxiety. Met also promoted antidepressant-like activities as observed with fluoxetine. In light of these data, we designed a modified HFD in which BCAA dietary supply was reduced by half. Deficiency in BCAAs failed to reverse HFD-induced metabolic impairments while producing antidepressant-like activity and enhancing the behavioral response to fluoxetine. Our results suggest that Met may act by decreasing circulating BCAAs levels to favor serotonergic neurotransmission in the hippocampus and promote antidepressant-like effects in mice fed an HFD. These findings also lead us to envision that a diet poor in BCAAs, provided either alone or as add-on therapy to conventional antidepressant drugs, could help to relieve depressive symptoms in patients with metabolic comorbidities.SIGNIFICANCE STATEMENT Insulin resistance in humans is associated with increased risk of anxiodepressive disorders. Such a relationship has been also found in rodents fed a high-fat diet (HFD). To determine whether insulin-sensitizing strategies induce anxiolytic- and/or antidepressant-like activities and to investigate the underlying mechanisms, we tested the effects of metformin, an oral antidiabetic drug, in mice fed an HFD. Metformin reduced levels of circulating branched-chain amino acids, which regulate tryptophan uptake within the brain. Moreover, metformin increased hippocampal serotonergic neurotransmission while promoting anxiolytic- and antidepressant-like effects. Moreover, a diet poor in these amino acids produced similar beneficial behavioral property. Collectively, these results suggest that metformin could be used as add-on therapy to a conventional antidepressant for the comorbidity between metabolic and mental disorders.

Elevation of branched-chain amino acid levels in diabetes and NAFL and changes with antidiabetic drug treatment.

Diabetes mellitus (DM), non-alcoholic fatty liver (NAFL), and obesity are associated with elevated branched-chain amino acid (BCAA) levels, but the mechanism and significance of this has not been elucidated. Eighty-four subjects were enrolled including 43 with DM. Serum BCAA levels were positively correlated with waist-hip ratio and ALT. Serum BCAA levels in subjects with DM were higher than non-DM and those in subjects with NAFL were also higher than non-NAFL. Treatment with pioglitazone and alogliptin (19 of 43 DM subjects) improved serum haemoglobin A1c and decreased BCAA levels. The decrease in BCAAs with improved glucose metabolism suggests that abnormal glucose metabolism is also a factor in elevated BCAA levels.

Growth inhibition of pathogenic bacteria by sulfonylurea herbicides.

Emerging resistance to current antibiotics raises the need for new microbial drug targets. We show that targeting branched-chain amino acid (BCAA) biosynthesis using sulfonylurea herbicides, which inhibit the BCAA biosynthetic enzyme acetohydroxyacid synthase (AHAS), can exert bacteriostatic effects on several pathogenic bacteria, including Burkholderia pseudomallei, Pseudomonas aeruginosa, and Acinetobacter baumannii. Our results suggest that targeting biosynthetic enzymes like AHAS, which are lacking in humans, could represent a promising antimicrobial drug strategy.

Role of exogenously supplied ferulic and p-coumaric acids in mimicking the mode of action of acetolactate synthase inhibiting herbicides.

Chlorsulfuron and imazethapyr (herbicides that inhibit acetolactate synthase; ALS, EC 4.1.3.18) produced a strong accumulation of hydroxycinnamic acids that was related to the induction of the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 2.5.2.54). The exogenous application of two hydroxycinnamic acids, ferulic and p-coumaric acids, to pea plants resulted in their internal accumulation, arrested growth, carbohydrate and quinate accumulation in the leaves, and the induction of ethanolic fermentation. These effects resemble some of the physiological effects detected after acetolactate synthase inhibition and suggest important roles for ferulic and p-coumaric acids in the mode of action of herbicides inhibiting the biosynthesis of branched chain amino acids.

Unraveling the role of fermentation in the mode of action of acetolactate synthase inhibitors by metabolic profiling.

Herbicides that inhibit branched chain amino acid biosynthesis induce aerobic fermentation. The role of fermentation in the mode of action of these herbicides is not known, nor is the importance of this physiological response in the growth inhibition and the lethality caused by them. Metabolic profiling was used to compare the effects of the herbicide imazethapyr (IM) on pea plants with two other treatments that also induce fermentation: hypoxia and the exogenous supply pyruvate for seven days. While hypoxic roots did not show internal anoxia, feeding pyruvate or applying IM to the roots led to internal anoxia, probably related to the respiratory burst detected. The three treatments induced ethanol fermentation, but fermentation induced following herbicide treatment was earlier than that following pyruvate supply and was not associated with a decrease in the energy status. No striking changes were detected in the metabolic profiling of hypoxic roots, indicating that metabolism was only slightly impaired. Feeding pyruvate resulted in marked succinate accumulation and a general amino acid accumulation. IM-treated roots showed a general accumulation of glycolytic metabolites upstream of pyruvate, a decrease in some TCA intermediates and an increase in the free amino acid pool sizes. All treatments caused GABA and putrescine accumulation. Our results indicate that IM supply impairs carbon/nitrogen metabolism and this impaired metabolism is likely to be related to the growth arrest detected. As growth is arrested, carbohydrates and glycolytic intermediates accumulate and energy becomes more available.

Protein synthesis is severely diminished following a simulated upper GI bleed in patients with cirrhosis.

BACKGROUND/AIMS: An upper gastrointestinal (GI) bleed in cirrhotic patients has been hypothesised to induce catabolism due to the absence of the essential branched chain amino acid (BCAA) isoleucine and an abundance of the BCAA leucine in haemoglobin. We tested whether an upper GI bleed produces hypoisoleucinemia via BCAA antagonism and impairs protein synthesis. METHODS: Isoleucine turnover and oxidation was studied in 5 metabolically stable patients with cirrhosis during a 4-h period of intragastric saline infusion followed by a 4-h period in which an upper GI bleed was simulated by an amino acid solution mimicking haemoglobin. RESULTS: The simulated upper GI bleed induced hypoisoleucinemia (26% of initial values) and an increase in leucine (400%) and valine (350%) concentrations. Isoleucine flux and isoleucine oxidation decreased to a third of initial values following a simulated bleed, but the fraction of isoleucine flux used for oxidation did not change. Consequently, the non-oxidative portion of isoleucine flux, representing protein synthesis, decreased similarly. CONCLUSIONS: The present study shows that a simulated upper GI bleed induces hypoisoleucinemia and decreases protein synthesis markedly. The fact that the percentage of isoleucine flux that was oxidized was not influenced by the hypoisoleucinemic state can only be explained by BCAA antagonism.

Regulation of taste-active components of meat by dietary branched-chain amino acids; effects of branched-chain amino acid antagonism.

1. The effects of dietary branched-chain amino acids (BCAAs) including leucine (Leu), isoleucine (Ile) and valine (Val) on taste-active components, especially free glutamate (Glu), in meat were investigated. 2. Broiler chickens (28 d old) were given varied dietary BCAA levels for 10 d before marketing. Dietary BCAA content ratios were either 100:100:100 (Low Leu group), 150:100:100 (Control group) or 150:150:150 (High Ile + Val group) for Leu:Ile:Val (% of each BCAA requirement according to NRC, 1994). Taste-related components of meat (free amino acids and ATP metabolites) and sensory scores of meat soup were estimated. 3. Free Glu content, the main taste-active component of meat, was significantly increased by dietary BCAA. Compared to the Control group, free Glu content increased by 30% in the High Ile + Val group. However, the inosine monophosphate (IMP) content in meat did not change among groups. 4. Sensory evaluation of meat soups showed that Control and High Ile + Val groups had different meat flavours. The sensory score of overall taste intensity was significantly higher in the High Ile + Val group. 5. These results suggest that dietary BCAA concentrations regulate free Glu in meat. Increasing dietary Ile + Val induces an increase in free Glu content of meat, improves meat taste and is more effective for increasing free Glu content in meat than decreasing dietary Leu level.

Nitrogen assimilation studies using 15N in soybean plants treated with imazethapyr, an inhibitor of branched-chain amino acid biosynthesis.

The pattern of nitrogen assimilation in soybean plants treated with a herbicide that inhibits branched-chain amino acid biosynthesis was evaluated by (15)N isotopic analysis. The herbicide imazethapyr caused a strong decrease in nitrate uptake by roots, partly due to a reduced stomatal conductance. The inhibition of (15)N uptake was accompanied by a decrease in the (15)N content in the plant and, concomitantly, an inhibition of translocation to the shoot. Imazethapyr inhibited nitrate reductase activity in leaves and roots. Among all parameters studied, "de novo" synthesis of proteins was the first parameter of the N assimilation metabolism affected by the herbicide. These results show that this class of herbicides totally damages N metabolism and indicates a regulatory effect on N uptake and translocation that would be mediated by the increase in free amino acid pool provoked by the inhibition of branched-chain amino acid biosynthesis.

Changes in mitochondrial electron partitioning in response to herbicides inhibiting branched-chain amino acid biosynthesis in soybean.

The adaptation of the respiratory metabolism in roots of soybean (Glycine max L. Merr. cv Ransom) treated with herbicides that inhibit the enzyme acetolactate synthase (ALS) was analyzed. A new gas phase dual-inlet mass spectrometry system for simultaneous measurement of 34O2 to 32O2 and O2 to N2 ratios has been developed. This system is more accurate than previously described systems, allows measurements of much smaller oxygen gradients, and, as a consequence, works with tissues that have lower respiration rates. ALS inhibition caused an increase of the alternative oxidase (AOX) protein and an accumulation of pyruvate. The combination of these two effects is likely to induce the activation of the alternative pathway and its participation in the total respiration. Moreover, the start of the alternative pathway activation and the increase of AOX protein were before the decline in the activity of cytochrome pathway. The possible role of AOX under ALS inhibition is discussed.

Utility of hemodialysis in maple syrup urine disease.

Maple syrup urine disease (MSUD) is an inborn error of metabolism stemming from a deficiency in 2-ketoacid dehydrogenase and resulting in the systemic accumulation of branched chain amino acids (BCAAs). Affected children may suffer profound developmental and cognitive impairment from exposure to high levels of BCAA and their associated neurotoxic metabolites. Endogenous renal clearance of BCAA is limited and several therapeutic modalities including intensive nutritional regimens, exchange transfusions, peritoneal dialysis, and continuous hemofiltration have been utilized in neonates with MSUD, all of which have had varying success in reducing systemic BCAA levels. In this report, a symptomatic 7-day-old 3-kg neonate with MSUD underwent treatment with a combination of early hemodialysis and aggressive enteral feedings of a metabolically appropriate formula. This approach results in a 75% reduction of systemic toxin levels within 3 h. When compared to other reported modalities of therapy for symptomatic neonates with MSUD, this approach appears to be most efficacious. Moreover, by minimizing the amount of time that an affected neonate is exposed to neurotoxic levels of BCAAs, long-term developmental and cognitive capabilities may be preserved.

Role of specific aminotransferases in de novo glutamate synthesis and redox shuttling in the retina.

In this study aminotransferase inhibitors were used to determine the relative importance of different aminotransferases in providing nitrogen for de novo glutamate synthesis in the retina. Aminooxyacetate, which inhibits all aminotransferases, blocked de novo glutamate synthesis from H(14)CO(3)(-) by more than 60%. Inhibition of neuronal cytosolic branched chain amino acid transamination by gabapentin or branched chain amino acid transport by the L-system substrate analog, 2-amino-bicyclo-(2,2,1)-heptane-2-carboxylic acid, lowered total de novo synthesis of glutamate by 30%, suggesting that branched chain amino acids may account for half of the glutamate nitrogen contributed by transamination reactions. L-cycloserine, an inhibitor of alanine aminotransferase, inhibited glutamate synthesis less than 15% when added in the presence of 5 mM pyruvate but 47% in the presence of 0.2 mM pyruvate. Although high levels of pyruvate blunted the inhibitory effectiveness of L-cycloserine, the results indicate that, under physiological conditions, alanine as well as branched chain amino acids are probably the predominant sources of glutamate nitrogen in ex vivo retinas. The L-cycloserine results were also used to evaluate activity of the malate/aspartate shuttle. In this shuttle, cytosolic aspartate (synthesized in mitochondria) generates cytosolic oxaloacetate that oxidizes cytosolic NADH via malate dehydrogenase. Because L-cycloserine inhibits cytosolic but not mitochondrial aspartate aminotransferase, L-cycloserine should prevent the utilization of aspartate but not its generation, thereby increasing levels of (14)C-aspartate. Instead, L-cycloserine caused a significant decline in (14)C-aspartate. The results suggest the possibility that shuttle activity is low in retinal Müller cells. Low malate/aspartate shuttle activity may be the molecular basis for the high rate of aerobic glycolysis in retinal Müller cells.

Inhibitors of branched-chain amino acid biosynthesis as potential antituberculosis agents.

Leucine auxotrophs of Mycobacterium bovis (BCG) were found to have a reduced ability to survive in spleens and lungs of mice. This indicated that inhibitors of branched-chain amino acid biosynthesis could possibly be used as antituberculosis agents. Herbicides that inhibit plant branched-chain amino acid biosynthetic enzymes were tested for inhibition of Mycobacterium tuberculosis growth in vitro. Sulphometuron methyl (SM) and metsulphuron methyl, inhibitors of acetolactate synthase (ALS), had a modest effect on growth of M. tuberculosis strain ATCC 35801 (inhibitory concentrations <20 microM). Two inhibitors of ketol acid reductoisomerase (KARI) were ineffective against growth of strain ATCC 35801 in vitro. On the other hand, ALS and KARI inhibitors were more effective against growth of clinical drug-resistant isolates than against strain ATCC 35801. Mouse model studies of tuberculosis infection showed that high doses of SM significantly prevented growth of M. tuberculosis strain ATCC 35801 in the lungs but did not affect the level of infection in the spleen. The results suggest that inhibitors of branched-chain amino acid biosynthesis may be useful as new antituberculosis agents.

Leucine affects the metabolism of valine by isolated perfused rat hearts: relation to branched-chain amino acid antagonism.

This study was conducted to determine the effects of different concentrations of leucine on the transport, transamination and oxidation of valine and on incorporation of valine into heart proteins in the isolated perfused rat heart. Valine metabolism was studied in rat hearts perfused with medium containing glucose and graded levels of L-leucine. In transport studies L-phenylalanine was also tested. Uptake of L-[1-14C]valine (0.2 mmol/L) was significantly reduced (-50%) by inclusion of 0.2 mmol/L phenylalanine or leucine, and by -70% by inclusion of 1.0 mmol/L phenylalanine or leucine in the perfusate. Transamination of valine decreased by 37 and 48%, and oxidation of valine by 53 and 71%, respectively, when 0.2 or 1.0 mmol/L leucine was included in the perfusate. Tissue concentrations of valine decreased by 43, 48 and 62% in the presence of 0.2, 0.5 and 1.0 mmol/L leucine, respectively; tissue concentrations of leucine, glutamate and alanine increased approximately 11-fold, 1.2-fold and 0.5-fold, respectively, when 1.0 mmol/L leucine was present in the perfusate. Addition of 0.2-1.0 mmol/L leucine did not affect incorporation of valine into heart proteins. We conclude that 1) competition among large neutral amino acids for transport into heart occurs at physiological concentrations of these amino acids in plasma; 2) inhibition of valine uptake by leucine can limit the rate of valine catabolism in heart; and 3) depletion of tissue valine concentration by an excess of leucine did not affect the rate of protein synthesis.

Separate and shared lysosomal transport of branched and aromatic dipolar amino acids.

Transport systems analogous to the T and L carriers for aromatic and bulky dipolar amino acids in plasma membranes have been characterized in the membranes of intact lysosomes isolated from human fetal skin fibroblasts. While system L appears ubiquitous in plasma membranes, system T has previously been discriminated only in the plasmalemma of human red blood cells and freshly isolated rat hepatocytes. Our findings with the lysosomal systems, provisionally designated t and l, reveal both shared and dissimilar properties with the plasma membrane systems. These properties include a lack of dependency on extralysosomal Na+, differential sensitivities to the classical system L analog, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), and the system T analog, D-tryptophan, as well as susceptibility to thiol modification at the membrane by reactivity with N-ethylmaleimide. A transport system in lysosomes from the FRTL-5 rat thyroid cell line has been described by Bernar et al. ((1986) J. Biol. Chem. 261, 17107-17112) resembles a composite of both carrier systems reported in this work.