Genetic separation of high- and low-affinity transport systems for branched-chain amino acids in Escherichia coli K-12.

The Escherichia coli K-12 mutant strain AE4107 (livH::Mu) is defective in the high-affinity binding protein-mediated uptake system for L-leucine, L-valine, and L-isoleucine (LIV-I). We have used this strain to produce mutations in the residual LIV-II membrane-bound branched-chain amino acid uptake system. Mutants selected for their inability to utilize exogenous L-leucine were found to be defective in the LIV-II system and fell into two classes. One class, represented by strain AE410709 (livP9), showed a complete loss of saturable uptake for L-leucine, L-valine, and L-isoleucine up to 50 muM, and a second class, represented by strain AE4017012 (liv-12), showed a residual component of saturable leucine uptake with increased Km. These mutations, livP9 and liv-12, were closely linked and mapped in the 74 to 78 min region of the E. coli genetic map. Strains constructed so that they lacked both LIV-I and LIV-II transport systems excreted leucine. Strains of the genotype livH+ livP were found to have normal high-affinity binding protein-mediated transport (LIV-I and leucine specific), whereas the low-affinity (LIV-II) transport was completely missing. We concluded from these studies that the high-affinity binding protein-mediated transport systems (LIV-I and leucine specific) can operate independently of the membrane-bound LIV-II system.     

Solubilization and reconstitution of sodium-dependent transport system for branched-chain amino acids from Pseudomonas aeruginosa

The sodium-dependent transport system for branched-chain amino acids of Pseudomonas aeruginosa was solubilized with n-octyl-beta-D-glucopyranoside and reconstituted into liposomes by a detergent-Sephadex G-50 gel filtration procedure. The reconstituted proteoliposomes exhibited Na+-dependent counterflow and Na+-gradient-driven transport of L-leucine, L-isoleucine, and L-valine. The leucine counterflow was specifically inhibited by only branched-chain amino acids and the uphill transport of two species of amino acids among the three was induced by counterflow of the other substrate. These results show that the transport system for branched-chain amino acids was reconstituted into liposomes from P. aeruginosa cells and strongly suggest that three branched-chain amino acids are transported by a common carrier system.     

Purification of branched-chain amino acid aminotransferase from <Emphasis Type="Italic">Helicobacter pylori</Emphasis> NCTC 11637

Summary. Branched-chain amino acid aminotransferase was purified by several column chromatographies from Helicobacter pylori NCTC 11637, and the N-terminal amino acid sequence was analyzed. The enzyme gene was sequenced based on a putative branched-chain amino acid aminotransferase gene, ilvE of H. pylori 26695, and the whole amino acid sequence was deduced from the nucleotide sequence. The enzyme existed in a homodimer with a calculated subunit molecular weight (MW) of 37,539 and an isoelectric point (pI) of 6.47. The enzyme showed high affinity to 2-oxoglutarate (K _m = 0.085 mM) and L-isoleucine (K _m = 0.34 mM), and V _max was 27.3 μmol/min/mg. The best substrate was found to be L-isoleucine followed by L-leucine and L-valine. No activity was shown toward the D-enantiomers of these amino acids. The optimal pH and temperature were pH 8.0 and 37 °C, respectively.     

Conversion of ammonia or urea into essential amino acids, L-leucine, L-valine, and L-isoleucine, using artificial cells containing an immobilized multienzyme system and dextran-NAD+. 2. Yeast alcohol dehydrogenase for coenzyme recycling.

Semipermeable nylon-polyethylenimine artificial cells containing leucine dehydrogenase (EC 1.4.1.9), alcohol dehydrogenase (EC 1.1.1.1), urease (EC 3.5.1.5), and dextran-NAD+ were prepared. Artificial cells could convert ammonia or urea into L-leucine, L-valine, and L-isoleucine. For batch conversion in 20.0 mM of ammonium acetate substrate solutions, in 2 h 0.2 ml of artificial cells could produce 4.48 mumol of L-leucine, 9.98 mumol of L-valine, or 5.96 mumol of L-isoleucine. The corresponding conversion ratios were 22.4, 49.9, and 29.8%. In 20.0 mM of urea substrate solutions, 13.71 mumol of L-leucine, 16.12 mumol of L-valine, or 13.44 mumol of L-isoleucine was produced and the conversion ratios were 68.6, 80.6, and 67.2%. The substrate specificity of leucine dehydrogenase for the reductive amination was determined. Of the three branched-chain amino acids produced, the production rates of L-valine were the highest. The apparent Km values were as follows: 0.32 mM for alpha-ketoisocaproate, 1.63 mM for alpha-ketoisovalerate, and 0.73 mM for Dl-alpha-keto-beta-methyl-n-valerate. The leucine dehydrogenase multienzyme system had a good storage stability. It retained 72.0% of the original activity with artificial cells were stored at 4 degrees C for 6 weeks. The optimum conversion pH and temperature were 8.5-9.0 and 35-40 degrees C. The effects of urea and ammonium salts on conversion rate were also studied. The relative activities in ammonium salts solutions were 45.1-75.9% of those in urea solutions.     

Functional differences in the catabolism of branched-chain L-amino acids in cultured normal and maple syrup urine disease fibroblasts

Possible functional differences in the catabolism of the four branched-chain L-amino acids in maple syrup urine disease were assessed using cultured human skin fibroblast stains. Transamination and oxidative decarboxylation were comparatively studied in 90-min incubations with 1 mmole/liter of 1-14C-labeled substrates. In normal cell strains (n = 5), apparent transamination rates (sum of branched-chain 2-oxo[14C]acid and 14CO2 release; means expressed in nmole/90 min/mg of cell protein) were in the order L-leucine (32) greater than L-valine (17) greater than or equal to L-isoleucine (14) greater than L-allo-isoleucine (8); 14CO2 production was in the order L-valine (9) greater than L-isoleucine (6) greater than or equal to L-leucine (5) greater than L-allo-isoleucine (2). In variant (n = 5) as well as classical (n = 2) MSUD cell lines, branched-chain 2-oxo-[14C]acid release rates were generally comparable to the control values. As compared to the 14CO2 release in controls (= 100%), branched-chain 2-oxo acid dehydrogenase activity in MSUD fibroblasts was individually reduced and varied considerably between strains (residual activity 2-38%). Within individual strains, only small differences in the residual decarboxylation activity were observed in incubations with L-valine, L-leucine, and L-isoleucine. It was remarkably high, however, when L-allo-isoleucine was applied as a substrate. With the exception of L-allo-isoleucine, apparent total transamination rates of branched-chain L-amino acids were therefore distinctly lower in MSUD cells than in normal cells.     

An enzymatic method for the estimation of L-leucine in rat blood.

A specific enzymatic method for the routine measurement of L-leucine in blood samples is presented. The method uses a commercial preparation of tRNAs and amino acetyl-tRNA synthetases for the specific loading of L-leucine into the tRNA(Leu) present, competing with carrier-free L-[U-14C]leucine. The radioimmunoassay-like plot of radioactivity found in the acid-insoluble (tRNA) fraction was used to determine the amount of unlabelled L-leucine of the samples when compared against a standard curve. The interference of L-isoleucine, L-valine and L-alanine was very low.     

Regulation of acetohydroxyacid synthetase in Bacillus subtilis

Summary In Bacillus subtilis, the activity of aceto hydroxyacid synthetase is inhibited by L-valine. The valine effect is antagonized by the simultaneous addition of L-isoleucine and L-leucine. Repression of enzyme formation required an excess of leucine and valine in the growth medium.     

Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of L-valine

Addition of L-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 DeltailvA DeltapanBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of the ilvB protein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations of L-isoleucine could relieve the valine effect on VAL1 whereas L-leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to the ilvA deletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids. Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ. Interestingly, addition of external valine stimulated valine production by VAL1. This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression of ilvBN, indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain.     

Glycine and L-Alanine as Antagonists of Growth Inhibition by the Branched-Chain Amino Acids in Spirodela polyrhiza

Growth inhibition of the duckweed Spirodela polyrhiza (L.) Schleidenby exogenously supplied L-leucine, L-valine, or L-isoleucinewas antagonized by simultaneously supplied glycine or L-alanine.Calculations, using the kinetic parameters for the uptake ofthese amino acids, indicated that the antagonisms to a considerabledegree resulted from competitive inhibition of the uptake ofthe growth-inhibiting amino acids. However, the antagonismsresulted partly from an interaction between growth inhibitorand antagonist inside the cells.     

Effects of amino acids on Thiobacillus acidophilus. I. Growth studies with special reference to valine.

The heterotrophic growth of Thiobacillus acidophilus was inhibited by branched-chain amino acids; valine, isoleucine, and leucine. The inhibition by valine and leucine were partially reversed by isoleucine, and the inhibition by isoleucine was partially reversed by valine. Inhibitions by methionine or threonine were partially reversed when both amino acids were present in the growth medium. Inhibition by tyrosine was increased by phenylalanine or tryptophan. Cystine completely inhibited growth. Other amino acids tested produced little or no inhibition. Acetohydroxy acid synthetase (AHAS) activity was demonstrated in crude extracts of T. acidophilus. In crude extracts the optimum pH was 8.5 with a shift to 9.0 in the presence of valine. Valine was the only branched-chain amino acid which inhibited the AHAS activity. The presence of only one peak of AHAS activity upon centrifugation in linear glycerol density gradients demonstrated that the AHAS activity sediments as one component.     

Repression and inhibition of transport systems for branched-chain amino acids in Salmonella typhimurium.

Kinetics of the transport systems common for entry of L-isoleucine, L-leucine, and L-valine in Salmonella typhimurium LT2 have been analyzed as a function of substrateconcentration in the range of 0.5 to 45 muM. The systems of transport mutants, KA203 (ilvT3) and KA204 (ilvT4), are composed of two components; apparent Km values for uptake of isoleucine, leucine, and valine by the low Km component are 2 muM, 2 to 3 muM, and 1 muM, respectively, and by the high Km component 30 muM, 20 to 40 muM, and 0.1 mM, respectively. The transport system(s) of the wild type has not been separated into components but rather displays single Km values of 9 muM for isoleucine, 10 muM for leucine, and 30 muM for valine. The transport activity of the wild type was repressed by L-leucine, alpha ketoisocaproate, glycyl-L-isoleucine, glycyl-L-leucine, and glycyl-L-methionine. That for the transport mutants was repressed by L-alanine, L-isoleucine, L-methionine, L-valine, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, glycyl-L-alanine, glycyl-L-threonine, and glycyl-L-valine, in addition to the compounds described above. Repression of the mutant transport systems resulted in disappearance of the low Km component for valine uptake, together with a decrease in Vmax of the high Km component; the kinetic analysis with isoleucine and leucine as substrates was not possible because of poor uptake. The maximum reduction of the transport activity for isoleucine was obtained after growing cells for two to three generations in a medium supplemented with repressor, and for the depression, protein synthesis was essential after removal of the repressor. The transport activity for labeled isoleucine in the transport mutant and wild-type strains was inhibited by unlabeled L-alanine, L-cysteine, L-isoleucine, L-leucine, L-methionine, L-threonine, and L-valine. D-Amino acids neither repressed nor inhibited the transport activity of cells for entry of isoleucine.     

Sulfonylurea-resistant mutants and natural tolerance of cyanobacteria

The herbicide sulfometuron methyl (SM) inhibited the growth of the cyanobacterium Synechococcus sp. PCC7942, but not of Synechocystis sp. PCC6714. The inhibitory effect was alleviated by the simultaneous addition of valine, leucine and isoleucine. SM resistant mutants were isolated from Synechococcus 7942, two types of which were further analysed. In these mutants, SM3/20 and SM2/32, the activity of acetolactate synthase (ALS) — a key enzyme in the biosynthesis of branched-chain amino acids —appeared 2600- and 300-fold, respectively, more resistant to SM than that of their wild type. Strain SM2/32 also exhibited a low level of ALS activity. Although the growth of the latter mutant was extremely inhibited by valine, the sensitivity of its ALS activity to feed-back inhibition by the amino acid was unaltered. At high concentrations valine inhibited growth of the wild type strains and of the mutant SM3/20. Isoleucine alleviated the valine-induced growth inhibition. Unlike that of Synechococcus 7942, the ALS activity of Synechocystis was found to tolerate high concentrations (100-fold) of the herbicide. The study confirms that the SM mutations are correlated with a cyanobacterial ilv gene.Abbreviations ALS acetolactate synthase; ile, isoleucine - leu leucine - NTG N-methyl-N-nitro-N-nitrosoguanidine - SM sulfometuron methyl - SM^r sulfometuron methyl resistant - val valine     

Studies on the regulation of leucine catabolism in the liver. Stimulation by pyruvate and dichloroacetate

The rate of oxidation of L-[1-14C]leucine to 14CO2 by isolated rat hepatocytes is increased by pyruvate and dichloroacetate. This effect is specific for L-leucine, not being observed for L-valine, L-isoleucine, or D-leucine. Transamination, the rate-limiting step of L-leucine catabolism in the liver, is the site of stimulation, because uptake of L-leucine by the cells and the oxidation of its transamination product, alpha-ketoisocaproate, are not increased. Measurement of steady state levels of alpha-ketoisocaproate indicate that both pyruvate and dichloroacetate promote the transamination of L-leucine, thereby increasing the availability of substrate for decarboxylation by the alpha-ketoisocaproate dehydrogenase complex (EC 1.2.4.3). Pyruvate stimulation of transamination is secondary to the provision of keto acid acceptors for the amino group of L-leucine. The mechanism of the effect of dichloroacetate remains unknown.     

In-vivo control of valine and leucine synthesis in the duckweed Spirodela polyrhiza

Duckweed colonies were grown on 1 l of nutrient solution supplied with 10 M l-[¹⁴C]leucine or with 25 M l-[¹⁴C]valine. Under these conditions the exogenously supplied amino acid did not inhibit growth, but caused in the plants a moderately increased pool of that amino acid, which remained essentially constant during the culture period. The effect of the increased pool of valine or leucine on the biosynthesis of these amino acids was determined from isotope dilution in the protein-bound valine and-or leucine. An increase in the leucine pool from 1.1 to 5.0 nmol mg^–1 dry weight resulted in a 21% reduction of metabolite flow through the common part of the valine-leucine biosynthetic pathway; leucine synthesis was reduced by 35%, but valine synthesis by only 5% and isoleucine synthesis was apparently unaffected. An increase in the valine pool from 3.2 to 6.6 nmol mg^–1 dry weight reduced the metabolite flow through the valine-leucine pathway by 48%, valine synthesis by 70%, and leucine synthesis from pyruvate by 29%, which was compensated by leucine synthesis from exogenous valine, whereas the synthesis of isoleucine was not changed. It is concluded that the biosynthesis of valine and leucine is mainly controlled by feedback inhibition of acetohydroxyacid synthetase. In vivo, the feedback inhibition can be exerted in such a way that synthesis of acetolactate (the precursor of valine and leucine) is appreciably reduced, whereas synthesis of acetohydroxybutyrate (the isoleucine precursor) is not inhibited.     

Purification of an inducible L-valine dehydrogenase of Streptomyces coelicolor A3(2)

Valine dehydrogenase (VDH) from Streptomyces coelicolor A3(2) was purified from cell-free extracts to apparent homogeneity. The enzyme had an Mr 41,000 in denaturing conditions and an Mr 70,000 by gel filtration chromatography, indicating that it is composed of two identical subunits. It oxidized L-valine and L-alpha-aminobutyric acid efficiently, L-isoleucine and L-leucine less efficiently, and did not act on D-valine. It required NAD+ as cofactor and could not use NADP+. Maximum dehydrogenase activity with valine was at pH 10.5 and the maximum reductive amination activity with 2-oxoisovaleric acid and NH4Cl was at pH 9. The enzyme exhibited substrate inhibition in the forward direction and a kinetic pattern with NAD+ that was consistent with a sequential ordered mechanism with non-competitive inhibition by valine. The following Michaelis constants were calculated from these data: L-valine, 10.0 mM; NAD+, 0.17 mM; 2-oxoisovalerate, 0.6 mM; and NADH, 0.093 mM. In minimal medium, VDH activity was repressed in the presence of glucose and NH4+, or glycerol and NH4+ or asparagine, and was induced by D- and L-valine. The time required for full induction was about 24 h and the level of induction was 2- to 23-fold.     

E.S.R. of spin-trapped radicals in gamma-irradiated polycrystalline amino acids. Chromatographic separation of radicals

The free radicals produced by gamma-radiolysis of polycrystalline amino acids (L-valine, L-leucine, L-isoleucine and L-proline) at room temperature in the absence of air were investigated by spin trapping with 2-methyl-2-nitrosopropane (MNP). The spin adducts produced by dissolving the irradiated solids in aqueous MNP solutions were separated by high-performance liquid chromatography and then identified by e.s.r. Deamination (ring-opening reaction for L-proline) was observed for all amino acid. For L-valine and L-leucine, H-abstraction from the tertiary carbon in the side chains occurred. For isoleucine, H-abstractions from the alpha-carbon of the amino acid and from a non-terminal carbon in the side chain were found.     

The sulfonylurea herbicide sulfometuron methyl is an extremely potent and selective inhibitor of acetolactate synthase in Salmonella typhimurium

The sulfonylurea herbicide sulfometuron methyl inhibits the growth of several bacterial species. In the presence of L-valine, sulfometuron methyl inhibits Salmonella typhimurium, this inhibition can be reversed by L-isoleucine. Reversal of growth retardation by L-isoleucine, accumulation of guanosine 5'-diphosphate 3'-diphosphate (magic spot), and relA mutant hypersensitivity suggest sulfometuron methyl interference with branched-chain amino acid biosynthesis. Growth inhibition of S. typhimurium is mediated by sulfometuron methyl's inhibition of acetolactate synthase, the first common enzyme in the branched-chain amino acid biosynthetic pathway. Sulfometuron methyl exhibits slow-binding inhibition of acetolactate synthase isozyme II from S. typhimurium with an initial Ki of 660 +/- 60 nM and a final, steady-state Ki of 65 +/- 25 nM. Inhibition of acetolactate synthase by sulfometuron methyl is substantially more rapid (10 times) in the presence of pyruvate with a maximal first-order rate constant for conversion from initial to final steady-state inhibition of 0.25 +/- 0.07 min-1 (minimal half-time of 2.8 min). Mutants of S. typhimurium able to grow in the presence of sulfometuron methyl were obtained. They have acetolactate synthase activity that is insensitive to sulfometuron methyl because of mutations in or near ilvG, the structural gene for acetolactate synthase isozyme II.     

Interconversion of valine and leucine by Clostridium sporogenes.

Clostridium sporogenes has been found to require L-leucine and L-valine for growth in a minimal medium, although valine can be replaced by isobutyrate and leucine by isovalerate. Cells grown in minimal media incorporated significant 14C from [14C]valine into leucine and from [14C]leucine into valine. Growth with [4,5-3H]leucine also resulted in the incorporation of 3H into valine. These results indicate that these bacteria can interconvert leucine and valine.     

Deregulated branched-chain amino acid synthesis in a Nicotiana plumbaginifolia cell line resistant to valine

A Nicotiana plumbaginifolia cell line able to grow in the presence of high doses of valine was isolated following -rays mutagenesis. The selected clone, named D5R5, showed a growth rate higher than that of wild-type. It was less sensitive also to an equimolar mixture of the three branched-chain amino acids, but did not display cross-resistance to isoleucine and leucine. The increased tolerance was due to neither a reduced valine uptake, nor a modification in the level or sensitivity to feed-back inhibition by valine of the first common enzyme (and the main regulative site) in isoleucine, leucine and valine synthesis, acetohydroxyacid synthase (AHAS). When wild-type cells were fed with valine or equimolar mixtures of the three aminoacids, a decrease in AHAS level was found. On the contrary, the level of extractable AHAS activity from D5R5 cells was significantly less affected by similar treatments, suggesting that some alteration in enzyme modulation mechanism(s) could account for valine resistance.Abbreviations AHAS acetohydroxyacid synthase - BCAA branched-chain amino acid - FAD flavin adenine dinucleotide - ILV equimolar mixture of isoleucine, leucine and valine - TPP thiamine pyrophosphate