Pathway Of Branched-chain Amino Acids Research Articles

Leucine biosynthesis in fungi: entering metabolism through the back door.

After exploring evolutionary aspects of branched-chain amino acid biosynthesis, the review focuses on the extended leucine biosynthetic pathway as it operates in Saccharomyces cerevisiae. First, the genes and enzymes specific for the leucine pathway are considered: LEU4 and LEU9 (encoding the alpha-isopropylmalate synthase isoenzymes), LEU1 (isopropylmalate isomerase), and LEU2 (beta-isopropylmalate dehydrogenase). Emphasis is given to the unusual distribution of the branched-chain amino acid pathway enzymes between mitochondrial matrix and cytosol, on the newly defined role of Leu5p, and on regulatory mechanisms governing gene expression and enzyme activity, including new evidence for the metabolic importance of the regulation of alpha-isopropylmalate synthase by coenzyme A. Next, structure-function relationships of the transcriptional regulator Leu3p are addressed, defining its dual role as activator and repressor and discussing evidence in support of the self-masking model. Recent data pointing at a more extended Leu3p regulon are discussed. An overview of the layered controls of the extended leucine pathway is provided that includes a description of the newly recognized roles of Ilv5p and Bat1p in maintaining mitochondrial integrity. Finally, branched-chain amino acid biosynthesis and its regulation in other fungi are summarized, the question of leucine as metabolic signal is addressed, and possible directions of future research in this area are outlined.

Crystallization and preliminary X-ray crystallographic analysis of acetohydroxy acid isomeroreductase from Pseudomonas aeruginosa.

Acetohydroxy acid isomeroreductase (AHIR) is involved in the biosynthetic pathway of branched-chain amino acids in microorganisms and plants. AHIR from Pseudomonas aeruginosa has been overexpressed in Escherichia coli and crystallized at 297 K using potassium/sodium tartrate as a precipitant. X-ray diffraction data have been collected to 2.0 A resolution at 100 K using synchrotron radiation. The crystals belong to the cubic space group P2(1)3, with unit-cell parameters a = b = c = 184.38 A, alpha = beta = gamma = 90 degrees. Six monomers are present in the asymmetric unit, giving a V(M) of 2.34 A(3) Da(-1) and a solvent content of 47.4%.

Isolation and Characterization of cDNA Clones for the E1β and E2 Subunits of the Branched-chain α-Ketoacid Dehydrogenase Complex in Arabidopsis

Branched-chain alpha-ketoacid dehydrogenase (BCKDH) has been known in mammals to be a key enzyme of the catabolic pathway of branched-chain amino acids. We have isolated two cDNA clones encoding the E1beta and E2 subunits of BCKDH, respectively, from Arabidopsis thaliana. Proteins encoded in these cDNA sequences had putative mitochondrial targeting sequences and conserved domains reported for their mammalian counterparts. Northern blot and immunoblot analyses showed that transcripts from the respective genes and E2 protein markedly accumulated in leaves kept in the dark. We found that the activity of BCKDH in the leaf extracts also increased when plants were placed in the dark. Addition of sucrose to detached leaves inhibited the accumulation of transcripts, whereas application of a photosynthesis inhibitor strongly induced the expression of these genes even under light illumination. These observations indicate that the cellular sugar level is likely responsible for the dark-induced expression of these genes. The transcript levels of these genes were also high in senescing leaves, in which photosynthetic activity is low and free amino acids from degraded protein are likely to serve as an alternative energy source.

Primary metabolism and its control in streptomycetes: A most unusual group of bacteria

Streptomycetes are Gram-positive bacteria with a unique capacity for the production of a multitude of varied and complex secondary metabolites. They also have a complex life cycle including differentiation into at least three distinct cell types. Whilst much attention has been paid to the pathways and regulation of secondary metabolism, less has been paid to the pathways and the regulation of primary metabolism, which supplies the precursors. With the imminent completion of the total genome sequence of Streptomyces coelicolor A3(2), we need to understand the pathways of primary metabolism if we are to understand the role of newly discovered genes. This review is written as a contribution to supplying these wants. Streptomycetes inhabit soil, which, because of the high numbers of microbial competitors, is an oligotrophic environment. Soil nutrient levels reflect the fact that plant-derived material is the main nutrient input; i.e. it is carbon-rich and nitrogen- and phosphate-poor. Control of streptomycete primary metabolism reflects the nutrient availability. The variety and multiplicity of carbohydrate catabolic pathways reflects the variety and multiplicity of carbohydrates in the soil. This multiplicity of pathways has led to investment by streptomycetes in pathway-specific and global regulatory networks such as glucose repression. The mechanism of glucose repression is clearly different from that in other bacteria. Streptomycetes feed by secreting complexes of extracellular enzymes that break down plant cell walls to release nutrients. The induction of these enzyme complexes is often coordinated by inducers that bear no structural relation to the substrate or product of any particular enzyme in the complex; e.g. a product of xylan breakdown may induce cellulase production. Control of amino acid catabolism reflects the relative absence of nitrogen catabolites in soil. The cognate amino acid induces about half of the catabolic pathways and half are constitutive. There are reduced instances of global carbon and nitrogen catabolite control of amino acid catabolism, which again presumably reflects the relative rarity of the catabolites. There are few examples of feedback repression of amino acid biosynthesis. Again this is taken as a reflection of the oligotrophic nature of the streptomycete ecological niche. As amino acids are not present in the environment, streptomycetes have rarely invested in feedback repression. Exceptions to this generalization are the arginine and branched-chain amino acid pathways and some parts of the aromatic amino acid pathways which have regulatory systems similar to Escherichia coli and Bacillus subtilis and other copiotrophic bacteria.

Identification of QTLs controlling acylsugar fatty acid composition in an intraspecific population of Lycopersicon pennellii (Corr.) D’Arcy

Acylsugars exuded by type IV glandular trichomes are responsible for insect resistances found in many Lycopersicon pennellii accessions. Acylsugars are complex mixtures composed of polyacylated sugars (glucose or sucrose) esterified to branched and straight-chain 4 : 0 to 12 : 0 fatty acids. The biogeneses of these unusual fatty acid constituents have their origins in branched-chain amino acid pathways. However, the mechanism of fatty acid elongation in these systems and the genetic control of carbon flux from amino acid to fatty acid pathways remain unclear. In this study, we used an intraspecific F2 population derived from the cross between L. pennellii LA716 and L. pennellii LA1912 to examine the genetic basis of acylsugar fatty acid composition. Six QTLs were detected which, combined, explain 23–60% of the variance observed for each of the nine segregating fatty acid constituents. Both correlation data and QTL analysis data indicate that branched medium-chain fatty acids are synthesized through elongation of short-chain precursors in two-carbon increments. The proportion of iso-branched acylsugar fatty acids that have an even-carbon chain length was found to be primarily determined by a single locus that maps to a location 5.5 cM above TG117 on chromosome 8. QTL function in several cases can be inferred from discrete patterns of fatty acid composition; in other cases, control of acylsugar fatty acid composition appears to be complex.

In plants a putative isovaleryl-CoA-dehydrogenase is located in mitochondria.

In plants the degradation pathways of branched-chain amino acids have remained somewhat unclear with respect to both their biochemistry and their intracellular location. While biochemical evidence has localized some of the catabolic enzymes in peroxisomes/glyoxysomes, others cofractionate with mitochondria. We have now identified a candidate protein and corresponding cDNA for an enzyme of the leucine catabolic pathway, the isovaleryl-CoA-dehydrogenase (IVD). This polypeptide is a member of the acyl-CoA-dehydrogenase (ACDH) family and is encoded in the nuclear genome of Arabidopsis thaliana. Expression of the putative IVD gene in pea seedlings is documented by western blot analyses with an antibody against the mammalian IVD. Subcellular fractionation identifies the putative IVD enzyme in the mitochondrion. This localization suggests that in plants mitochondria contain at least part of the branched-chain amino acid degradation pathway(s).

Growth inhibition of pollen tubes by blocking the aromatic or branched-chain amino acid pathways of biosynthesis

Growth inhibition of pollen tubes by blocking the aromatic or branched-chain amino acid pathways of biosynthesis

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

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.

Mode of Growth Inhibitory Activity and Metabolism of Imazosulfuron in Excised Roots of Plants

Growth inhibition activity of imazosulfuron, 1-(2-chloroimidazo[1, 2-a] pyridin-3-ylsulfonyl)-3-(4, 6-dimethoxypyrimidin-2-yl)urea, against excised pea and soybean roots was investigated. Fifty percents growth inhibition concentrations of imazosulfuron (I 50 ) against pea and soybean roots were 17.6 and 54.2 ppb, respectively. When the excised pea roots were incubated in the culture medium containing 25 ppb of imazosulfuron and 1 mM of either branched-chain amino acids or intermediates of their biosynthetic pathways, the growth inhibition activity of excised roots was alleviated by the combination of isoleucine and valine or α-ketoisovalerate and α-keto-β-methyl-n-valerate. These findings suggest that imazosulfuron exhibits inhibition of acetolactate synthase which catalyses the biosynthetic pathways of branched-chain amino acids. Metabolic fate of imazosulfuron in excised pea roots was studied using the 14 C-labeled compounds. Imazosulfuron was predominantly demethylated to afford a monodemethyl derivative (HMS) in the roots, and hydrolytically cleaved at the sulfonylurea bond to give a sulfonamide (IPSN) and an aminopyrimidine (ADPM) in the culture medium. Since HMS did not inhibit the growth of excised roots, metabolic fate of imazosulfuron is involved in a selectivity between plants.

Isolation and kinetic properties of acetohydroxy acid isomeroreductase from spinach (Spinacia oleracea) chloroplasts overexpressed in Escherichia coli.

Acetohydroxy acid isomeroreductase catalyses a two-step reaction, an alkyl migration and a NADPH-dependent reduction, in the assembly of the carbon skeletons of branched-chain amino acids. Detailed investigations of acetohydroxy acid isomeroreductase aimed at elucidating the biosynthetic pathway of branched-chain amino acids and at designing new inhibitors of the enzyme having herbicidal potency have so far been conducted with the enzymes isolated from bacteria. To gain more information on a plant system, the gene encoding the mature acetohydroxy acid isomeroreductase from spinach (Spinacia oleracea) leaf chloroplasts has been used to transform Escherichia coli cells and to overexpress the enzyme. A rapid protocol is described that allows the preparation of large quantities of pure spinach chloroplast acetohydroxy acid isomeroreductase. Kinetic and structural properties of the plant enzyme expressed in Escherichia coli are compared with those reported in our previous studies on the native enzymes purified from spinach chloroplasts and with those reported for the corresponding enzymes isolated from Escherichia coli and Salmonella typhimurium. Both the plant and the bacterial enzymes obey an ordered mechanism in which NADPH binds first, followed by substrate (either 2-acetolactate or 2-aceto-2-hydroxybutyrate). Inhibition studies employing an inactive substrate analogue, 2-hydroxy-2-methyl-3-oxopentanoate, showed, however, that the binding of 2-hydroxy-2-methyl-3-oxopentanoate and NADPH occurs randomly, suggestive of some flexibility of the plant enzyme active site. The observed preference of the enzyme for 2-aceto-2-hydroxybutyrate over 2-acetolactate is discussed with regard to the contribution of acetohydroxy acid isomeroreductase activity in the partitioning between isoleucine and valine biosyntheses. Moreover, the kinetic properties of the chloroplast enzyme support the notion that biosynthesis of branched-chain amino acids in plants is controlled by light. As judged by analytical-ultracentrifugation and gel-filtration analyses the overexpressed plant enzyme is a dimer of identical subunits.

Modified branched‐chain amino acid pathways give rise to acyl acids of sucrose esters exuded from tobacco leaf trichomes

A major diversion of carbon from branched-chain amino acid biosynthesis/catabolism to form acyl moieties of sucrose esters (6-O-acetyl-2,3,4-tri-O-acyl-alpha-D-glucopyranosyl-beta-D- fructofuranosides) was observed to be associated with specialized trichome head cells which secrete large amounts of sucrose esters. Surface chemistry and acetyl and acyl substituent groups of tobacco (T.I. 1068) sucrose esters were identified and quantified by gas chromatography/mass spectrometry. Sucrose esters were prominent surface constituents and 3-methylvaleric acid, 2- and 3-methylbutyric acid, and methylpropionic acid accounted for 60%, 25% and 9%, respectively, of total C3--C7 acyl substituents. Radiolabeled Thr, Ile, Val, Leu, pyruvate and Asp, metabolites of branched-chain amino acid pathways, were compared with radioactively labeled acetate and sucrose as donors of carbon to sucrose, acetyl and acyl components of sucrose esters using epidermal peels with undisturbed trichomes. Preparations of biosynthetically competent trichome heads (site of sucrose ester formation) were also examined. Results indicate that 3-methylvaleryl and 2-methylbutyryl groups are derived from the Thr pathway of branched-chain amino acid metabolism, 3-methylbutyryl and methylpropionyl groups are formed via the pyruvate pathway, and that acetyl groups are principally formed directly via acetyl-CoA. Arguments are presented which rule out participation of fatty acid synthase in the formation of prominent acyl acids. Results suggest that the shunting of carbon away from the biosynthesis of Val, Leu and Ile may be due to a low level of amino acid utilization in protein synthesis in specialized glandular head cells of trichomes. This would result in the availability of corresponding oxo acids for CoA activation and esterification to form sucrose esters. Preliminary evidence was found for the involvement of cycling reactions in oxo-acid-chain lengthening and for utilization of pyruvate-derived 2-oxobutyrate to form straight-chain acyl substituents.

Isolation and Characterization of Cell Lines of Valeriana wallichii Resistant to Trifluoroleucine

Two cell lines of Valeriana wallichii DC resistant to the leucine analogue 5′,5′,5′-trifluoro-D,L-leucine have been isolated. The two lines (V125, V 500) accumulated higher amounts of aspartic acid (up to 5 fold) and glutamic acid (up to 57 fold) compared to the initial cultures; furthermore, V 500 accumulated comparatively high amounts of valine (R∼15 fold) and leucine (∼ 37 fold). The activity of acetohydroxyacid-synthetase, the key enzyme in the biosynthetic pathway of branched-chain amino acids, was little affected by these amino acids in V 500. In V 125 the enzyme showed no change in feedback control properties. While in the literature there is some evidence that leucine degradation products are incorporated into terpenoid compounds, the high pool of leucine in V 500 did not induce a higher accumulation of terpenoid derived compounds such as valepotriates, volatile terpenes and sterols.

Biosynthesis of branched-chain amino acids in Schizosaccharomyces pombe: regulation of the enzymes involved in isoleucine, valine, and leucine synthesis.

The activities and regulation of the enzymes of the synthetic pathway of branched-chain amino acids were investigated in the fission yeast, Schizosaccharomyces pombe. Previous studies had shown the presence of threonine deaminase (TD) and acetohydroxy acid synthetase (AHAS). The remaining isoleucine–valine enzymes, isomeroreductase (IR), dehydrase, and transaminase B, have now been characterized in cell-free extracts, indicating the presence in this yeast of the complete pathway as demonstrated in other microorganisms. α-Isopropylmalate synthetase (IPMS), the first enzyme of the leucine pathway, has properties of a typical regulatory enzyme; it is most active in the pH range 7.5–8.5, but is most sensitive to feedback inhibition by L-leucine at pH 6.5–7.0. Unlike the situation in baker's yeast, only AHAS and IR appeared to be subject to multivalent repression. TD was relatively resistant to any change in level, and AHAS was repressible by valine included in the growth medium. IPMS was repressed when cells were grown in complex medium; leucine alone did not cause repression, and in contrast with baker's yeast, neither did leucine plus threonine or a combination of all three branched-chain amino acids.

New amino acid regulatory locus having unusual properties in heterozygous merodiploids.

Spontaneous mutants of Escherichia coli B/r resistant to 5',5',5',-trifluoro-dl-leucine contain defects in a gene which maps to the left of the threonine region. Low-level constitutive expression of the isoleucine-valine and leucine operons is caused by this mutation in haploid strains. This is in contrast to extremely high levels of gene expression in the heterozygous merodiploids (F' wild type/mutant allele). The properties of these mutants define a new locus and suggest that it encodes a subunit protein which is involved in the repression of the structural genes for the branched-chain amino acid pathways.