Biosynthesis of Branched-Chain Amino Acids in Yeast: Regulation of Synthesis of the Enzymes of Isoleucine and Valine Biosynthesis

Regulation of the levels of the five enzymes required for the biosynthesis of isoleucine and valine was studied in a Saccharomyces sp. When a mixture of isoleucine, valine, and leucine was added to the medium, the enzymes in the wild-type strain were repressed from about 30% (transaminase B) to about 90% (acetohydroxy acid synthetase) relative to the level in minimal medium-grown cells. Repression was also observed when threonine replaced isoleucine in the mixture but not when it replaced the other two amino acids. Significant derepression relative to the level in minimal-grown cells was not obtained by growing suitably blocked auxotrophs on medium containing limiting amounts of valine, isoleucine, or leucine.     

Multivalent Repression of Isoleucine-Valine Biosynthesis in Saccharomyces cerevisiae

Regulation of the biosynthesis of four of the five enzymes of the isoleucine-valine pathway was studied in Saccharomyces cerevisiae. A method is described for limiting the growth of a leucine auxotroph by using valine as a competitor for the permease. Limitation for isoleucine and valine was accomplished by the use of peptides containing these amino acids conjugated with glycine as nutritional supplements for auxotrophs. The enzymes were repressed on synthetic medium containing isoleucine, valine, and leucine, as well as on broth supplemented with these amino acids. Limitation for any of the three branched-chain amino acids led to derepression of the isoleucine-valine biosynthetic pathway. Maximal derepression ranged from 3-fold for threonine deaminase to approximately 10-fold for acetohydroxyacid synthase. (Two of the enzymes, acetohydroxyacid synthase and dihydroxyacid dehydrase, may be controlled by a mechanism different from that regulating threonine deaminase.) Possible molecular mechanisms for multivalent repression are discussed.     

O-Methylthreonine Inhibition of Growth and of Threonine Deaminase in Escherichia coli

O-methylthreonine (OMT), an isosteric analogue of isoleucine, markedly inhibited growth of Escherichia coli 15. This inhibition was overcome most effectively by addition of isoleucine, valine, or leucine to the medium and less effectively by addition of threonine. The dipeptide, valylleucine, also relieved the OMT-induced inhibition but only after a lag period, suggesting that valine and leucine, liberated by dipeptidase action, compete with OMT for entry into the cell. OMT was activated and transferred to transfer ribonucleic acid (RNA) by isoleucyl-RNA synthetase in vitro. The rate of OMT incorporation into protein of intact cells was comparable to that of isoleucine. In contrast to isoleucine, very high concentrations of OMT were required to inhibit threonine deaminase, and the inhibition was strictly competitive with threonine. In addition, OMT inhibited a threonine deaminase preparation desensitized to isoleucine inhibition.     

Regulation of the formation of isoleucyl-tRNA-synthetase and the level of isoleucine biosynthetic enzymes in E. coli K12

Summary During derepression of threonine deaminase and acetolactate synthetase due to valine deficiency—initiated by -aminobutyric acid limited growth of E. coli K12 or by limited valine supply to an ilv/leu auxotroph of E. coli K12—no alteration of the specific activity of isoleucyl-tRNA-synthetase occurs. Leucine limited growth of the auxotroph, leading to an even higher derepression of the isoleucine biosynthetic enzymes, also does not affect the specific activity of isoleucyl-tRNA-synthetase. However, under growth conditions where the same degree of derepression of threonine deaminase is due to isoleucine deficiency, as in E. coli K12B or two valine resistant mutants thereof grown in the presence of valine, or in the auxotroph during growth-limiting isoleucine supply, a specific two- to three-fold derepression of the isoleucyl-tRNA-synthetase takes place. But there is no strict correlation between the degree of derepression of threonine deaminase due to isoleucine deficiency and the degree of derepression of isoleucyl-tRNA-synthetase, as especially shown in case of the valine resistant mutant Val R4 and Val R5 grown in the presence of valine.These results demonstrate that the rate of formation of isoleucyl-tRNA-synthetase and of threonine deaminase are not regulated by the same molecular devices and that a certain degree of isoleucine deficiency is a prerequisite for a derepression of isoleucyl-tRNA-synthetase.     

Involvement of threonine deaminase in repression of the isoleucine-valine and leucine pathways in Saccharomyces cerevisiae

l-Threonine deaminase (l-threonine dehydratase [deaminating], EC 4.2.2.16) has been shown to be involved in the regulation of three of the enzymes of isoleucine-valine biosynthesis in yeast. Mutations affecting the affinity of the enzyme for isoleucine also affected the repression of acetohydroxyacid synthase, dihydroxyacid dehydrase, and reductoisomerase. The data indicate that isoleucine must be bound for effective repression of these enzymes to take place. In a strain with a nonsense mutation midway in liv 1, the gene for threonine deaminase, starvation for isoleucine or valine did not lead to derepression of the three enzymes; starvation for leucine did. The effect of the nonsense mutation is recessive; it is tentatively concluded, therefore, that intact threonine deaminase is required for derepression by two of the effectors for multivalent repression, but not by the third. A model is presented which proposes that a regulatory species of leu tRNA(leu) is the key intermediate for repression and that threonine deaminase is a positive element, regulating the available pool of charged leu tRNA by binding it.     

Regulation of branched-chain amino acid transport in Escherichia coli.

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine, biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyl-transfer ribonucleic acid synthetase was also required for repression of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed.     

An efficient approach to identify ilvA mutations reveals an amino-terminal catalytic domain in biosynthetic threonine deaminase from Escherichia coli.

High-level expression of the regulatory enzyme threonine deaminase in Escherichia coli strains grown on minimal medium that are deficient in the activities of enzymes needed for branched-chain amino acid biosynthesis result in growth inhibition, possibly because of the accumulation of toxic levels of alpha-ketobutyrate, the product of the committed step in isoleucine biosynthesis. This condition affords a means for selecting genetic variants of threonine deaminase that are deficient in catalysis by suppression of growth inhibition. Strains harboring mutations in ilvA that decreased the catalytic activity of threonine deaminase were found to grow more rapidly than isogenic strains containing wild-type ilvA. Modification of the ilvA gene to introduce additional unique, evenly spaced restriction enzyme sites facilitated the identification of suppressor mutations by enabling small DNA fragments to be subcloned for sequencing. The 10 mutations identified in ilvA code for enzymes with significantly reduced activity relative to that of wild-type threonine deaminase. Values for their specific activities range from 40% of that displayed by wild-type enzyme to complete inactivation as evidenced by failure to complement an ilvA deletion strain to isoleucine prototrophy. Moreover, some mutant enzymes showed altered allosteric properties with respect to valine activation and isoleucine inhibition. The location of the 10 mutations in the 5' two-thirds of the ilvA gene is consistent with suggestions that threonine deaminase is organized functionally with an amino-terminal domain that is involved in catalysis and a carboxy-terminal domain that is important for regulation.     

Enhancement of isoleucine hydroxamate-mediated growth inhibition and improvement of isoleucine-producing strains of Serratia marcescens.

Growth inhibition by isoleucine hydroxamate in Serratia marcescens was significantly enhanced by adding valine plus leucine and by using glycerol as the carbon source. Isoleucine hydroxamate-resistant mutants were isolated under conditions in which growth inhibition was enhanced. One of the mutants, strain GIHVLr2179, lacked both feedback inhibition and repression of threonine deaminase. An alpha-aminobutyric acid-resistant mutant derived from strain GIHVLr2179, strain GIHVLAr2795, produced 12 mg of isoleucine per ml in the medium containing glucose and urea as carbon and nitrogen sources (a twofold increase over prior reports). This strain had increased activities of threonine deaminase, acetohydroxy acid synthase, aspartokinase, and homoserine dehydrogenase.     

Enhancement of isoleucine hydroxamate-mediated growth inhibition and improvement of isoleucine-producing strains of Serratia marcescens.

Growth inhibition by isoleucine hydroxamate in Serratia marcescens was significantly enhanced by adding valine plus leucine and by using glycerol as the carbon source. Isoleucine hydroxamate-resistant mutants were isolated under conditions in which growth inhibition was enhanced. One of the mutants, strain GIHVLr2179, lacked both feedback inhibition and repression of threonine deaminase. An alpha-aminobutyric acid-resistant mutant derived from strain GIHVLr2179, strain GIHVLAr2795, produced 12 mg of isoleucine per ml in the medium containing glucose and urea as carbon and nitrogen sources (a twofold increase over prior reports). This strain had increased activities of threonine deaminase, acetohydroxy acid synthase, aspartokinase, and homoserine dehydrogenase.     

Isoleucine and valine metabolism in Escherichia coli. XIX. Inhibition of isoleucine biosynthesis by glycyl-leucine

The inhibition of growth of the K-12 strain of Escherichia coli by glycyl-l-leucine observed originally by Simmonds and co-workers was investigated. The inhibition was reversed by isoleucine and those precursors of isoleucine beyond threonine in the biosynthetic pathway. Threonine reversed the inhibition poorly. With heavy cell suspensions, the inhibition was transient: the onset of growth followed the disappearance of the dipeptide from the medium and the appearance of glycine and leucine. Glycyl-leucine was shown to be an inhibitor of threonine deaminase (EC 4.2.1.16 l-threonine hydro-lyase [deaminating]). One kind of glycyl-leucine-resistant mutant had a threonine deaminase that was resistant to isoleucine and glycyl-leucine inhibition. The pattern of glycyl-leucine inhibition is compared with those of inhibition by isoleucine and by the weaker inhibitors leucine and valine.     

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.     

Isoleucine and valine metabolism in Escherichia coli. XXI. Mutations affecting derepression and valine resistance

The activity of acetohydroxy acid isomeroreductase, an essential enzyme for isoleucine and valine biosynthesis in Escherichia coli, was examined in a series of mutants containing derepressed levels of acetohydroxy acid synthetase activity but which differed from each other in the sensitivity of the synthetases to valine inhibition. The finding that isomeroreductase was highest in the strain with the synthetase that was least sensitive to valine inhibition supported the model of internal induction of the isomeroreductase by its acetohydroxy acid substrates. The mutation leading to the acetohydroxy acid synthetase least sensitive to valine was found to be unlinked to the ilv gene cluster and appeared to result in a synthetase that differed from the normal enzyme in several properties. The locus of this mutation is designated ilvF. The loci leading to derepression were designated azl. A pleiotropic, apparently single-step, mutation was found that led to restoration of end-product sensitivity to the synthetase, loss of end-product sensitivity of threonine deaminase [EC 4.2.1.16, l-threonine hydro-lyase (deaminating) and loss of isomeroreductase activity.     

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.     

Cysteine and growth inhibition of Escherichia coli: threonine deaminase as the target enzyme.

Cysteine has been shown to inhibit growth in Escherichia coli strains C6 and HfrH 72, but not M108A. Growth inhibition was overcome by inclusion of isoleucine, leucine, and valine in the medium. Isoleucine biosynthesis was apparently affected, since addition of this amino acid alone could alter the inhibitory effects of cysteine. Homocysteine, mercaptoethylamine, and mercaptoethanol inhibited growth to varying degrees in some strains, these effects also being prevented by addition of branched-chain amino acids. Cysteine, mercaptoethylamine, and homocysteine were inhibitors of threonine deaminase but not transaminase B, two enzymes of the ilvEDA operon. Cysteine inhibition of threonine deaminase was reversed by threonine, although the pattern of inhibition was mixed. These results suggest a relationship between the growth-inhibitory effects of cysteine and other sulfur compounds and the inhibition of isoleucine synthesis at the level of threonine deaminase.     

Multivalent repression and genetic depression of isoleucine-valine biosynthetic enzymes in Serratia marcescens

The regulation of the formation of isoleucine-valine biosynthetic enzymes was examined to elucidate the mechanism of isoleucine-valine accumulation by alpha-aminobutyric acid-resistant (abu-r) mutants of Serratia marcescens. In the isoleucine-valine auxotroph, l-threonine dehydratase, acetohydroxy acid synthetase, and transaminase B were repressed when isoleucine, valine, and leucine were simultaneously added to minimal medium. These enzymes were derepressed at the limitation of any single branched-chain amino acid. Pantothenate, which stimulated growth of this auxotroph, had no effect on the enzyme levels. It became evident from these results that in S. marcescens isoleucine-valine biosynthetic enzymes are subject to multivalent repression by three branched-chain amino acids. The abu-r mutants had high enzyme levels in minimal medium, with or without three branched-chain amino acids. Therefore, in abu-r mutants, isoleucine-valine biosynthetic enzymes are genetically derepressed. This derepression was considered to be the primary cause for valine accumulation and increased isoleucine accumulation.     

Separate regulation of transport and biosynthesis of leucine, isoleucine, and valine in bacteria.

Since both transport activity and the leucine biosynthetic enzymes are repressed by growth on leucine, the regulation of leucine, isoleucine, and valine biosynthetic enzymes was examined in Escherichia coli K-12 strain EO312, a constitutively derepressed branched-chain amino acid transport mutant, to determine if the transport derepression affected the biosynthetic enzymes. Neither the iluB gene product, acetohydroxy acid synthetase (acetolactate synthetase, EC 4.1.3.18), NOR THE LEUB gene product, 3-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate-nicotinamide adenine dinucleotide oxido-reductase, EC 1.1.1.85), were significantly affected in their level of derepression or repression compared to the parental strain. A number of strains with alterations in the regulation of the branched-chain amino acid biosynthetic enzymes were examined for the regulation of the shock-sensitive transport system for these amino acids (LIV-I). When transport activity was examined in strains with mutations leading to derepression of the iluB, iluADE, and leuABCD gene clusters, the regulation of the LIV-I transport system was found to be normal. The regulation of transport in an E. coli strain B/r with a deletion of the entire leucine biosynthetic operon was normal, indicating none of the gene products of this operon are required for regulation of transport. Salmonella typhimurium LT2 strain leu-500, a single-site mutation affecting both promotor-like and operator-like function of the leuABCD gene cluster, also had normal regulation of the LIV-I transport system. All of the strains contained leucine-specific transport activity, which was also repressed by growth in media containing leucine, isoleucine and valine. The concentrated shock fluids from these strains grown in minimal medium or with excess leucine, isoleucine, and valine were examined for proteins with leucine-binding activity, and the levels of these proteins were found to be regulated normally. It appears that the branched-chain amino acid transport systems and biosynthetic enzymes in E. coli strains K-12 and B/r and in S. typhimurium strain LT2 are not regulated together by a cis-dominate type of mechanism, although both systems may have components in common.     

Homocysteine toxicity in Escherichia coli is caused by a perturbation of branched-chain amino acid biosynthesis

In Escherichia coli the sulfur-containing amino acid homocysteine (Hcy) is the last intermediate on the methionine biosynthetic pathway. Supplementation of a glucose-based minimal medium with Hcy at concentrations greater than 0.2 mM causes the growth of E. coli Frag1 to be inhibited. Supplementation of Hcy-treated cultures with combinations of branched-chain amino acids containing isoleucine or with isoleucine alone reversed the inhibitory effects of Hcy on growth. The last intermediate of the isoleucine biosynthetic pathway, alpha-keto-beta-methylvalerate, could also alleviate the growth inhibition caused by Hcy. Analysis of amino acid pools in Hcy-treated cells revealed that alanine, valine, and glutamate levels are depleted. Isoleucine could reverse the effects of Hcy on the cytoplasmic pools of valine and alanine. Supplementation of the culture medium with alanine gave partial relief from the inhibitory effects of Hcy. Enzyme assays revealed that the first step of the isoleucine biosynthetic pathway, catalyzed by threonine deaminase, was sensitive to inhibition by Hcy. The gene encoding threonine deaminase, ilvA, was found to be transcribed at higher levels in the presence of Hcy. Overexpression of the ilvA gene from a plasmid could overcome Hcy-mediated growth inhibition. Together, these data indicate that in E. coli Hcy toxicity is caused by a perturbation of branched-chain amino acid biosynthesis that is caused, at least in part, by the inhibition of threonine deaminase.     

Isoleucine and Valine Metabolism of Escherichia coli XVI. Pattern of Multivalent Repression in Strain K-12

The levels of the five enzymes required for isoleucine and valine synthesis were examined under several growth conditions in strain K-12 of Escherichia coli and mutants derived from it. In strains with wild type repressibility, the same pattern of derepression was found on limiting isoleucine as is found to be constitutive in strain Tir-8, which has an altered isoleucine-activating enzyme. Homoserine dehydrogenase, which is essential for the biosynthesis of threonine and is normally derepressed on limiting isoleucine or threonine, is also derepressed in strain Tir-8. Threonine deaminase and homoserine dehydrogenase were partially repressed in strain Tir-8 by very high levels of isoleucine, but were not further derepressed over levels in minimal medium by limiting isoleucine.     

Derepression of Synthesis of the Aminoacyl-Transfer Ribonucleic Acid Synthetases for the Branched-Chain Amino Acids of Escherichia coli

The kinetics of derepression of valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined during valine-, isoleucine-, and leucine-limited growth. When valine was limiting growth, valyl-tRNA synthetase formation was maximally derepressed within 5 min, whereas the rates of synthesis of isoleucyl-, and leucyl-tRNA synthetases were unchanged. Isoleucine-restricted growth caused a maximal derepression of isoleucyl-tRNA synthetase formation in 5 min and derepression of valyl-tRNA synthetase formation in 15 min with no effect on leucyl-tRNA synthetase formation. When leucine was limiting growth, leucyl-tRNA synthetase formation was immediately derepressed, whereas valyl- and isoleucyl-tRNA synthetase formation was unaffected by manipulation of the leucine supply to the cells. These results support our previous findings that valyl-tRNA synthetase formation is subject to multivalent repression control by both isoleucine and valine. In contrast, repression control of iso-leucyl- and leucyl-tRNA synthetase formation is specifically mediated by the supply of the cognate amino acid.     

Isoleucine Auxotrophy Due to Feedback Hypersensitivity of Biosynthetic Threonine Deaminase

Isoleucine-deficient mutants of Salmonella typhimurium were isolated. Three groups of mutants can be discerned by their nutritional requirements and enzyme patterns. (i) Mutants which grow with isoleucine alone are devoid of biosynthetic threonine deaminase (TD). (ii) Mutants growing with isoleucine and valine are devoid of transaminase B. (iii) Mutants growing with either isoleucine or threonine have normal levels of TD. However, the sensitivity of this enzyme to feedback inhibition by isoleucine is greatly enhanced. The inhibitory effect of isoleucine can be counterbalanced by high concentrations of threonine. These results indicate that the production of isoleucine in the mutants is restricted to a low level not sufficient to support the growth of the cells. This hypothesis is confirmed by studies with revertants of an isoleucine-threonine mutant. In nine revertants, wild-type properties of TD have been restored. In four revertants, the hypersensitivity of TD is unchanged, but the strains produce a greatly enhanced quantity of threonine, which is excreted into the culture medium. It follows, that hypersensitivity of TD to inhibition by isoleucine is the cause of the nutritional requirement of isoleucine-threonine mutants.