Biosynthesis of Branched-Chain Amino Acids in Schizosaccharomyces pombe: Properties of Acetohydroxy Acid Synthetase1

The regulatory properties of acetohydroxy acid synthetase (AHAS), the first enzyme in the biosynthetic pathway to valine and the second in the isoleucine pathway, were investigated in the fission yeast Schizosaccharomyces pombe. The enzyme was partially purified from crude extracts by protamine sulfate treatment, ammonium sulfate fractionation, and gel filtration through Sephadex G-25. AHAS from S. pombe is unique in that its activity shows a single peak around pH 6.5; high sensitivity to feedback inhibition by valine at this pH (K(i) = 0.1 mM) indicates that the enzyme is involved in valine biosynthesis. Pyruvate saturation kinetics of AHAS extracted from cells grown on glycerol as sole carbon and energy source were normal and hyperbolic. In contrast, the enzyme from glucose-grown cells exhibited sigmoidal saturation kinetics, an effect which disappeared when the synthetase from such cells was partially purified. This phenomenon was shown to be due to competition for pyruvate between AHAS and pyruvate decarboxylase; the latter enzyme is present in large amounts in cells fermenting glucose. Valine inhibition is noncompetitive in nature, and this effector exhibits homotropic cooperative effects; isoleucine is a less-potent inhibitor of AHAS activity. Mercurial treatment reversibly desensitized the enzyme to valine inhibition. On the basis of these data, the S. pombe AHAS appears to be an allosteric regulatory enzyme with the properties of a negative V system.     

Relationships among the herbicide and functional sites of acetohydroxy acid synthase from Chlorella emersonii

The properties of acetohydroxy acid synthase (AHAS, EC 4.1.3.18) from wild-type Chlorella emersonii (var. Emersonii, CCAP-211/11n) and two spontaneous sulfometuron methyl (SMM)-resistant mutants were examined. The AHAS from both mutants was resistant to SMM and cross-resistant to imazapyr (IM) and the triazolopyrimidine sulfonanilide herbicide XRD-498 (TP). The more-SMM-resistant mutant had AHAS with altered catalytic parameters (K _m, specificity), but unchanged sensitivity to the feedback inhibitors valine and leucine. The second mutant enzyme was less sensitive to the feedback inhibitors, but had otherwise unchanged kinetic parameters. Inhibition-competition experiments indicated that the three herbicides (SMM, IM, TP) bind in a mutually exclusive manner, but that valine can bind simultaneously with SMM or TP. The three herbicide classes apparently bind to closely overlapping sites. We suggest that the results with C. emersonii and other organisms can all be explained if there are separate binding sites for herbicides, feedback inhibitors and substrates.Abbreviations AHAS acetohydroxy acid synthase - AL acetolactate - AHB acetohydroxybutyrate - IM imazapyr - TP triazolopyrimidine sulfonanilide herbicide XRD-498 - R enzyme specificity - SMM sulfometuron methyl This research was supported in part by the United States — Israel Binational Science Foundation (BSF), Jerusalem, Israel (Grant 86-00205) and the Fund for Basic Research, Israel Academy of Sciences.     

A comparison by means of antisera and lectins of surface structures ofNitrobacter winogradskyi andN. agilis

Nitrobacter winogradskyi was grown autotrophically on carbon dioxide and nitrite, mixotrophically on pyruvate and nitrite, and heterotrophically on acetate and pyruvate.N. agilis was grown autotrophically on carbon dioxide and nitrite and heterotrophically on pyruvate, acetate, and yeast extract-peptone. Antisera were then prepared against these cells. Strong cross-reactions occurred between all antisera raised againstNitrobacter agilis cells in the homologous and heterologous reactions with the differently grownNitrobacter agilis cells. Similar results were obtained withN. winogradskyi; but there were differences between the heterotrophically grown cells and the autotrophically and mixotrophically grown cells. The autotrophically grown cells ofN. winogradskyi andN. agilis had nearly no immunological differences, while the cross-reactions of the heterotrophically grown cells differed strongly. So, growth conditions are of considerable influence on the serological behavior of the two bacteria tested. Of twelve lectins tested, five (fromAaptos papillata, Axinella polypoides, Cerianthus sp.,Ulex europeus, and Anti-H specific agglutinin of eel serum) agglutinated cells ofN. agilis andN. winogradskyi, indicating thatN-acetyl-glucosamine, galactose, and fucose may be present in the bacterial cell wall. Immunodiffusion showed that cells of both nitrifying organisms grown under different conditions had at least one antigenic surface structure in common.     

1H, 13C, 15N assignments of the dimeric regulatory subunit (ilvN) of the E. coli AHAS I

Acetohydroxyacid synthase (AHAS) is an enzyme involved in the biosynthesis of the branched chain amino acids viz, valine, leucine and isoleucine. The activity of this enzyme is regulated through feedback inhibition by the end products of the pathway. Here we report the backbone and side-chain assignments of ilvN, the 22 kDa dimeric regulatory subunit of E. coli AHAS isoenzyme I, in the valine bound form. Detailed analysis of the structure of ilvN and its interactions with the catalytic subunit of E. coli AHAS I will help in understanding the mechanism of activation and regulation of the branched chain amino acid biosynthesis.     

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.     

Oxidative and reductive acetyl CoA/carbon monoxide dehydrogenase pathway in Desulfobacterium autotrophicum

It has been proposed that in some anaerobic facultatively autotrophic bacteria the acetyl CoA/CO dehydrogenase pathway is operating both in the reductive and in the oxidative direction, depending on the growth conditions. One of these anaerobes, the Gram-negative sulfate-reducing cubacterium Desulfobacterium autotrophicum, was examined for enzymes of the proposed pathway. All the required enzyme activities were present in sufficient amounts both in autotrophically and in heterotrophically grown cells, provided that the cellular tetrahydropterin rather than tetrahydrofolate was used as cosubstrate in some of the enzyme assays. The question arises whether two sets of enzymes are operating in the reductive and oxidative direction, respectively. The key enzyme of this pathway, CO dehydrogenase, which was reasonably oxygen stable, was analysed by native polyacrylamide gel electrophoresis and anaerobic activity staining. Extracts from heterotrophically grown cells exhibited five enzyme activity bands. Extracts from autotrophically grown cells showed the same pattern but an additional activity band appeared.     

Role of cysteine in activation and allosteric regulation of maize phosphoenolpyruvate carboxylase

The effect of 5-5′-dithiobis-2-nitrobenzoate (DTNB) on the kinetic parameters and structure of phosphoenolpyruvate carboxylase purified from maize (Zea mays L.) has been studied. The V_max is found to be independent of the presence of this thiol reagent. The K_m is increased upon oxidation of cysteines by DTNB. At a substrate concentration higher than K_m (3.1 millimolar Mgphosphoenolpyruvate), a significant reversible decrease of the activity is observed. Malate has little effect in preventing the modification of these cysteines. The V type inhibition by malate was also studied at a saturating phosphoenolpyruvate level (9.3 millimolar Mgphosphoenolpyruvate). In the presence of 50 micromolar DTNB, up to 60% inhibition is caused by 15 millimolar malate; however, in the presence of both 50 micromolar DTNB and 50 millimolar dithiothreitol (DTT) this inhibition is reduced to 20%. The presence of DTT alone increases the size of the phosphoenolpyruvate carboxylase molecule as determined by light scattering. The activity at nonsaturating substrate concentration is increased by 36% in the presence of DTT. The oligomerization equilibrium between the dimer and the tetrameric form of the enzyme is affected by cysteine. The K_m for the substrate, the sensitivity toward malate, and the size of the enzyme are found to be modified upon incubation in the presence of DTT.     

Characterization of acetohydroxyacid synthase from the hyperthermophilic bacterium Thermotoga maritima

Acetohydroxyacid synthase (AHAS) is the key enzyme in branched chain amino acid biosynthesis pathway. The enzyme activity and properties of a highly thermostable AHAS from the hyperthermophilic bacterium Thermotoga maritima is being reported. The catalytic and regulatory subunits of AHAS from T. maritima were over-expressed in Escherichia coli. The recombinant subunits were purified using a simplified procedure including a heat-treatment step followed by chromatography. A discontinuous colorimetric assay method was optimized and used to determine the kinetic parameters. AHAS activity was determined to be present in several Thermotogales including T. maritima. The catalytic subunit of T. maritima AHAS was purified approximately 30-fold, with an AHAS activity of approximately 160±27 U/mg and native molecular mass of 156±6 kDa. The regulatory subunit was purified to homogeneity and showed no catalytic activity as expected. The optimum pH and temperature for AHAS activity were 7.0 and 85 °C, respectively. The apparent K_m and V_max for pyruvate were 16.4±2 mM and 246±7 U/mg, respectively. Reconstitution of the catalytic and regulatory subunits led to increased AHAS activity. This is the first report on characterization of an isoleucine, leucine, and valine operon (ilv operon) enzyme from a hyperthermophilic microorganism and may contribute to our understanding of the physiological pathways in Thermotogales. The enzyme represents the most active and thermostable AHAS reported so far.     

Untersuchungen über die Aufnahme von Glykolat bei Chlorogonium

Summary The alga Chlorogonium was cultured either heterotrophically or autotrophically under different partial pressures of CO₂ by aerating with pure air of air enriched with 2% CO₂. Cells were harvested in the logarithmic phase, transferred to phosphate buffer containing 0.01 M 1C¹⁴-glycolate and incubated with shaking in the dark. Under these conditions the rate of glycolate uptake was higher when the cells had been grown in the light. Cells grown in the light at the lower CO₂-concentration took up more glycolate than those grown with 2% CO₂. Approximately 90% of the radioactivity taken up with the glycolate was released as CO₂. The radioactivity remaining in the algae was somewhat higher in those cells which had been cultured heterotrophically or autotrophically under air than in cells grown autotrophically under air enriched with 2% CO₂.Addition of glycolate increased the uptake of oxygen by the cells. The consumption of the oxygen was quantitatively correlated to the uptake of glycolate.     

A microcomputer-based image analysis system and applications to chlorophyll fluorescence studies

An IBM-PC based software system to quantify images of biological sytems is presented. To illustrate the potential of the imaging system described, UV light-induced chlorophyll fluorescence of Chlorella vulgaris cells was monitored using a video camera interfaced to a microscope. The photoinhibitor, DCMU (30 μM) inhibited the UV-induced fluorescence decay in heterotrophically grown cells but not in autotrophically grown cells. Algal cells were also incubated in various concentrations of ascorbic acid (500, 250, 100, 50, 10 and 0 mM) prior to UV light exposure. In the presence of 500, 250 and 100 mM ascorbic acid, the decay of chlorophyll fluorescence was significantly delayed in both heterotrophically and autotrophically grown cells. Heterotrophically grown cells were more sensitive to ascorbic acid than autotrophically grown cells since 10 nM ascorbic acid delayed fluorescence decay in heterotrophic cultures.     

Pyruvate decarboxylase activity of the acetohydroxyacid synthase of Thermotoga maritima

Acetohydroxyacid synthase (AHAS) catalyzes the production of acetolactate from pyruvate. The enzyme from the hyperthermophilic bacterium Thermotoga maritima has been purified and characterized (k_cat ~100 s^?1). It was found that the same enzyme also had the ability to catalyze the production of acetaldehyde and CO₂ from pyruvate, an activity of pyruvate decarboxylase (PDC) at a rate approximately 10% of its AHAS activity. Compared to the catalytic subunit, reconstitution of the individually expressed and purified catalytic and regulatory subunits of the AHAS stimulated both activities of PDC and AHAS. Both activities had similar pH and temperature profiles with an optimal pH of 7.0 and temperature of 85 °C. The enzyme kinetic parameters were determined, however, it showed a non-Michaelis-Menten kinetics for pyruvate only. This is the first report on the PDC activity of an AHAS and the second bifunctional enzyme that might be involved in the production of ethanol from pyruvate in hyperthermophilic microorganisms.     

Arabidopsis Acetohydroxyacid Synthase Expressed in Escherichia coli Is Insensitive to the Feedback Inhibitors

Acetohydroxyacid synthase (AHAS), the first enzyme unique to the biosynthesis of isoleucine, leucine, and valine, is the target enzyme for several classes of herbicides. The AHAS gene from Arabidopsis thaliana, including the chloroplast transit peptide, was cloned into the bacterial expression plasmid pKK233-2. The resulting plasmid was used to transform an AHAS-deficient Escherichia coli strain MF2000. The growth of the MF2000 strain of E. coli was complemented by the functional expression of the Arabidopsis AHAS. The AHAS protein was processed to a molecular mass of 65 kilodaltons that was similar to the mature protein isolated from Arabidopsis seedlings. The AHAS activity extracted from the transformed E. coli cells was inhibited by imidazolinone and sulfonylurea herbicides. AHAS activity extracted from Arabidopsis is inhibited by valine and leucine; however, this activity was insensitive to these feedback inhibitors when extracted from the transformed E. coli.     

AN HERBICIDE (SULFOMETURON METHYL) RESISTANT MUTANT IN PORPHYRIDIUM (RHODOPHYTA)1

The unicellular red alga Porphyridium sp. is, like many bacteria, fungi and higher plants, sensitive to the sulfonylurea herbicide sulfometuron methyl (SMM). However, the minimal inhibitory concentration for SMM in Porphyridium (55 μM) is higher than in green plants. We isolated a spontaneous SMM-resistant mutant SMR1 of Porphyridium sp. by use of a double-layered agar technique. The mutation frequency and the similarity of the mutant's morphology and growth patterns to the parent strain strongly suggest that SMR1 is a single gene mutation. The activity of the enzyme acetohydroxy acid synthase (AHAS) in crude extracts of the mutant SMR1 is at least two orders of magnitude less sensitive to SMM than that of the parent strain, which indicates that AHAS is the target of SMM (as has been shown in a variety of organisms). We propose that such a mutation, the first isolated in a unicellular rhodophyte, would be a useful marker for genetic studies of Porphyridium. It may also be useful for maintaining unialgal cultures in large scale open ponds.     

Isolation and characterization of dihydrodipicolinate synthase from maize

Dihydrodipicolinate synthase (EC 4.2.1.52), the first enzyme specific to lysine biosynthesis in plants, was purified from maize (Zea mays L.) cell suspension cultures and leaves. The subunit molecular weight of maize dihydrodipicolinate synthase was estimated to be 38,000 based on SDS-PAGE. The condensation of l-aspartate semialdehyde and pyruvate by highly purified dihydrodipicolinate synthase exhibited kinetics characteristic of a Ping Pong Bi Bi ordered reaction in which pyruvate binds first to the enzyme. Substrate inhibition evident at higher concentrations of l-aspartate semialdehyde was partially alleviated by increasing concentrations of pyruvate. Pyruvate binding exhibited cooperativity with an apparent number of 2 and 1.86 millimolar concentration required for 50% of maximal activity. The K_m for aspartate semialdehyde was estimated to be 0.6 millimolar concentration. Lysine was an allosteric cooperative inhibitor of dihydrodipicolinate synthase with an estimated Hill number of 4 and 23 micromolar concentration required for 50% inhibition. The physical and kinetic data are consistent with a homotetramer model for the native enzyme.     

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 ΔilvA ΔpanB 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.     

Imidazolinone-induced loss of acetohydroxyacid synthase activity in maize is not due to the enzyme degradation

Acetohydroxyacid synthase (AHAS), the first enzyme leading to the biosynthesis of valine, leucine, and isoleucine, is inhibited by different chemical classes of herbicides. There is a loss in the extractable AHAS activity in imidazolinone-treated plants. Immunological studies using a monoclonal antibody against AHAS revealed no degradation of AHAS protein in imidazolinone-treated maize (Zea mays) plants. Therefore, the loss in AHAS activity is not due to the loss of AHAS protein.     

Enzymic comparisons of the inorganic sulfur metabolism in autotrophic and heterotrophic Thiobacillus ferrooxidans.

Activities of enzymes which mediate the oxidation of thiosulfate to sulfate and the assimilation of sulfate to sulfide were assayed in various cell-free fractions of Thiobacillus ferrooxidans grown autotrophically on either ferrous iron or thiosulfate or heterotrophically on glucose. There was no activity of the thiosulfate-oxidizing enzyme in extracts of bacteria grown with ferrous iron. Comparable activities for ATP-sulfurylase (EC 2.7.7.4), ADP-sulfurylase (EC 2.7.7.5), and adenylate kinase (EC 2.7.4.3) were found in the bacteria grown autotrophically with either Fe2+ or S2O32- or heterotrophically with glucose.     

Carbon metabolism inSulfobacillus thermosulfidooxidans subsp.asporogenes, strain 41

The activities of carbon metabolism enzymes were determined in cellular extracts of the moderately thermophilic, chemolithotrophic, acidophilic bacteriumSulfobacillus thermosulfidooxidans subsp.asporogenes, strain 41, grown either at an atmospheric content of CO₂ in the gas phase (autotrophically, heterotrophically, or mixotrophically) or autotrophically at a CO₂ content increased to 5–10%. Regardless of the growth conditions, all TCA cycle enzymes (except for 2-oxoglutarate dehydrogenase), one glyoxylate bypass enzyme (malate synthase), and some carboxylases (ribulose bisphosphate carboxylase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase) were detected in the cell-free extracts of strain 411. During autotrophic cultivation of strains 41 and 1269, the increase in the CO2 content of the supplied air to 5–10% resulted in the activation of growth and iron oxidation, a 20–30% increase in the cellular content of protein, enhanced activity of the key TCA enzymes (citrate synthase and aconitase), and, in strain 41, a decrease in the activity of carboxylases.     

Oligomeric forms of plant acetolactate synthase depend on flavin adenine dinucleotide

Acetolactate synthase (ALS, EC 4.1.3.18) has been extracted and partially purified from etiolated barley shoots (Hordeum vulgare L.). Multiple forms of this enzyme were separated by gel filtration and/or anion-exchange chromatography using fast protein liquid chromatography. It could be demonstrated that these two species are in equilibrium, which strongly depends on the structural role of flavin adenine dinucleotide and pyruvate. With 50 micromolar of flavin adenine dinucleotide in the medium most of the ALS aggregates as a high molecular weight form (M_r = 440,000), while 50 millimolar pyruvate facilitates dissociation into the smaller form (M_r = 200,000). Data are presented to show that two enzymatically active forms are not isozymes but different oligomeric species or aggregates of the basic 58-kilodalton subunit of ALS. These different ALS species exhibit little difference in feedback inhibition by valine, leucine and isoleucine or in inhibition by the sulfonylurea herbicide chlorsulfuron. Both aggregation forms show a broad pH-optimum between 6.5 and 7. Furthermore, the affinity for pyruvate and the amount of directly-formed acetoin indicate similar properties of these separated ALS forms.     

Regulation of the pyruvate kinase from Alcaligenes eutrophus H 16 in vitro and in vivo

The biosynthesis of the enzyme pyruvate kinase (E.C. 2.7.1.40) of Alcaligenes eutrophus (Hydrogenomonas eutropha) H 16 was influenced by the carbon and energy source. After growth on gluconate the specific enzyme activity was high while acetate grown cells exhibited lower activities (340 and 55 μmoles/min·g protein, respectively). The pyruvate kinase from autotrophically grown cells was purified 110-fold. The enzyme was characterized by homotropic cooperative interactions with the substrate phosphoenolpyruvate, the activators AMP, ribose-5-phosphate, glucose-6-phosphate and the inhibitor ortho-phosphate. In addition to phosphate ATP caused inhibition but in this case non-sigmoidal kinetics was obtained. The half maximal substrate saturation constant S_0.5 for phosphoenolpyruvate in the absence of any effectors was 0.12 mM, in the presence of 1 mM ribose-5-phosphate 0.07 mM, and with 9 mM phosphate 0.67 mM. The corresponding Hill values were 0.96, 1.1 and 2.75. The ADP saturation curve was hyperbolic even in the presence of the effectors, the K _m value was 0.14 mM ADP. When the known intracellular metabolite concentrations in A. eutrophus H 16 were compared with the regulatory sensitivity of the enzyme, it appeared that under the conditions in vivo the inhibition by ATP was more important than the regulation by the allosteric effectors.