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.     

Regulation of the Pool Size of Valine in Escherichia coli K-12

Three mutations (ilvH611, ilvH612, and ilvH613) are described which make Escherichia coli K-12 resistant to valine inhibition and are located near leu. The expression of the ilv genes appears to be normal in these mutants since the isoleucine-valine biosynthetic enzymes are not derepressed relative to the wild type. The intracellular concentration of valine is, however, higher in the mutants than in the isogenic ilvH(+) strain. These mutants also excrete valine, probably because of the high intracellular concentration of this amino acid. The pool size of valine is regulated independently from that of isoleucine and leucine. The increased intracellular concentration of valine is due to a decreased feedback inhibition that valine exerts on its own biosynthetic pathway. In fact, acetolactate synthase activity assayed in extracts of ilvH612 and ilvH613 mutants is more resistant to valine inhibition than the activity assayed in the ilvH(+) isogenic strain. Two forms of acetolactate synthase activity can be separated from these extracts by adsorption and elution on hydroxylapatite. One of them is as sensitive to valine inhibition as that of the wild type, the other is more resistant to valine inhibition.     

New acetohydroxy acid synthase activity from mutational activation of a cryptic gene in Escherichia coli K-12

Summary A mutation in an allele identified as ilvJ662 causes the expression of acetohydroxy acid synthase activity that is resistant to feedback inhibition by L-valine. The ilvJ662 allele was transduced as an unselected marker into a strain, CU1126 (ilvB, ilvHI), deficient in acetohydroxy acid synthase activity. The ilvJ662 allele appears to code for a new acetohydroxy acid synthase activity (acetohydroxy acid synthase IV), with physical, kinetic, and physiological properties distinct from the other three isozymes.The catalytic function of acetohydroxy acid synthase IV is highly stable at 37° C in the presence or absence of ethylene glycol. However, sensitivity to feedback inhibition by valine is rapidly lost at 37° C, but this property is somewhat stabilized by ethylene glycol. The rate of synthesis of acetohydroxy acid synthase IV is uniquely repressed by either leucine or isoleucine. These results suggest that the ilvJ ⁺ allele is cryptic for acetohydroxy acid synthase IV, an isozyme distinct from the other acetohydroxy acid synthases.     

Mutations in genes cpxA and cpxB of Escherichia coli K-12 cause a defect in isoleucine and valine syntheses.

Mutations in two chromosomal genes of Escherichia coli, cpxA and cpxB, produced a temperature-sensitive growth defect that was remedied specifically by the addition of isoleucine and valine to the minimal medium. This auxotrophy was manifested only when the medium contained exogenous leucine, suggesting that mutant cells fail to elaborate active acetohydroxy acid synthase, isozyme I. In the presence of leucine, this enzyme was required to catalyze the first reaction common to the biosynthesis of isoleucine and valine. Measurements of enzyme activity in crude extracts showed that mutant cells were seven- to eightfold deficient in active isozyme I when the cells were grown in the presence of leucine. When grown in the absence of leucine, mutant cells contained more acetohydroxy acid synthase activity. We attribute this activity to isozyme III, the product of the ilvHI genes, which are derepressed in the absence of exogenous leucine. The cpxA and cpxB mutations appear to affect the production of active isozyme I, rather than its activity, since (i) neither the cpxA nor the cpxB gene mapped near the structural gene for isozyme I (ilvB), (ii) the growth of mutant cells shifted from the permissive (34 degrees C) to the nonpermissive (41 degrees C) temperature did not immediately cease, but declined gradually over a period corresponding to several normal generation times, and (iii) the enzyme from mutant cells grown at 34 degrees C was as stable at 41 degrees C as the enzyme from cpx+ cells.     

Reversion of the effects of a threonine deaminase regulatory mutant by a mutation in ilvH in Escherichia coli K-12

In a strain carrying an ilvA538 mutation, the ilvGEDA operon expression is decreased (hyperattenuated) and the activity and/or expression of isoleucyl- and valyl- tRNA synthetases is decreased. We have isolated two revertants of ilvA538 owing to mutations in the ilvH gene, whose product is acetohydroxy acid synthase III. The regulatory properties of these revertants are consistent with a dual role for threonine deaminase as an effector of the ilvGEDA operon and the isoleucyl- and valyl- tRNA synthetase structural genes.     

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.     

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.     

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.     

Inhibition of acetohydroxy acid synthase by leucine

The enzymatic reaction of acetohydroxy acid synthase in crude extracts of Escherichia coli K-12 is inhibited by leucine. Inhibition is most pronounced at low pH values and is low at pH values higher than 8.0. Both isoenzymes of acetohydroxy acid synthase present in E. coli K-12 (isoenzyme I and isoenzyme III) are inhibited by leucine. Isoenzyme I, which is responsible for the majority of acetohydroxy acid synthase activity in E. coli K-12 at physiological pH, is inhibited almost completely by 30 mM leucine at pH 6.25-7.0 and is not affected at all at pH values higher than 8.4. Inhibition of isoenzyme I by leucine is a mixed noncompetitive process. Leucine inhibition of isoenzyme III is pH-independent and reaches only 40% at 30 mM leucine. The inhibition of acetohydroxy acid synthase by leucine at physiological pH, observed in vitro in this study, correlates with the idea that acetohydroxy acid synthase is a target for the toxicity of the abnormally high concentrations of leucine in E. coli K-12.     

Feedback-resistant acetohydroxy acid synthase increases valine production in Corynebacterium glutamicum

Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Km(r)). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum DeltailvA DeltapanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.     

Subunit association in acetohydroxy acid synthase isozyme III.

Acetohydroxy acid synthase isozyme III (AHAS III) from Escherichia coli is composed of large and small subunits (encoded by the genes ilvI and ilvH) in an alpha 2 beta 2 structure. The large (61-kDa) subunit apparently contains the catalytic machinery of the enzyme, while the small (17-kDa) subunit is required for specific stabilization of the active conformation of the large subunit as well as for valine sensitivity. The interaction between subunits has been studied by using purified enzyme and extracts containing subcloned subunits. The association between large and small subunits is reversible, with a dissociation constant sufficiently high to have important experimental consequences: the activity of the enzyme shows a concentration dependence curve which is concave upward, and this dependence becomes linear upon the addition of excess large or small subunits. We estimate that at a concentration of 10(-7) M for each subunit (7 micrograms of enzyme ml-1), the large subunits are only half associated as the I2H2 active holoenzyme. This dissociation constant is high enough to cause underestimation of the activity of AHAS III in bacterial extracts. The true activity of this isozyme in extracts is observed in the presence of excess small subunits, which maintain the enzyme in its associated form. Reexamination of an E. coli K-12 ilvBN+ ilvIH+ strain grown in glucose indicates that AHAS III is the major isozyme expressed. As an excess of small subunits does not influence the apparent Ki for valine inhibition of the purified enzyme, it is likely that valine binds to and inhibits I2H2 rather than inducing dissociation. AHAS I and II seem to show a much lower tendency to dissociate than does AHAS III.     

Acetohydroxy acid synthase activity from a mutation at ilvF in Escherichia coli K-12.

Examination of the ilvF locus at 54 min on the Escherichia coli K-12 chromosome revealed that it is a cryptic gene for expression of a valine-resistant acetohydroxy acid synthase (acetolactate synthase; EC 4.1.3.18) distinct from previously reported isozymes. A spontaneous mutation, ilvF663, yielded IlvF+ enzyme activity that was multivalently repressed by all three branched-chain amino acids, was completely insensitive to feedback inhibition, was highly stable at elevated temperatures, and expressed optimal activity at 50 degrees C. The IlvF+ enzyme activity was expressed in strains in which isozyme II was inactive because of the ilvG frameshift in the wild-type strain K-12 and isozymes I and III were inactivated by point mutations or deletions. Tn5 insertional mutagenesis yielded two IlvF- mutants, with the insertion in ilvF663 in each case. These observations suggest that the ilvF663 locus may be a coding region for a unique acetohydroxy acid synthase activity.     

抗甲磺隆假单胞菌的分离及其乙酰乳酸合酶的大小亚基ilvIH基因的克隆和表达

乙酰乳酸合酶(也称乙酰羟酸合酶acetohydroxyacid synthase,AHAS)是植物、真菌和细菌细胞内支链氨基酸Val、Leu、Ile生物合成过程中关键酶,是乙酰乳酸合酶抑制剂类除草剂如磺酰脲类、咪唑啉酮类、嘧啶水杨酸和磺酰氨类的作用靶标.[目的]获得抗甲磺隆的乙酰乳酸合酶基因,构建其表达载体,并分析基因中的位点突变与乙酰乳酸合酶对磺酰脲类除草剂抗性产生原因.[方法]从长期使用甲磺隆的土壤中分离到l株抗甲磺隆的菌株Lm10,利用PCR技术从Lm10总DNA中克隆到乙酰乳酸合酶的大小亚基基因ilvIH,对ilvIH氨基酸序列进行比对分析.分别将ilvI和ilvH分别连接到表达载体pET29a( )多克隆位点,转化大肠杆菌(Escherichia coli)获得转化子BL21(pET-I)和BL21(pET-H),并诱导表达.[结果]菌株Lm10鉴定为假单孢菌(Pseudomonas sp.),对甲磺隆的最高耐受浓度达到14000 μmol/L,且对各种乙酰乳酸合酶抑制剂类除草剂具有交叉抗性.Lm10与甲磺隆敏感菌株KT2440的小亚基氨基酸序列完全相同,而大亚基有6个氨基酸位点发生变异.转化子在IPTG诱导下,乙酰乳酸合酶的大小亚基的蛋白成功表达,粗酶液酶活试验结果表明Lm10的ilvI基因表达的乙酰乳酸合酶大亚基对甲磺隆有很强的抗性.[结论]发现菌株Lm10的乙酰乳酸合酶大亚基对甲磺隆有很强的抗性,抗甲磺隆菌株Lm10与敏感菌株KT2440的ilvI有6个氨基酸位点差异,这些位点突变可能是乙酰乳酸合酶对甲磺隆抗性产生的原因.     

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.     

Branched-Chain Amino Acid Biosynthesis in Salmonella typhimurium: a Quantitative Analysis

We report here the first quantitative study of the branched-chain amino acid biosynthetic pathway in Salmonella typhimurium LT2. The intracellular levels of the enzymes of the pathway and of the 2-keto acid intermediates were determined under various physiological conditions and used for estimation of several of the fluxes in the cells. The results led to a revision of previous ideas concerning the way in which multiple acetohydroxy acid synthase (AHAS) isozymes contribute to the fitness of enterobacteria. In wild-type LT2, AHAS isozyme I provides most of the flux to valine, leucine, and pantothenate, while isozyme II provides most of the flux to isoleucine. With acetate as a carbon source, a strain expressing AHAS II only is limited in growth because of the low enzyme activity in the presence of elevated levels of the inhibitor glyoxylate. A strain with AHAS I only is limited during growth on glucose by the low tendency of this enzyme to utilize 2-ketobutyrate as a substrate; isoleucine limitation then leads to elevated threonine deaminase activity and an increased 2-ketobutyrate/2-ketoisovalerate ratio, which in turn interferes with the synthesis of coenzyme A and methionine. The regulation of threonine deaminase is also crucial in this regard. It is conceivable that, because of fundamental limitations on the specificity of enzymes, no single AHAS could possibly be adequate for the varied conditions that enterobacteria successfully encounter.     

Acetohydroxy acid synthase isoenzymes of Escherichia coli K-12: A trans-acting regulatory locus for ilvHI gene expression

Summary We isolated an Escherichia coli K-12 regulatory mutation affecting the acetohydroxy acid synthase III isoenzyme. This mutation was found to lie outside the structural genes ilvHI for this isoenzyme and was designated lrs-1. A strain carrying this mutation was found to be altered in the leucine-mediated control of ilvHI mRNA and acetohydroxy acid synthase III synthesis observed in the isogenic lrs ⁺ strain. These alterations appeared to be a consequence of a reduced intracellular concentration of a single one of five tRNA^Leu isoaccepting species.     

Characterisation of the enzyme activities involved in the valine biosynthetic pathway in a valine-producing strain of Corynebacterium glutamicum

The enzyme activities of the valine biosynthetic pathway and their regulation have been studied in the valine-producing strain, Corynebacterium glutamicum 13032DeltailvApJC1ilvBNCD. In this micro-organism, this pathway might involve up to five enzyme activities: acetohydroxy acid synthase (AHAS), acetohydroxy acid isomeroreductase (AHAIR), dihydroxyacid dehydratase and transaminases B and C. For each enzyme, kinetic parameters (optimal temperature, optimal pH and affinity for substrates) were determined. The first enzyme of the pathway, AHAS, was shown to exhibit a weak affinity for pyruvate (K(m)=8.3 mM). It appeared that valine and leucine inhibited the three first steps of the pathway (AHAS, AHAIR and DHAD). Moreover, the AHAS activity was inhibited by isoleucine. Considering the kinetic data collected during this work, AHAS would be a key enzyme for further strain improvement intending to increase the valine production by C. glutamicum.     

Overproduction of noncanonical amino acids by <Emphasis Type="Italic">Escherichia coli</Emphasis> 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.     

Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during isoleucine formation from α-hydroxybutyric acid

When Corynebacterium glutamicum ATCC 14310 (leu^-) was cultured with 200 mg/l leucine and 150 mM -hydroxybutyric acid the acetohydroxy acid synthase activity was increased to 0.17 U/mg as compared to 0.03 U/mg in the wildtype. This increase was a combined effect of the limiting amounts of leucine added, together with an apparent additional internal leucine/valine shortage resulting from accumulated -ketobutyric acid (5 mM) and the kinetic characteristics of the acetohydroxy acid synthase. The increase in the specific AHAS activity by the appropriate amino acid limitation resulted in an increased isoleucine yield of 71 mmol/l as compared to 27 mmol/l obtained under non-limiting conditions.Abbreviation AHAS Acetohydroxy acid synthase     

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.