Dual role of alpha-acetolactate decarboxylase in Lactococcus lactis subsp. lactis.

The alpha-acetolactate decarboxylase gene aldB is clustered with the genes for the branched-chain amino acids (BCAA) in Lactococcus lactis subsp. lactis. It can be transcribed with BCAA genes under isoleucine regulation or independently of BCAA synthesis under the control of its own promoter. The product of aldB is responsible for leucine sensibility under valine starvation. In the presence of more than 10 microM leucine, the alpha-acetolactate produced by the biosynthetic acetohydroxy acid synthase IlvBN is transformed to acetoin by AldB and, consequently, is not available for valine synthesis. AldB is also involved in acetoin formation in the 2,3-butanediol pathway, initiated by the catabolic acetolactate synthase, AlsS. The differences in the genetic organization, the expression, and the kinetics parameters of these enzymes between L. lactis and Klebsiella terrigena, Bacillus subtilis, or Leuconostoc oenos suggest that this pathway plays a different role in the metabolism in these bacteria. Thus, the alpha-acetolactate decarboxylase from L. lactis plays a dual role in the cell: (i) as key regulator of valine and leucine biosynthesis, by controlling the acetolactate flux by a shift to catabolism; and (ii) as an enzyme catalyzing the second step of the 2,3-butanediol pathway.     

Salmonella typhimurium mutants defective in acetohydroxy acid synthases I and II.

An analysis of transposon-induced mutants shows that Salmonella typhimurium possesses two major isozymes of acetohydroxy acid synthase, the enzymes which mediate the first common step in isoleucine and valine biosynthesis. A third (minor) acetohydroxy acid synthase is present, but its significance in isoleucine and valine synthesis may be negligible. Mutants defective in acetohydroxy acid synthase II (ilvG::Tn10) require isoleucine, alpha-ketobutyrate, or threonine for growth, a mutant defective in acetohydroxy acid synthase I (ilvB::Tn5) is a prototroph, and a double mutant (ilvG::Tn10 ilvB::Tn5) requires isoleucine plus valine for growth.     

Absence of acetohydroxy acid synthetase in a clinical isolate of Neisseria gonorrhoeae requiring isoleucine and valine

A clinical isolate of Neisseria gonorrhoeae with an unusual growth requirement for isoleucine and valine lacked the activity of acetohydroxy acid synthetase, one of the enzymes required for the biosynthesis of these amino acids. A spontaneous mutant which no longer required isoleucine and valine had acquired this enzymatic activity.     

Effect of a leu-linked mutation on the valine sensitivity of acetohydroxy acid synthase activity in Escherichia coli.

A spontaneous leu-linked mutation (ilvH2015) in Escherichia coli K-12 made the strain resistant to 1 mM valine and l mM glycylvaline (Val-r) and caused the isoleucine and valine biosynthetic enzyme, acetohydroxy acid synthase, to be less sensitive to feedback inhibition by valine than the wild-type enzyme. Transfer of the ilvDAC deletion into a strain carrying ilvH2015 abolished the effect of the marker on the acetohydroxy acid synthase and rendered it as sensitive to valine as the enzyme in the isogenic control strain without the Val-r marker under both repressing and limiting conditions. In contrast, auxotrophy caused by transfer of an ilvC lesion into the Val-r strain did not interfere with the effect of ilvH2015 on valine sensitivity of acetohydroxy acid synthase. In addition, the presence of the Val-r marker produced minor but significant pleiotropic effects on several other isoleucine and valine biosynthetic enzymes but did not cause derepression of the ilv gene cluster. These studies suggest some type of interaction between a product produced by a gene close to leu and the isoleucine and valine biosynthetic enzymes.     

The existence of three types of acetohydroxy acid synthetase in an isoleucine-requiring mutant of Aerobacter aerogenes.

The synthesis of the three types of acetolactate synthase (EC 4.1.3.18) which are responsible for the biosynthesis os isoleucine and valine, was observed in Aerobacter aerogenes I-12, an isoleucine-requiring mutant, when grown on the four kinds of media. When the cells were grown on isoleucine-rich medium, acetolactate synthase sensitive to feedback inhibition and having an optimum pH at 8.0 was formed. By increasing the amount of potassium phosphate in the medium, the catabolite repression of the enzyme having an optimum pH at 6.0 and which is insensitive to feedback inhibition, was released. In contrast, acetolactate synthase having an optimum pH at 8.0 and insensitive to feedback inhibition was formd when isoleucine was limited, irrespective of phosphate concentrations. Two insensitive enzymes were not regulated by isoleucine, leucine and valine, although sensitive pH 8.0 enzyme was repressed by them. Thus, it may be assumed that the synthesis of insensitive pH 8.0 enzyme were repressed by limiting the amount of isoleucine is still open.     

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.     

Control of acetohydroxy acid synthetase in Escherichia coli 9723.

A method by which three acetohydroxy acid synthetase activities are separated from extracts of Escherichia coli 9723 has been developed. Isoleucine specifically represses synthesis of one of the enzymes, which is not sensitive to valine inhibition, and isoleucine also simultaneously enhances the production of a second activity, which is valine inhibitable. The valine-inhibitable activity is repressed by leucine and valine, a combination of which is more effective than either alone. The third acetohydroxy acid synthetase, which is more active at pH 6 than at 8, is not controlled by the branched-chain amino acids. In a mutant of E. coli 9723 selected for the ability of valine to inhibit growth, the isoleucine-repressible acetohydroxy acid synthetase activity was no longer present, but isoleucine addition still resulted in enhanced production of the valine-inhibitable activity.     

Cyclic AMP can replace the relA-dependent requirement for derepression of acetohydroxy acid synthase in E. coli K-12.

Derepression of the isoleucine and valine biosynthetic enzymes was strongly impaired in a relA strain of E. coli K-12 grown in an amino acid-glucose medium. The expression of the isoleucine and valine operons during leucine starvation was markedly defective in the relA mutant as compared to an isogenic rel⁺ strain. Downshift to a poor carbon and energy source or the addition of cyclic AMP to the glucose medium allowed normal derepression in the relA mutant of one of the isoleucine and valine enzymes, acetohydroxy acid synthase. The other isoleucine and valine enzymes failed to derepress under these conditions, in contrast to the high enzyme levels in the rel⁺ parent. No increase in acetohydroxy acid synthase was found in relA cya or relA crp strains during glycerol or pyruvate downshift. Cyclic AMP allowed derepression in the relA cya mutant but not in the relA crp strain. These data strongly suggest that the relA requirement for normal expression of acetohydroxy acid synthase can be replaced by cyclic AMP.     

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.     

Regulation of expression of the ilvB operon in Salmonella typhimurium

The ilvB gene of Salmonella typhimurium encodes the valine-sensitive form of acetohydroxy acid synthase, acetohydroxy acid synthase I, which catalyzes the first step in the parallel biosynthesis of isoleucine and valine. Although nearly all of the other genes involved in this pathway are clustered at minute 83, ilvB was found to lie at minute 80.5. Expression of ilvB was shown to be nearly completely repressed by the end products leucine and valine. Studies in which we used strains with mutations in cya (adenylate cyclase) and crp (cAMP receptor protein) demonstrated that synthesis of acetohydroxy acid synthase I is enhanced by the cAMP-cAMP receptor protein complex. Although no stimulation was achieved by growth on poor carbon sources, introduction of crp on a multicopy plasmid led to markedly increased expression. Strains of S. typhimurium lacking valine-resistant acetohydroxy acid synthase II (ilvG) are like Escherichia coli K-12 in that they are not able to grow in the presence of L-valine owing to a conditional isoleucine auxotrophy. The valine toxicity of these ilvG mutants of S. typhimurium was overcome by increasing the level of acetohydroxy acid synthase I. Enzyme activity could be elevated either by maximally derepressing expression with severe leucine limitation, by introduction of either ilvB or crp on a multicopy plasmid, or by the presence of the ilv-513 mutation. This mutation, which is closely linked to genes encoding the phosphoenol pyruvate:sugar phosphotransferase system (pts), causes highly elevated expression of ilvB that is refractory to repression by leucine and valine, as is the major ilv operon. The response of ilvB to the cAMP-cAMP receptor protein complex was not affected by this lesion. Data obtained by using this mutant led us to propose that the two modes of regulation act independently. We also present some evidence which suggests that ilvB expression may be affected by the phosphoenol pyruvate:sugar phosphotransferase system.     

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.     

Enhanced acetohydroxy acid synthase III activity in an ilvH mutant of Escherichia coli K-12.

The acetohydroxy acid synthase III isozyme, which catalyzes the first common step in the biosynthesis of isoleucine, leucine, and valine in Escherichia coli K-12, is composed of two subunits, the ilvI and ilvH gene products. A missense mutation in ilvH (ilvH612), which reduced the sensitivity of the enzyme to the end product inhibition by valine, also increased its specific activity and lowered the Km for alpha-acetolactate synthesis. The mutation increased the sensitivity of acetohydroxy acid synthase III to dialysis and heat treatment and reduced the requirement for thiamine pyrophosphate addition to the assay mixture for activity. A strain carrying the ilvH612 mutation grew better than a homologous ilvH+ strain in the presence of leucine. The data indicate that this is a consequence of a more active acetohydroxy acid synthase III isozyme rather than the result of an alteration of the leucine-mediated repression of the ilvIH operon.     

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.     

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.     

Overproduction of noncanonical amino acids by Escherichia coli cells

Overproduction of noncanonical amino acids norvaline and norleucine by Escherichia coli with inactivated acetohydroxy acid synthases was demonstrated. The cultivation conditions for the overproduction of noncanonical amino acids were studied. The effect of the restoration of acetohydroxy acid synthase activity, increased expression of the leuABCD operon, and inactivation of the biosynthetic threonine deaminase on norvaline and norleucine synthesis was studied. When grown under valine limitation, E. coli cells with inactivated acetohydroxy acid synthases and an elevated level of expression of the valine operon were shown to accumulate norvaline and norleucine (up to 0.8 and 4 g/l, respectively). These results confirm the existing hypothesis of norvaline and norleucine formation from 2-ketobutyrate by leucine biosynthesis enzymes.     

A comparative study of the acetohydroxy acid synthase isoenzymes of Escherichia coli K-12

We studied the properties of the two acetohydroxy acid synthase isoenzymes expressed in wild type Escherichia coli K-12 in two isogenic strains, PS1035 (containing only acetohydroxy acid synthase III) and PS1036 (containing only acetohydroxy acid synthase I). The pH dependence is different for the two enzymes: acetohydroxy acid synthase I shows optimum activity at neutral pH, while acetohydroxy acid synthase III is most active at alkaline pH. Both activities require Mg²⁺ and thiamine pyrophosphate, but acetohydroxy acid synthase I, as compared to acetohydroxy acid synthase III, has a specific requirement for flavin adenine dinucleotide. Acetohydroxy acid synthase I is also more resistant to valine inhibition but more sensitive to inactivating conditions such as dialysis and temperature. The catalytic role of acetohydroxy acid synthase I in the synthesis of α-acetolactate is characterized by a higher affinity for pyruvate and a lower sensitivity to inhibition by α-ketobutyrate.     

The acetolactate synthase isoenzymes of Escherichia coli K-12.

Summary Strains of Escherichia coli K-12 possessing only one of the three genes coding for acetolactate synthetase activity present either in the wild type or in its ilv0603 derivative were prepared and analyzed. Extracts prepared from these strains show different values of acetolactate synthase specific activity and different sensitivity to valine inhibition. These strains show a unique pattern of growth inhibition by different substances.Temperature sensitive (ts) mutations in the ilvB and ilvG genes, have been isolated and characterized. Extracts of these strains were found to have an acetolactate synthase activity more heat labile than that of a strain containing the corresponding wild type allele. We conclude that ilvB and ilvG are the structural genes for two different forms of acetolactate synthase activity, most likely two isoenzymes. Moreover, since the strains containing a ts mutation show a temperature sensitive auxotrophy for isoleucine and valine, these two acetolactate synthases participate in isoleucine and valine biosynthesis. Similar evidence for a third acetolactate synthase, the product of the ilvHI genes, has been reported previously.We propose the following names for the acetolactate synthase isoenzymes: acetolactate synthase I (AHAS I), the product of the ilvB gene; acetolactate synthase II (AHAS II), the product of ilvG gene; and acetolactate synthase III (AHAS III), the product of the ilvHI genes.     

A single amino acid change in acetolactate synthase confers resistance to valine in tobacco

The metabolic control of branched chain amino acid (BCAA) biosynthesis involves allosteric regulation of acetolactate synthase (ALS) by the end-products of the pathway, valine, leucine and isoleucine. We describe here the molecular basis of valine resistance. We cloned and sequenced an ALS gene from the tobacco mutant Val^r-1 and found a single basepair substitution relative to the wild-type allele. This mutation causes a serine to leucine change in the amino acid sequence of ALS at position 214. We then mutagenized the wild-type allele of the ALS gene ofArabidopsis and found that it confers valine resistance when introduced into tobacco plants. Taken together, these results suggest that the serine to leucine change at position 214 of ALS is responsible for valine resistance in tobacco.This paper is dedicated to the memory of Jean-Pierre Bourgin, who died on October 29, 1994, at the age of 50     

Repression by glucose of acetohydroxy acid synthetase in Escherichia coli B

Acetolactate formation in Escherichia coli B results from the activity of a single system, acetohydroxy acid synthetase, which has a pH optimum of 8.0 and is sensitive to end-product inhibition by l-valine. Acetohydroxy acid synthetase was found to be subject to catabolite repression, and the nature and concentration of the carbon source had a greater effect on the formation of the enzyme than had the known end products (valine, isoleucine, leucine and pantothenate) of the biosynthetic pathways of which this enzyme is a member. The results suggest that acetohydroxy acid synthetase may play an amphibolic role in E. coli B.