Isoleucine-valine enzyme, biosynthetic

Singer, P. A., Levinthal, M., Williams, L. S. (1984). Synthesis of the isoleucyl- and valyl-tRNA synthetases and the isoleucine-valine biosynthetic enzymes in a threonine deaminase regulatory mutant of Escherichia coli K-12. J. Mol. Biol. 175, 39-55. 

The most unexpected feature of the leu S mutants at the time they were first encountered was the effect of the mutation on the enzymes of the isoleucine-valine biosynthetic pathway (Scheme 2). As shown in... 

Table II, in these mutants, but not in the leu mutants, the isoleucine-vafine biosynthetic enzymes were also derepressed. At that time, however, it was already known that the repression of these enzymes is multivalent, that is, the enzymes are repressed only if isoleucine, valine, and leucine are in excess [16]. Thus, it was clear that the leu S function needed in generating the leucine excess signal for repression of the...

Derepression of Isoleucine-Valine Biosynthetic Enzymes in Escherichia coli Relative Specific Activities ... 

The results obtained by Blatt [20] studying ilv S mutants of S. typhi-murium have thus far offered a simpler picture. The ilv S mutants, as mentioned above, were selected as isoleucine auxotrophs from strains diploid for the ilv region. In the mutants, the K for isoleucine of the isoleucyl tRNA synthetase is at least 100-fold greater than is that of the parent strain. The mutants will grow slowly in minimal medium, but in doing so they exhibit increased levels of all five isoleucine-valine biosynthetic enzymes as well as increased intracellular pool levels of isoleucine, valine, and leucine. All three amino acids are also found to accumulate in the culture fluids in amounts greatly in excess of the levels found in fluids of the parent strains. 

Effect of an ilv S Mutation on Isoleucine Valine Biosynthetic Enzymes Relative Specific Activity"... 

Because of the superficial similarity of the azaleucine-resistant (azlO E. coli strains that had derepressed levels of all four leucine and four of the five isoleucine-valine biosynthetic enzymes to the leu S mutants of S. typhimurium, they have been examined in greater detail than the other azl" strains. However, examination has shown that these mutants have normal levels of leucyl tRNA synthetase [2-7]. In view of the fact that some of the lesions leading to derepression of the his operon appear to affect the levels of IRNA , the kind and amounts of tRNA species... 

For the isoleucine-valine system, there is evidence for the involvement of valyl-tRNA synthetase (Eidlic and Neidhardt [97]) and isoleucyl-tRNA synthetase (Blatt and Umbarger [98]) in repression. Likewise, leucyl-tRNA synthetase has been implicated in the regulation of the formation of the leucine biosynthetic enzymes (Calvo et al. [99]). In multivalent repression of the branched-chain amino acids [100], all three aminoacyl-tRNA synthetases appear to play a role. Leucyl-tRNA has been proposed as a regulatory element (Hatfield and Burns [101]). 

Capsaicinoids are synthesized by the condensation of vanillylamine with a short chain branched fatty acyl CoA. A schematic of this pathway is presented in Fig. 8.4. Evidence to support this pathway includes radiotracer studies, determination of enzyme activities, and the abundance of intermediates as a function of fruit development [51, 52, 57-63], Differential expression approaches have been used to isolate cDNA forms of biosynthetic genes [64-66], As this approach worked to corroborate several steps on the pathway, Mazourek et al. [67] used Arabidopsis sequences to design primers to clone the missing steps from a cDNA library. They have expanded the schema to include the biosynthesis of the key precursors phenylalanine and leucine, valine and isoleucine. Prior to this study it was not clear how the vanillin was produced, and thus the identification of candidate transcripts on the lignin pathway for the conversion of coumarate to feruloyl-CoA and the subsequent conversion to vanillin provide key tools to further test this proposed pathway. 

Branched Chain Amino Acid Biosynthesis. The branched chain amino acids, leucine, isoleucine and valine, are produced by similar biosynthetic pathways (Figure 2.11). In one pathway, acetolactate is produced from pyruvate and in the other acetohydroxybutyrate is produced from threonine. Both reactions are catalysed by the same enzyme that is known as both acetolactate synthase (ALS) and acetohy-droxy acid synthase (AHAS). 

The aspartate and pyruvate families together contain 11 amino acids. Because of the reactions involved in its synthesis, isoleucine is considered a member of both families. Isoleucine and valine use four enzymes in common in their biosynthetic pathways. 

ALS is the first common enzyme in the biosynthetic route to valine, leucine and isoleucine. It is the site of action for the triazolopyrimidine (TP) herbicides as well as the sulfonylureas (SU) and imidazolinones (IM). These compounds act on the meristem and are slow to bring about plant death. 

Acetolactate synthase (ALS, EC 4.1.3.18) is the first common enzyme in the biosynthetic route to the branched chain amino acids, valine, leucine and isoleucine. It is the primary target site of action for at least three structurally distinct classes of herbicides, the imidazolinones (IM), sulfonylureas (SU), and triazolopyrimidines (TP) (Figure 1). SU and IM were discovered in greenhouse screening programs whereas TP was subsequently targeted as a herbicide. Numerous substitution patterns can be incorporated into the basic structure of all three classes of herbicides to provide crop selectivity as well as broad spectrum weed control. This is amply demonstrated in the seven products based on SU and four based on IM already in the market. A number of others are in various stages of development. The rapid success of ALS inhibitors as herbicidal products has attracted a world-wide research commitment. Not since the photosystem II... 

The sulfonylurea herbicides are a new family of chemical compounds, some of which are selectively toxic to weeds but not to crops. The selectivity of the sulfonylureas results from their metabolism to non-toxic compounds by particular crops, but not by weeds. In addition to efficient weed control, the sulfonylurea herbicides provide environmentally desirable properties such as field use rates as low as two grams/hectare and very low toxicity to mammals. The high specificity of the herbicides for their molecular target contributes to both of these properties. In addition, the low toxicity to mammals results from their lack of the target enzyme for the herbicides. Sulfonylureas inhibit the enzyme acetolactate synthase (ALS), also known as acetohydroxyacid synthase (AHAS), which catalyzes the first common step in the biosynthesis of the branched chain amino acids leucine, isoleucine and valine. In mammals these are three of the essential amino acids which must be obtained through dietary intake because the biosynthetic pathway for the branched chain amino acids is not present. The prototype structure of a sulfonylurea herbicide is shown in Figure 1. 

The valine and isoleucine biosynthetic pathways share four enzymes. Isoleucine synthesis begins with the reaction of a-ketobutyrate (a derivative of threonine) with pyruvate. In valine synthesis the condensation of two pyruvate molecules is the first step. Leucine is produced by a series of reactions that begin with a-ketoisovalerate, an intermediate in valine synthesis. 

By using colistine for the enrichment procedure, many auxotrophic mutants defective in the biosynthetic pathway of valine and isoleucine have been isolated. From an isoleucine-requiring mutant, defective in threonine desaminase, a prototrophic revertant has been isolated. The threonine desaminase of this revertant differs from the wild type enzyme in that its affinity for isoleucine is diminished. This revertant excretes isoleucine. Another revertant of an isoleucine-deficient mutant was obtained which formed the enzyme acetohydroxy add synthetase constitutively. During heterotrophic growth with fructose or lactate as substrates, valine, isoleucine and leucine were excreted into the culture medium. Approximately 0.6 g of amino acids were produced per liter suspension when lactate was supplied as a substrate under autotrophic conditions the excretion was negligible (Reh, 1970 Fig. 12). 

As expected, among the mutants excreting leucine there was a mutant constitutively derepressed with respect to the formation of the enzyme a-isopropylmalate synthetase, which is the first enzyme in the leucine biosynthetic pathway. In another mutant, this enzyme is insensitive to endproduct inhibition by leucine. However, contrary to our expectations, we found mutants carrying regulatory defects in the control of the valine-isoleucine biosynthetic pathway several mutants are constitutively derepressed with respect to the formation of aceto-hydroxy acid synthase and in one mutant this enzyme is insensitive to endproduct inhibition by valine. The selection and the existence of... 

The first committed step in the biosynthetic pathway of the branched chain amino acids is catalyzed by the enzyme acetohydroxyacid synthase (AHAS, EC 2.2.1.6), which is also referred to as acetolactate synthase (ALS). As depicted in Fig. 2.1.1, the pathway leading to valine and leucine begins with the condensation of two molecules of pyruvate accompanied by loss of carbon dioxide to give (S)-2-acetolactate. A parallel reaction leading to isoleucine involves the condensation of pyruvate with 2-ketobutyrate to afford (S)-2-aceto-2-hydroxybutyrate after loss of carbon dioxide. Both reactions are catalyzed by AHAS, which requires the cofactors thiamin diphosphate (ThDP) and flavin adenine dinudeotide (FAD). A divalent metal ion, most commonly is also required. Several excellent reviews... 

Fig. 1. Biosynthetic relationships among the aspartate family and branched-chain amino acids. Compounds which serve as branch point metabolites are bracketed and abbreviated as follows ASA, aspartate semialdehyde PHS, O-phosphohomoserine OlV, 2-oxoisovaierate. Each arrow represents an enzyme catalyzed reaction, and the details of the pathways for threonine, lysine, isoleucine, and valine, and leucine biosynthesis are presented in Figs. 2, 3, 4, and 5, respectively.

Although each of the branched-chain amino acids will inhibit plant growth to some extent, the combination of leucine plus valine or leucine plus isoleucine is particularly effective (Table VII,B). In contrast, the combination of all three branched-chain amino acids or valine plus isoleucine is only slightly inhibitory. An explanation of these effects is provided by examination of the known regulatory mechanisms associated with the biosynthetic pathways (Fig. 8). Leucine plus valine cooperatively inhibits both acetohy-droxyacid synthase and plant growth (Miflin, 1969a,b). Inhibition of the enzyme would result in an isoleucine limitation. Since inhibition of the enzyme by either leucine or valine may be enhanced by the presence of isoleucine, the combination of leucine plus isoleucine would tend to limit valine biosyn-... 

The effect of different amino acids supplements on the synthesis of PHB by recombinant E. coli was evaluated by Mahishi and Rawal. The study revealed that when the basal medium is supplemented with amino acids, except glycine and valine, all other amino acid supplements enhanced PHB accumulation in recombinant E. coli harboring PHB synthesizing genes from S. aureqfaciens. Cysteine, isoleucine, or methionine supplementation increased PHB accumulation by 60, 45, and 61%, respectively. Amino acid biosynthetic enzyme activities in several pathways are repressed by end produa supplementation. End product inhibition in the cysteine biosynthetic pathway controls the carbon flow due to sensitivity of serine transacetylase to cysteine. Hence, supplementation of cysteine favors a change in carbon flux that eliminates the requirement of acetyl-CoA for serine transacetylation which in turn provides more carbon source and acetyl-CoA for PHB synthesis. Degradation of methionine and isoleucine yields succinyl CoA, an intermediate of tricarboxylic acid cycle and allows more acetyl-CoA to enter the PHB biosynthetic pathway. 

Three enzymes are involved in the synthesis of 2,3-BD a-acetolactate synthase (EC 4.1.3.18), a-acetolactate decarboxylase (EC 4.1.1.5), and butanediol dehydrogenase (also known as diacetyl [acetoin] reductase Larsen and Stormer 1973 Johansen et al. 1975 Stormer 1975). Two different enzymes form acetolactate from pyruvate. The first, termed catabolic a-acetolactate synthase, has a pH optimum of 5.8 in acetate and is part of the butanediol pathway. The other enzyme, termed anabolic a-acetolactate synthase or acetohydroxyacid synthetase, has been well studied and characterized and will not be discussed here. This enzyme is part of the biosynthetic pathway for isoleucine, leucine, and valine and is coded for by the ilvBN, ilvGM, and ilvH genes in E. colt and Salmonella typhimurium (Bryn and Stormer 1976). 

---