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.

Biosynthesis of 2-aceto-2-hydroxy acids: acetolactate synthases and acetohydroxyacid synthases.

Two groups of enzymes are classified as acetolactate synthase (EC 4. 1.3.18). This review deals chiefly with the FAD-dependent, biosynthetic enzymes which readily catalyze the formation of acetohydroxybutyrate from pyruvate and 2-oxobutyrate, as well as of acetolactate from two molecules of pyruvate (the ALS/AHAS group). These enzymes are generally susceptible to inhibition by one or more of the branched-chain amino acids which are ultimate products of the acetohydroxyacids, as well as by several classes of herbicides (sulfonylureas, imidazolinones and others). Some ALS/AHASs also catalyze the (non-physiological) oxidative decarboxylation of pyruvate, leading to peracetic acid; the possible relationship of this process to oxygen toxicity is considered. The bacterial ALS/AHAS which have been well characterized consist of catalytic subunits (around 60 kDa) and smaller regulatory subunits in an alpha2beta2 structure. In the case of Escherichia coli isozyme III, assembly and dissociation of the holoenzyme has been studied. The quaternary structure of the eukaryotic enzymes is less clear and in plants and yeast only catalytic polypeptides (homologous to those of bacteria) have been clearly identified. The presence of regulatory polypeptides in these organisms cannot be ruled out, however, and genes which encode putative ALS/AHAS regulatory subunits have been identified in some cases. A consensus sequence can be constructed from the 21 sequences which have been shown experimentally to represent ALS/AHAS catalytic polypeptides. Many other sequences fit this consensus, but some genes identified as putative 'acetolactate synthase genes' are almost certainly not ALS/AHAS. The solution of the crystal structures of several thiamin diphosphate (ThDP)-dependent enzymes which are homologous to ALS/AHAS, together with the availability of many amino acid sequences for the latter enzymes, has made it possible for two laboratories to propose similar, reasonable models for a dimer of catalytic subunits of an ALS/AHAS. A number of characteristics of these enzymes can now be better understood on the basis of such models: the nature of the herbicide binding site, the structural role of FAD and the binding of ThDP-Mg2+. The models are also guides for experimental testing of ideas concerning structure-function relationships in these enzymes, e.g. the nature of the substrate recognition site. Among the important remaining questions is how the enzyme suppresses alternative reactions of the intrinsically reactive hydroxyethylThDP enamine formed by the decarboxylation of the first substrate molecule and specifically promotes its condensation with 2-oxobutyrate or pyruvate.

On the mechanism of leftward frameshifting at several hungry codons.

We have used lacZ reporter genes to assess leftward ribosome frameshifting on sequences containing the quadruplet U UUC followed by several different triplets coding for lysine, isoleucine, or leucine. Limitation for lysine-tRNA provokes leftward frameshifting when the slippery quadruplet is followed by either lysine codon aag or aaa, but not when followed by an isoleucine or leucine codon. Limitation for isoleucine provokes frameshifting when the quadruplet is followed by either isoleucine codon aua or auc, but not when it is followed by a lysine codon. We conclude that the quadruplet promotes shifting when the ribosome is stalled at any "hungry" codon immediately after it. Changing the quadruplet to U AGC, at which peptidyl-tRNA cognate to the AGC triplet will be mismatched at all three anticodon positions if it slips left, abolishes frameshifting when the ribosome is stalled at the next position. We conclude that the U UUC quadruplet promotes frameshifting by virtue of its ability to pair with a left-slipped peptidyl-tRNA. The frameshift promoted by isoleucine-tRNA limitation of the U UUC aua sequence was analyzed by amino acid sequencing of the protein product. It occurs through reading of the Cau histidine codon overlapping the hungry codon from the left. This result rules out a "simultaneous slippage" type of mechanism. It strongly suggests instead that starvation-promoted frameshifting occurs primarily by slippage of peptidyl-tRNA just upstream of the stall site, followed by decoding of the triplet overlapping the stall site from the left or 5' side. A secondary finding is that the last base of the "hungry" codon has a moderate effect on its shiftiness, aag being shiftier than aaa, and aua being shiftier than auc.

Enhanced ribosome frameshifting in stationary phase cells.

We have examined the effect of growth phase in Escherichia coli on the translation of a plasmid-borne lacZ gene in which active enzyme synthesis requires a leftward frameshift. During the log phase of growth, the differential rate of enzyme synthesis is very low. It increases by about two orders of magnitude during the small amount of protein synthesis which occurs at the end of log phase and the early part of stationary phase. The increase is sufficient to increase the enzyme's specific activity in crude extracts to 30 times more than it would be if the log-phase differential rate continued unchanged. No such large increase is observed with a zero-frame lacZ+ control gene on the same plasmid under the control of the same promoter; a significant but much smaller increase is observed with a zero-frame control containing an in-frame terminator triplet in the region of the required frameshift. Protein sequence analysis of the enzyme made from the frameshift reporter in stationary cells shows that the increased enzyme synthesis is due to frameshifting, and not due to termination and reinitiation. The frameshift occurs at or right after the sequence U UUC AAG, an intrinsically shifty site.

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.

Characterization of acetohydroxy acid synthase activity in the archaeon Haloferax volcanii.

Whereas the biochemistry of acetohydroxy acid synthase has been extensively studied in bacteria and eukaryotes, relatively little is known about the enzyme in archaea, the third kingdom of life. The present study biochemically characterizes acetohydroxy acid synthase activity in the halophilic archaea Haloferax volcanii. In addressing ion requirements, enzyme inhibition and antibody labeling, the results reveal that, except for its elevated salt requirements, the haloarchaeal enzyme is remarkably similar to its bacterial counterpart.

Raising activity of Bacillus thuringiensis var. israelensis against Anopheles stephensi larvae by encapsulation in Tetrahymena pyriformis (Hymenostomatida:Tetrahymenidae).

Toxicity of Bacillus thuringiensis var israelensis (B.t.i.) against surface-feeding mosquito larvae of Anopheles stephensi was enhanced by encapsulation in the protozoan Tetrahymena pyriformis. In the laboratory, larvae died about 8 times faster when exposed to protozoan cells filled with B.t.i. than when exposed to the same concentrations of B.t.i. alone. Best larvicidal activities were achieved with ratios of 1:200-1:500 T. pyriformis cells to B.t.i. spores. The concentration of B.t.i. needed to kill 50% of exposed populations was 4-fold lower with T. pyriformis than with B.t.i. alone in 100 ml-test cups. Toxicity enhancement is very likely a consequence of concentrating B.t.i. insecticidal crystal proteins in T. pyriformis cells and floating them to the water surface in the larval feeding zone. Reduction in the exposure time of B.t.i. to unfavorable field conditions, as a result of the decrease in larval mortality time, might improve the persistence of this biological control agent in nature.

Specialized peptide transport system in Escherichia coli.

Trileucine is utilized as a source of leucine for growth of strains of Escherichia coli K-12 that are deficient in the oligopeptide transport system (Opp). Trithreonine is toxic to E. coli K-12. Opp- mutants of E. coli K-12 retain complete sensitivity to this tripeptide. Moreover, E. coli W, which is resistant to trithreonine, can utlize this tripeptide as a threonine source and this capability is fully maintained in E. coli W (Opp-). A spontaneous trithreonine-resistant mutant of E. coli K-12 (Opp-) has been isolated that has an impaired growth response to trileucine and is resistant to trithreonine. Trileucine competes with the uptake of trithreonine as measured by its ability to relieve trithreonine toxicity in E. coli K-12. It is concluded that trileucine as well as trithreonine are transported into E. coli K-12 or W by a common uptake system that is distinct from the Opp system. Trimethionine can act as a competitor of trileucine or trithreonine-supported growth and as an antagonist of trithreonine toxicity in Opp- mutants. It is concluded that trimethionine is recognized by the trileucine-trithreonine transport system. Trithreonine, trimethionine, and trileucine are also transported by the Opp system, as they all relieve triornithine toxicity towards E. coli W and compete with tetralysine utilization as lysine source for growth of a lysine auxotroph of this strain.

Fate of Bacillus thuringiensis subsp. israelensis under Simulated Field Conditions.

The fate of Bacillus thuringiensis subsp. israelensis in a natural aquatic habitat was studied in a model system by using laboratory-simulated field waters and a mutant of the bacterium resistant to three antibiotics. Contact with mud of a sporal culture of the mutant resulted in an immediate disappearance of the larvicidal activity but had no influence on viability. The cessation of toxicity was caused by bacterial adsorption on soil particles, since 99.8% of the bacteria was found in the mud fraction within 45 min, with concurrent disappearance from the supernatant. When the mud was stirred, the bacteria could be redetected. The viability count of the mud suspension remained practically constant for at least 22 days, indicating that the spores were still fully viable but were incapable of germinating and multiplying in the mud under our experimental conditions. Approximately 8% of the colony forming ability of the bacteria could be separated from the mud by vigorous mixing followed by immediate filtration. The filtrated spores retained their toxicity, killing 90% of the larval populations even after 22 days incubation in the soil. The inactivation of the toxic activity of B. thuringiensis subsp. israelensis in the mud was therefore a reversible process and was probably due to masking of the bacteria, thus making the bacteria and their toxin inaccessible to the larvae. In the simulated field waters without mud, we observed only a very slow inhibition of the larvicidal activity. In contrast to the activity in the mud suspension, this activity could not be restored.

Acetohydroxy acid synthase is a target for leucine containing peptide toxicity in Escherichia coli.

Acetohydroxy acid synthase from a mutant resistant to leucine-containing peptides was insensitive to leucine inhibition. It is concluded that acetohydroxy acid synthase is a target for the toxicity of the high concentrations of leucine brought into Escherichia coli K-12 by leucine-containing peptides.

Toxicity of leucine-containing peptides in Escherichia coli caused by circumvention of leucine transport regulation.

A variety of leucine-containing peptides (LCP), Phe-Leu, Gly-Leu, Pro-Leu, Ala-Leu, Ala-Leu-Lys, Leu-Phe-Ala, Leu-Leu-Leu, and Leu-Gly-Gly, inhibited the growth of a prototrophic strain of Escherichia coli K-12 at concentrations between 0.05 and 0.28 mM. Toxicity requires normal uptake of peptides. When peptide transport was impaired by mutations, strains became resistant to the respective LCP. Inhibition of growth occurred immediately after the addition of LCP, and was relieved when 0.4 mM isoleucine was added. The presence of Gly-Leu in the medium correlated with the inhibition of growth, and the bacteria began to grow at the normal rate 70 min after Gly-Leu became undetectable. Disappearance of the peptide corresponded with the appearance of free leucine and glycine in the medium. The concentration of leucine inside the LCP-treated bacteria was higher than that in the leucine-treated and the control cultures. We suggest that entry of LCP into the cells via peptide transport systems circumvents the regulation of leucine transport, thereby causing abnormality high concentrations of leucine inside the cells. This accumulation of leucine interferes with the biosynthesis of isoleucine and inhibits the growth of the bacteria.

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.

Protozoan-enhanced toxicity of Bacillus thuringiensis var. israelensis delta-endotoxin against Aedes aegypti larvae.

The toxicity of Bacillus thuringiensis var. israelensis (Bti) in mosquito larvae was enhanced by encapsulation in the protozoan Tetrahymena pyriformis. Aedes aegypti larvae which fed on T. pyriformis loaded with Bti died about three times faster than when fed on the same concentrations of Bti alone due to ingestion of higher toxin concentrations, reflected by shorter death times of exposed populations. The best larvicidal activities were achieved at ratios of cell/spore numbers in the range of 1:200 to 1:500. This enhancement of mortality by preincubation with T. pyriformis was higher at low Bti concentrations or in late third-instar larvae. Ninety minutes of preincubation yielded the best enhancement effect. Toxicity enhancement is very likely a consequence of concentrating large quantities of Bti spores and crystals (containing delta-endotoxin) by T. pyriformis cells and delivering them to the larvae. Shortening larval mortality time by encapsulation in T. pyriformis should reduce the exposure time of Bti to unfavorable field conditions that inactivate its larvicidal activity. Whether this method will indeed improve Bti efficacy is still to be determined.

A method for simultaneous determination of the two possible products of acetohydroxy acid synthase.

A method for the simultaneous assay of 2-acetolactate and 2-aceto-2-hydroxybutyrate formation catalyzed by acetohydroxy acid synthase in the presence of its substrates pyruvate and 2-ketobutyrate is described. The method, appropriate for the study of the physiologically and mechanistically significant competition between the two reactions, involves oxidative decarboxylation of the acetohydroxy acids to the corresponding 2,3-diketones, transfer of the volatile diketones to methanol, and gas chromatographic analysis with electron-capture detection. Oxidative decarboxylation by air requires catalytic activation, and addition of iron salts is crucial to the success of the method with purified enzymes.