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.     

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.     

Structural Genes for a Newly Recognized Acetolactate Synthase in Escherichia coli K-12

Evidence is reported that shows the presence in Escherichia coli K-12 of a newly found acetolactate synthase. This enzyme is the product of two genes, ilvH and ilvI, both located very close to leu. Amber mutations have been found in both genes and therefore their products are polypeptides. Mutations in the ilvH gene cause the appearance of an acetolactate synthase activity which is relatively resistant to valine inhibition and can be separated by adsorption on hydroxylapatite from another activity present in the extract and more sensitive to valine inhibition than the former. A mutant altered in the ilvI gene was isolated among the revertants sensitive to valine inhibition of an ilvH mutant. Such a mutant lacks the resistant acetolactate synthase. A temperature-sensitive revertant of the ilvI mutant contained a temperature-sensitive acetolactate synthase. Thus ilvI is the structural gene for a specific acetolactate synthase. The activity of the ilvH gene product has been measured by adding an extract containing it to a purified ilvI acetolactate synthase, which, upon incubation, became more sensitive to valine inhibition. Conversely, a valine-sensitive acetolactate synthase (the product of the ilvH and the ilvI genes) became more resistant to valine inhibition upon incubation with an extract of a strain containing a missense ilvH gene product.     

Sulfonylurea-resistant mutants and natural tolerance of cyanobacteria

The herbicide sulfometuron methyl (SM) inhibited the growth of the cyanobacterium Synechococcus sp. PCC7942, but not of Synechocystis sp. PCC6714. The inhibitory effect was alleviated by the simultaneous addition of valine, leucine and isoleucine. SM resistant mutants were isolated from Synechococcus 7942, two types of which were further analysed. In these mutants, SM3/20 and SM2/32, the activity of acetolactate synthase (ALS) — a key enzyme in the biosynthesis of branched-chain amino acids —appeared 2600- and 300-fold, respectively, more resistant to SM than that of their wild type. Strain SM2/32 also exhibited a low level of ALS activity. Although the growth of the latter mutant was extremely inhibited by valine, the sensitivity of its ALS activity to feed-back inhibition by the amino acid was unaltered. At high concentrations valine inhibited growth of the wild type strains and of the mutant SM3/20. Isoleucine alleviated the valine-induced growth inhibition. Unlike that of Synechococcus 7942, the ALS activity of Synechocystis was found to tolerate high concentrations (100-fold) of the herbicide. The study confirms that the SM mutations are correlated with a cyanobacterial ilv gene.Abbreviations ALS acetolactate synthase; ile, isoleucine - leu leucine - NTG N-methyl-N-nitro-N-nitrosoguanidine - SM sulfometuron methyl - SM^r sulfometuron methyl resistant - val valine     

Acetolactate Synthase Activity in Developing Maize (Zea mays L.) Kernels

Acetolactate synthase (EC 4.1.3.18) activity was examined in maize (Zea mays L.) endosperm and embryos as a function of kernel development. When assayed using unpurified homogenates, embryo acetolactate synthase activity appeared less sensitive to inhibition by leucine + valine and by the imidazolinone herbicide imazapyr than endosperm acetolactate synthase activity. Evidence is presented to show that pyruvate decarboxylase contributes to apparent acetolactate synthase activity in crude embryo extracts and a modification of the acetolactate synthase assay is proposed to correct for the presence of pyruvate decarboxylase in unpurified plant homogenates. Endosperm acetolactate synthase activity increased rapidly during early kernel development, reaching a maximum of 3 micromoles acetoin per hour per endosperm at 25 days after pollination. In contrast, embryo activity was low in young kernels and steadily increased throughout development to a maximum activity of 0.24 micromole per hour per embryo by 45 days after pollination. The sensitivity of both endosperm and embryo acetolactate synthase activities to feedback inhibition by leucine + valine did not change during kernel development. The results are compared to those found for other enzymes of nitrogen metabolism and discussed with respect to the potential roles of the embryo and endosperm in providing amino acids for storage protein synthesis.     

Properties of mutant acetolactate synthases resistant to triazolopyrimidine sulfonanilide

Triazolopyrimidine sulfanilides are a class of highly active herbicides whose primary target is acetolactate synthase. Spontaneous mutants of tobacco (Nicotiana tabacum) (KS-43) and cotton (Gossypium hirsutum) (PS-3 and DO-2) resistant to triazolopyrimidine sulfonanilide were selected in tissue culture. Acetolactate synthase partially purified from the three mutants were 80- to 1000-fold less sensitive to inhibition by the compound compared with the corresponding wild-type enzyme. The mutants also varied in the cross-resistance pattern to other acetolactate synthase inhibiting herbicides in the sulfonylurea, imidazolinone, and pyrimidyl-oxy-benzoate chemical families. Thus, acetolactate synthase from KS-43, PS-3, and DO-2 cultures have different mutations. The affinities for pyruvate, thiamine pyrophosphate, as well as the activity of the mutant enzymes were found to be comparable to the corresponding wild-type enzymes. However, the enzyme from PS-3 was highly resistant to feedback inhibition by valine and leucine. In contrast, acetolactate synthase from KS-43 and DO-2 were inhibited by valine and leucine to nearly the same extent as the wild-type enzymes. Also, PS-3 cultures accumulated much higher levels of the branched chain amino acids compared to the wild-type cotton culture. The mutation in the PS-3 enzyme has therefore rendered it insensitive to feedback regulation by valine and leucine.     

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.     

Role of small subunit (IlvN polypeptide) of acetohydroxyacid synthase I from Escherichia coli K-12 in sensitivity of the enzyme to valine inhibition.

Most of the coding sequence for the IlvN polypeptide subunit of acetohydroxyacid synthase I was deleted from the ilvB+ ilvN+ plasmid pTCN12 by in vitro methods. Several ilvB+ delta ilvN derivatives of pTCN12 were identified among transformants of a strain otherwise lacking any acetohydroxyacid synthase. Deletion derivatives produced an enzymatically active IlvB polypeptide, as shown by the Ilv+ phenotype of transformed cells and by immunologic and enzymatic assays. However, whereas the growth of pTCN12 transformants was sensitive to valine inhibition, growth of the ilvB+ delta ilvN transformants was relatively resistant. Moreover, in vitro analyses confirmed that both acetolactate and acetohydroxybutyrate synthesis in extracts of the ilvB+ delta ilvN transformants was resistant to valine inhibition, in comparison with that in extracts of pTCN12 transformants or with that catalyzed by purified acetohydroxyacid synthase I. The IlvN polypeptide had a minimal effect, if any, on IlvB polypeptide accumulation as measured by immunoprecipitation, but its absence resulted in a greater than 10-fold reduction in enzyme specific activity.     

Expression of a valine-resistant acetolactate synthase activity mediated by the ilv O and ilv G genes of Escherichia coli K-12

Summary A strain carrying the ilv0603 mutation has been isolated in E. coli K-12 and its characteristics were found to be very similar to those previously reported by Ramakrishnan and Adelberg (1965a) for other ilv0 mutants.The strain carrying the ilv0603 mutation is resistant to valine inhibition (Val^r) and we show that this resistance depends on the expression of a newly recognized gene, ilvG, which is located at min 75, between ilvE and ilvD on the E. coli K-12 map. The ilvG gene causes the expression of a Val^r acetolactate synthase, which is detectable only when the ilv0603 mutation is also present in cis on the same chromosome. Under these conditions the Val^r acetolactate synthase activity is eluted, on a hydroxylapatite column, at an ionic strength slightly lower than that required for elution of the remaining acetolactate synthase activity (sensitive to valine inhibition). The Val^r peak is missing in a strain carrying an ilvG (amber) mutation.     

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.     

Role of the C-terminal domain of the regulatory subunit of AHAS isozyme III: use of random mutagenesis with in vivo reconstitution (REM-ivrs)

In order to clarify the role of the C-terminal domain of the ilvH protein (the regulatory subunit of enterobacterial AHAS isozyme III, whose structure has been solved and reported by Kaplun et al., J Mol Biol 357, 951, 2006) in the process of valine inhibition of AHAS III, we developed a procedure that randomly mutagenizes a specific segment of a gene through error-prone PCR and screens for mutants on the basis of the properties of the holoenzymes reconstituted in vivo (REM-ivrs). Previous work showed that the N-terminal domain includes the valine-binding ACT domain of the regulatory subunit and is sufficient to completely activate the catalytic subunit, but that this domain cannot confer valine sensitivity on the reconstituted enzyme. It appeared that the C-terminal domain of the ilvH is involved in some way in "signal transmission" of the inhibition by valine. As knowledge of the structure of AHAS holoenzymes and the interactions between the catalytic and regulatory subunits is very limited, a procedure that focuses on the C-terminal domain in the ilvH gene could add to the understanding of the mechanism by which the binding of valine to the regulatory subunit is coupled to inhibition of the catalytic activity. In the REM-ivrs procedure, a medium copy (~40 copies) plasmid expressing ilvH with a Val(r) mutation confers the Val(r) phenotype upon bacteria. All the single missense mutations produced by REM-ivrs were found to be localized to the interface between the C-terminal domains of two monomers in the ilvH dimer. The loss of specific contacts involved in inter-monomer interactions in this region might conceivably disrupt the structure of the C-terminal domain itself. Biochemical study of an isolated Val(r) mutant elicited by the REM-ivrs method detected no binding of radioactively labeled valine, as previously found in a truncation mutant. The idea that the C-terminal domain has a specific "signal-transmission" role was also contradicted by examination of the thermal stability of the Val(r) REM-ivrs variants by the Thermofluor method, which does not detect any signs of biphasic melting behavior for any of the mutants. We propose that the mutants of ilvH isolated by the REM-ivrs method differ from the wild-type in the equilibrium between two states of the enzyme. Without the specific interdomain contacts of the wild-type ilvH protein, the holoenzyme reconstituted from mutant regulatory subunits is apparently in a state with uninhibited activity and low affinity for valine.     

Acetohydroxyacid synthase: a proposed structure for regulatory subunits supported by evidence from mutagenesis

Valine inhibition of acetohydroxyacid synthase (AHAS) plays an important role in regulation of biosynthesis of branched-chain amino acids in bacteria. Bacterial AHASs are composed of separate catalytic and regulatory subunits; while the catalytic subunits appear to be homologous with several other thiamin diphosphate-dependent enzymes, there has been no model for the structure of the small, regulatory subunits (SSUs). AHAS III is one of three isozymes in Escherichia coli. Its large subunit (encoded by ilvI) by itself has 3-5 % activity of the holoenzyme and is not sensitive to inhibition by valine. The SSU (encoded by ilvH) associates with the large subunit and is required for full catalytic activity and valine sensitivity. The isolated SSU binds valine. The properties of several mutant SSUs shed light on the relation between their structure and regulatory function. Three mutant SSUs were obtained from spontaneous Val(R) bacterial mutants and three more were designed on the basis of an alignment of SSU sequences from valine-sensitive and resistant isozymes, or consideration of the molecular model developed here. Mutant SSUs N11A, G14D, N29H and A36V, when reconstituted with wild-type large subunit, lead to a holoenzyme with drastically reduced valine sensitivity, but with a specific activity similar to that of the wild-type. The isolated G14D and N29H subunits do not bind valine. Mutant Q59L leads to a valine-sensitive holoenzyme and isolated Q59L binds valine. T34I has an intermediate valine sensitivity. The effects of mutations on the affinity of the large subunits for SSUs also vary. D. Fischer's hybrid fold prediction method suggested a fold similarity between the N terminus of the ilvH product and the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase. On the basis of this prediction, together with the properties of the mutants, a model for the structure of the AHAS SSUs and the location of the valine-binding sites can be proposed.     

Effect of valine and the herbicide sulfometuron methyl on acetolactate synthase activity in nuclear and plasmid-borne sulphometuron methyl resistant Saccharomyces cerevisiae strains

Acetolactate synthase (ALS) specific activity was evaluated in isogenic lines of Saccharomyces cerevisiae carrying the wild-type ILV2 gene or mutations in this gene for resistance to the herbicide sulfometuron methyl (SM). Statistical comparisons were made between two nuclear alleles and among five alleles borne on a YE chimaeric plasmid transformed into a strain carrying a 1.5-kilobase deletion in the nuclear ILV2 gene. Decreased ALS activity of plasmid-borne SM-resistant mutations was shown not to be caused by copy number effects. ALS-specific activity in strains carrying the wild-type ILV2 allele exhibited strong feedback inhibition by valine and was sensitive to SM. All nuclear and plasmid-borne SM-resistance alleles resulted in ALS-specific activity highly resistant to SM and resistant to valine feedback inhibition.     

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.     

Mutations in two distinct regions of acetolactate synthase regulatory subunit from Streptomyces cinnamonensis result in the lack of sensitivity to end-product inhibition

Acetolactate synthase small subunit encoding ilvN genes from the parental Streptomyces cinnamonensis strain and mutants resistant either to valine analogues or to 2-ketobutyrate were cloned and sequenced. The wild-type IlvN from S. cinnamonensis is composed of 175 amino acid residues and shows a high degree of similarity with the small subunits of other valine-sensitive bacterial acetolactate synthases. Changes in the sequence of ilvN conferring the insensitivity to valine in mutant strains were found in two distinct regions. Certain point mutations were located in the conserved domain near the N terminus, while others resulting in the same phenotype shortened the protein at V(104) or V(107). To confirm whether the described mutations were responsible for the changed biochemical properties of the native enzyme, the wild-type large subunit and the wild-type and mutant forms of the small one were expressed separately in E. coli and combined in vitro to reconstitute the active enzyme.     

Homoserine kinase and threonine synthase in methionine-overproducing soybean tissue cultures

To gain understanding of the regulation of methionine level in plants, we assayed homoserine kinase and threonine synthase in extracts of wild type and several methionine-overproducing soybean [Glycine max (L.) Merr.] callus lines. The specific activity of homoserine kinase was depressed by 45–73%, and that of threonine synthase by 26–43% in the high methionine lines. Cysteine inhibited threonine synthase in wild type and variant lines. Threonine synthase in two variant lines showed significantly less inhibition by cysteine and in one line was inhibited by threonine. Depressed threonine synthase activity may increase the availability of homoserine phosphate to the competing methionine biosynthetic pathway.Abreviations MOPS morpholinopropane-sulfonate - EDTA ethylenediamine-tetraacetate - DTE dithioerythritol - AdoMet S-adenosylmethionine     

Mutations in Chlamydomonas reinhardtii Conferring Resistance to the Herbicide Sulfometuron Methyl

Chlamydomonas reinhardtii mutants resistant to the herbicide sulfometuron methyl (SM) were isolated and characterized. Growth of C. reinhardtii is sensitive to inhibition by SM at a concentration of 1 micromolar. Four mutants resistant to 10- to 100-fold higher concentrations were isolated. All possess a form of acetolactate synthase (ALS) whose specific activity in cell extracts is 100- to 1000-fold more resistant to SM than is the specific activity of wild-type enzyme. Only one mutant had abnormally low ALS specific activity in the absence of SM. All mutations were inherited as single lesions in the nuclear genome and were expressed in heterozygous diploids. The mutations in two strains mapped to linkage group IX about 30 centimorgans from streptomycin resistance and on the same side of the centromere, and in genetic crosses between mutants no segregation was observed. Accordingly, all mutations are tentatively assigned to gene smr-1. Herbicide resistance appears to be suitable as a selectable marker for molecular transformation in this organism.     

Growth inhibition of Escherichia coli K-12 by L-valine: a consequence of a regulatory pattern.

Summary We studied the production of the ilvG gene product, the valine resistant acetolactate synthase isoenzyme II, in an ilvO ⁺ G ⁺ ilvB ilvHI derivative of Escherichia coli K-12. This strain contains mutations in the structural genes for the valine sensitive acetolactate synthase isoenzymes I and III. We find that the ilvG gene is not expressed in this strain when grown with either isoleucine and valine or with isoleucine, leucine and valine, or when limited for either isoleucine or valine. Since we previously found that the ilvG gene is expressed in an ilvO603 containing strain (Favre et al., 1976), we presume that the mechanism by which E. coli K-12 regulates the ilv gene cluster is responsible for the lack of ilvG expression in the ilvO ⁺ strain. The valine sensitivity of E. coli K-12 is a consequence of this regulatory pattern.     

Mutant of Escherichia coli K-12 Missing Acetolactate Synthase Activity

A mutant requiring isoleucine and valine for growth, because of the absence of acetolactate synthase activity, has been isolated. At least one of three different genes (ilvG, ilvB, ilvI) is required for the expression of acetolactate synthase activity, thus suggesting the presence of three different acetolactate synthase isoenzymes.     

ilvB-encoded acetolactate synthase is resistant to the herbicide sulfometuron methyl.

The herbicide sulfometuron methyl is a potent inhibitor of the branched-chain amino acid biosynthetic enzyme acetolactate synthase (ALS) isolated from bacteria, fungi, and plants. However, it did not prevent growth of wild-type Salmonella typhimurium LT2 or Escherichia coli K-12. These species each contain two acetolactate synthase isozymes. Growth of S. typhimurium and E. coli mutants lacking ALS I was prevented by the herbicide, suggesting that activity of the remaining ALS isoenzyme (II or III, respectively) was stopped by sulfometuron methyl. Synthesis of ALS I requires either an relA function or an elevated cyclic AMP level. A relA mutant of S. typhimurium was inhibited by sulfometuron methyl on rich carbon sources that display a basal cyclic AMP level but not on poor carbon sources where the cyclic AMP concentration is elevated. When L-valine, which allosterically inhibits ALS I activity, was added, growth retardation of the relA- strain by sulfometuron methyl was observed on both poor and rich carbon sources. Enzymological analyses indicated that ALS I activities derived from both species were resistant to the herbicide. In contrast, activities of S. typhimurium ALS II and E. coli ALS III were abolished by sulfometuron methyl.