Regulation of serine transhydroxymethylase activity in Salmonella typhimurium

The regulation of serine transhydroxymethylase (EC 2.1.2.1.; l-serine:tetrahydrofolic-5,10-hydroxymethyltransferase) has been investigated in Salmonella typhimurium LT2. Our results indicate that limitation of a methionine auxotroph for methionine does not cause derepression of this enzyme as reported for Escherichia coli. However, a sixfold decrease in specific activity was observed when S. typhimurium cells were grown in glucose minimal medium supplemented with serine, glycine, methionine, adenine, guanine, and thymine. None of these compounds added to the growth medium individually produced more than a 42% reduction of wild-type enzyme activity. This enhanced repression by the combination of compounds suggests a form of cumulative repression of this enzyme. Growth of serine and thymine auxotrophs, with the respective requirement of each limiting, did not result in increased enzyme activity. However, growth of a purine auxotroph with a limiting amount of either guanine or inosine resulted in a five- to sevenfold increase in enzyme activity. A second condition causing significant derepression (fourfold increase) above the levels observed with cells grown in minimal medium was the addition of 0.5 mug of trimethoprim per ml, an inhibitor of the dihydrofolate reductase activity. (A partial report on this work was presented at 1974 meeting of the American Society for Microbiology.)     

Threonine production by ethionine-resistant mutants of Serratia marcescens.

Ethionine reduced both the growth rate and the final growth level of Serratia marcescens Sr41. Growth inhibition was completely reversed by methionine. Strain D-315, defective in homoserine dehydrogenase I, was more sensitive to ethionine-mediated growth inhibition than was the wild-type strain. Ethionine-resistant mutants were isolated from cultures of strain D-316, which was derived from strain D-315 as a threonine deaminase-deficient mutant. Of 60 resistant colonies, 7 excreted threonine on minimal agar plates. One threonine-excreting strain, ETr17, was highly resistant to ethionine and, moreover, insensitive to methionine-mediated growth inhibition, whereas the parent strain was sensitive. When cultured in minimal medium with or without excess methionine, strain ETr17 had a higher homoserine dehydrogenase level than did strain D-316. The homoserine dehydrogenase activity was not inhibited by threonine or methionine. Transductional analysis revealed that the ethionine-resistant (etr-1) mutation carried by strain ETr17 was located in the metBM-argE region and caused the derepressed synthesis of homoserine dehydrogenase II. Strain ETr17 had a higher aspartokinase level than did the parent strain. By transductional cross with the argE+ marker, the etr-1 mutation was transferred into strain D-562 which was derived from D-505, a strain defective in aspartokinases I and III. The constructed strain had a higher aspartokinase level than did strain D-505 in medium with or without excess methionine, indicating that the etr-1 mutation led to the derepressed synthesis of aspartokinase II. Strain ETr17 produced about 8 mg of threonine per ml of medium containing sucrose and urea.     

Characterization of yeast homoserine dehydrogenase, an antifungal target: the invariant histidine 309 is important for enzyme integrity

Fungal homoserine dehydrogenase (HSD) is required for the biosynthesis of threonine, isoleucine and methionine from aspartic acid, and is a target for antifungal agents. HSD from the yeast Saccharomyces cerevisiae was overproduced in Escherichia coli and 25 mg of soluble dimeric enzyme was purified per liter of cell culture in two steps. HSD efficiently reduces aspartate semialdehyde to homoserine (Hse) using either NADH or NADPH with kcat/Km in the order of 10(6-7) M(-1) x s(-1) at pH 7.5. The rate constant of the reverse direction (Hse oxidation) was also significant at pH 9.0 (kcat/Km approximately 10(4-5) M(-1) x s(-1)) but was minimal at pH 7.5. Chemical modification of HSD with diethyl pyrocarbonate (DEPC) resulted in a loss of activity that could be obviated by the presence of substrates. UV difference spectra revealed an increase in absorbance at 240 nm for DEPC-modified HSD consistent with the modification of two histidines (His) per subunit. Amino acid sequence alignment of HSD illustrated the conservation of two His residues among HSDs. These residues, His79 and His309, were substituted to alanine (Ala) using site directed mutagenesis. HSD H79A had similar steady state kinetics to wild type, while kcat/Km for HSD H309A decreased by almost two orders of magnitude. The recent determination of the X-ray structure of HSD revealed that His309 is located at the dimer interface [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. The His309Ala mutant enzyme was found in very high molecular weight complexes rather than the expected dimer by analytical gel filtration chromatography analysis. Thus the invariant His309 plays a structural rather than catalytic role in these enzymes.     

Regulation of aspartate-derived amino acid biosynthesis in the yeastSaccharomyces cerevisiae

The activity of three enzymes, aspartokinase, homoserine dehydrogenase, and homoserine kinase, has been studied in the industrial strainSaccharomyces cerevisiae IFI256 and in the mutants derived from it that are able to overproduce methionine and/or threonine. Most of the mutants showed alteration of the kinetic properties of the enzymes aspartokinase, which was less inhibited by threonine and increased its affinity for aspartate, and homoserine dehydrogenase and homoserine kinase, which both lost affinity for homoserine. Furthermore, they showed in vitro specific activities for aspartokinase and homoserine kinase that were higher than those of the wild type, resulting in accumulation of aspartate, homoserine, threonine, and/or methionine/S-adenosyl-methionine (Ado-Met). Together with an increase in the specific activity of both aspartokinase and homoserine kinase, there was a considerable and parallel increase in methionine and threonine concentration in the mutants. Those which produced the maximal concentration of these amino acids underwent minimal aspartokinase inhibition by threonine. This supports previous data that identify aspartokinase as the main agent in the regulation of the biosynthetic pathway of these amino acids. The homoserine kinase in the mutants showed inhibition by methionine together with a lack or a reduction of the inhibition by threonine that the wild type undergoes, which finding suggests an important role for this enzyme in methionine and threonine regulation. Finally, homoserine dehydrogenase displayed very similar specific activity in the mutants and the wild type in spite of the changes observed in amino acid concentrations; this points to a minor role for this enzyme in amino acid regulation.     

Metabolism of aspartate in Mycobacterium smegmatis

Mycobacterium smegmatis grows best on L-asparagine as a sole nitrogen source; this was confirmed. [14C]Aspartate was taken up rapidly (46 nmol.mg dry cells-1.h-1 from 1 mM L-asparagine) and metabolised to CO2 as well as to amino acids synthesised through the aspartate pathway. Proportionately more radioactivity appeared in the amino acids in bacteria grown in medium containing low nitrogen. Activities of aspartokinase and homoserine dehydrogenase, the initial enzymes of the aspartate pathway, were carried by separate proteins. Aspartokinase was purified as three isoenzymes and represented up to 8% of the soluble protein of M. smegmatis. All three isoenzymes contained molecular mass subunits of 50 kDa and 11 kDa which showed no activity individually; full enzyme activity was recovered on pooling the subunits. Km values for aspartate were: aspartokinases I and III, 2.4 mM; aspartokinase II, 6.4 mM. Aspartokinase I was inhibited by threonine and homoserine and aspartokinase III by lysine, but aspartokinase II was not inhibited by any amino acids. Aspartokinase activity was repressed by methionine and lysine with a small residue of activity attributable to unrepressed aspartokinase I. Homoserine dehydrogenase activity was 96% inhibited by 2 mM threonine; isoleucine, cysteine and valine had lesser effects and in combination gave additive inhibition. Homoserine dehydrogenase was repressed by threonine and leucine. Only amino acids synthesised through the aspartate pathway were tested for inhibition and repression. Of these, only one, meso-diaminopimilate, had no discernable effect on either enzyme activity.     

Immunochemical properties of lipoprotein lipase. Development of an immunoassay applicable to several mammalian species

Reuber hepatoma H-35 was found to retain the activity of carbamoyl-phosphate synthetase I. The content of this enzyme in H-35 grown in Eagle's minimal essential medium was about half that in rat liver. The enzyme from H-35 was the same as that from rat liver in molecular weight estimated by SDS-polyacrylamide gel electrophoresis, specific enzyme activity, kinetic parameters for ATP and N-acetyl-L-glutamate, and immunological crossreactivity. The enzyme in H-35 was induced by dexamethasone (1.4-fold) but not by glucagon or dibutyryl cAMP. Incorporation of [35S] methionine into the enzyme indicated that the effect of dexamethasone was due to increased synthesis of the enzyme protein (2.1-fold). By labeling with [35S]methionine, the precursor and the mature forms of carbamoyl-phosphate synthetase I were observed in the post-mitochondrial and mitochondrial fractions, respectively. By chasing the labeled cells with unlabeled methionine and cycloheximide, it was observed that the rate of translocation of the precursor into mitochondria is not affected by dexamethasone.     

Role of methionine in the regulation of serine hydroxymethyltransferase in Eschericia coli.

Significant derepression of serine hydroxymethyltransferase is observed when metE or metF mutants of Escherichia coli K-12 are grown on D-methionine sulfoxide instead of L-methionine. The derepression is not prevented by addition of glycine, adenosine, guanosine, guanosine, and thymidine to the growth medium of methionine-limited metF cells showing that the effect is not due to a secondary deficiency of these nutrients. On the other hand, methionine-limited growth of a metA mutant leads to derepression of met regulon enzymes, but only a marginal increase in serine hydroxymethyltransferase activity. A prototrophic metJ strain grown on minimal medium has about the same serine hydroxymethyltransferase as the wild type. The enzyme activity of the metJ strain is not influenced by methionine, but it is partially repressed by glycine, adenosine, and thymidine. metK strains have about twice as much serine hydroxymethyltransferase activity as wild-type cells when grown on minimal medium; but when both types of cells are grown on medium supplemented with glycine, adenosine, guanosine, and thymidine, their enzyme activities are about the same. The results show that methionine limitation can lead to depression of serine hydroxymethyltransferase, but that the regulatory system is different from the one which controls the methionine regulon.     

Partial purification and some properties of homoserine O-acetyltransferase of a methionine auxotroph of Saccharomyces cerevisiae.

A wild-type strain and six methionine auxotrophs of Saccharomyces cerevisiae were cultured in a synthetic medium supplemented with 0.1 mM L-cysteine or L-methionine and analyzed for the synthesis of homoserine O-acetyltransferase (EC 2.3.1.31). Among them, four mutant strains exhibited enzyme activity in cell extracts. Methionine added to the synthetic medium at concentrations higher than 0.1 mM repressed enzyme synthesis in two of these strains. The enzyme was partially purified (3,500-fold) from an extract of a mutant strain through ammonium sulfate fractionation and chromatography on columns of DEAE-cellulose, Phenyl-Sepharose C1-4B, and Sephadex G-150. The enzyme exhibited optimal pH at 7.5 for activity and at 7.8 for stability. The reaction product was ascertained to be O-acetyl-L-homoserine by confirming that it produced L-homocysteine in an O-acetyl-L-homoserine sulfhydrylase reaction. The Km for L-homoserine was 1.0 mM, and for acetyl coenzyme A it was 0.027 mM. The molecular weight of the enzyme was estimated to be approximately 104,000 by Sephadex G-150 column chromatography and 101,000 by sucrose density gradient centrifugation. The isoelectric point was at pH 4.0. Of the hydroxy amino acids examined, the enzyme showed reactivity only to L-homoserine. Succinyl coenzyme A was not an acyl donor. In the absence of L-homoserine, acetyl coenzyme A was deacylated by the enzyme, with a Km of 0.012 mM. S-Adenosylmethionine and S-adenosylhomocysteine slightly inhibited the enzyme, but methionine had no effect.     

Regulation of homoserine dehydrogenase in developing organs of soybean seedlings

The regulation of homoserine dehydrogenase (HSD) activity (EC 1.1.1.3) by L-threonine, L-cysteine and K⁺ was examined using extracts of organs of soybean seedlings harvested 3, 6, 11, and 19 days after germination. K⁺ stimulated HSD activity from each source at least 2-fold. HSD activity was completely inhibited by 10 mM L-cysteine while 10 mM D-cysteine was not inhibitory. A progressive decrease in sensitivity of NAD-dependent HSD to inhibition by 10 mM L-threonine occurred in all organs except the leaf during the sampling period. This progressive decrease in sensitivity of the HSD to threonine inhibition was detected only when K⁺ was present in the assay mixtures. Four major molecular forms, including one rapidly migrating form (form I) and three more slowly migrating forms (forms II, III, IV) of HSD, were identified in extracts of soybean organs by polyacrylamide electrophoresis. Chromatographic and electrophoretic data indicate that form I, which was not inhibited by threonine or stimulated by K⁺, was of lower MW than forms II, III and IV which were of similar MW. These latter 3 forms were inhibited by threonine and stimulated by K⁺. During soybean seedling development form II increased in amount and forms I and IV decreased in amount. This alteration in the amounts of the forms of HSD occurred during the same period as the decrease in the amount of threonine inhibition. Since K⁺ stimulation of HSD decreased during soybean organ development and K⁺ enhanced threonine inhibition, this might account for the observed decrease in threonine inhibition.     

Identification of a monofunctional aspartate kinase gene of Arabidopsis thaliana with spatially and temporally regulated expression

We screened a gene trap library of Arabidopsis thaliana and isolated a line in which a gene encoding a homologue of monofunctional aspartate kinase was trapped by the reporter gene. Aspartate kinase (AK) is a key enzyme in the biosynthsis of aspartate family amino acids such as lysine, threonine, isoleucine, and methionine. In plants, two types of AK are known: one is AK which is sensitive to feedback inhibition by threonine and carries both AK and homoserine dehydrogenase (HSD) activities. The other one is monofunctional, sensitive to lysine and synergistically S-adenosylmethionine, and has only AK activity. We concluded that the trapped gene encoded a monofunctional aspartate kinase and designated as AK-lys3, because it lacked the HSD domain and had an amino acid sequence highly similar to those of the monofunctional aspartate kinases ofA. thaliana. AK-lys3 was highly expressed in xylem of leaves and hypocotyls and stele of roots. Significant expression of this gene was also observed in trichomes after bolting. Slight expression of AK-lys3 was detected in vascular bundles and mesophyll cells of cauline leaves, inflorescence stems, sepals, petals, and stigmas. These results indicated that this aspartate kinase gene was not expressed uniformly but in a spatially specific manner.     

Regulation of enzymes of lysine biosynthesis in Corynebacterium glutamicum

The regulation of the six enzymes responsible for the conversion of aspartate to lysine, together with homoserine dehydrogenase, was studied in Corynebacterium glutamicum. In addition to aspartate kinase activity, the synthesis of diaminopimelate decarboxylase was also found to be regulated. The specific activity of this enzyme was reduced to one-third in extracts of cells grown in the presence of lysine. Aspartate-semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, and diaminopimelate dehydrogenase were neither influenced in their specific activity, nor inhibited, by any of the aspartate family of amino acids. Homoserine dehydrogenase was repressed by methionine (to 15% of its original activity) and inhibited by threonine (4% remaining activity). Inclusion of leucine in the growth medium resulted in a twofold increase of homoserine dehydrogenase specific activity. The flow of aspartate semialdehyde to either lysine or homoserine was influenced by the activity of homoserine dehydrogenase or dihydrodipicolinate synthase. Thus, the twofold increase in homoserine dehydrogenase activity resulted in a decrease in lysine formation accompanied by the formation of isoleucine. In contrast, repression of homoserine dehydrogenase resulted in increased lysine formation. A similar increase of the flow of aspartate semialdehyde to lysine was found in strains with increased dihydrodipicolinate synthase activity, constructed by introducing the dapA gene of Escherichia coli (coding for the synthase) into C. glutamicum.     

Regulation of the methionine feedback-sensitive enzyme in mutants of Salmonella typhimurium

Assay of the first enzyme unique to methionine biosynthesis, homoserine-O-transsuccinylase, in metJ and metK regulatory mutants of Salmonella typhimurium showed that synthesis of the enzyme was derepressed seven- and fourfold, respectively. The possibility of noncoordinate regulation of the methionine enzymes is discussed. In metA feedback-resistant mutants, the enzyme activity can be inhibited in vitro by 10 mmS-adenosylmethionine but not by 10 mm l-methionine; hence, the synergistic inhibition found for the wild-type enzyme is not effective in these latter mutants.     

Comparison of homoserine dehydrogenase from different plant sources

Homoserine dehydrogenase (HSD) was partially purified from castor bean, pea and wheat seedlings. The enzyme from pea had a MW of 75 000 and no sensitivity to threonine when measured in the direction of homoserine formation (forward reaction). The enzyme purified from castor bean had a MW of 290 000–350 000 and exhibited an almost complete inhibition by 1 mM threonine. Furthermore, this enzyme exhibited a polymeric nature as shown by polyacrylamide electrophoresis of the desensitized preparation and by SDS electrophoresis of the native enzyme. In wheat two isoenzymes were separated by gel filtration on Sephadex G 200. The fast-moving fraction (HSD I) was completely inhibited by threonine and exhibited a MW of 280 000, while the slow-moving fraction (HSD II) was insensitive to threonine and had a MW of 75 000. The sensitive enzyme from wheat and castor bean showed an almost absolute requirement for K⁺. The enzyme from pea and the insensitive form from wheat did not show a requirement for K⁺. For the wheat enzyme the effect of several amino acids and the main kinetic constants were studied.     

Inhibition of growth and aspartokinase activity of Salmonella typhimurium by thialysine.

Thialysine (S-2-aminoethyl cysteine) is an analog of lysine and has been reported to inhibit the lysyl-tRNA synthetase activity of Escherichia coli. This analog inhibits the growth of Salmonella typhimurium when added to glucose minimal medium at concentrations of 1.25 mM or greater. The addition of lysine with thialysine restores the normal growth rate, whereas, methionine, valine, or leucine each enhances the growth inhibition casued by thialysine. Enzyme assays demonstrate that thialysine inhibits not only the lysyl-tRNA synthetase from S. typhimurium, but also the aspartokinase activity. Lysine and thialysine appear to inhibit the same 40% of the total aspartokinase because simultaneous addition of the two compounds to the reaction mixture does not increase the inhibition caused by either alone. Furthermore, the slow growth of cells in the presence of 2.5 mM thialysine decreases the level of aspartokinase activity, suggesting that thialysine causes repression of enzyme synthesis as well as inhibition of activity.     

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.     

Expression of the metA gene of Escherichia coli K-12 in recombinant plasmids

Abstract The expression of the metA gene coding for the first enzyme in the methionine biosynthethic pathway was studied in wild-type and in deregulated strains of Escherichia coli K-12 carrying the gene on multicopy plasmids.
We looked at (a) in vitro activity of the metA product—The enzyme homoserine transsuccinylase (HTS); (b) resistance of cells carrying metA plasmids to the analogue α-methylmethionine which specifically inhibits HTS, and (c) the metA polypeptide in mini cells.
The results indicate that the M _r value of the polypeptide synthesized by the metA gene is 40 000. The synthesis of HTS, even when the metA gene is cloned on a multicopy plasmid, is under the negative control of the regulatory metJ gene.     

Activities and regulation of the enzymes involved in the first and the third steps of the aspartate biosynthetic pathway in Enterococcus faecium

The enzymes aspartokinase and homoserine dehydrogenase catalyze the reaction at key branching points in the aspartate pathway of amino acid biosynthesis. Enterococcus faecium has been found to contain two distinct aspartokinases and a single homoserine dehydrogenase. Aspartokinase isozymes eluted on gel filtration chromatography at molecular weights greater than 250,000 and about 125,000. The molecular weight of homoserine dehydrogenase was determined to be 220,000. One aspartokinase isozyme was slightly inhibited by meso-diaminopimelic acid. Another aspartokinase was repressed and inhibited by lysine. Although the level of diaminopimelate-sensitive (DAP^s) enzyme was not much affected by growth conditions, the activity of lysine-sensitive (Lys^s) aspartokinase disappeared rapidly during the stationary phase and was depressed in rich media. The synthesis of homoserine dehydrogenase was controlled by threonine and methionine. Threonine also inhibited the specific activity of this enzyme. The regulatory properties of aspartokinase isozymes and homoserine dehydrogenase from E. faecium are discussed and compared with those from Bacillus subtilis.     

Growth inhibition by alpha-aminoadipate and reversal of the effect by specific amino acid supplements in Saccharomyces cerevisiae

The growth of Saccharomyces cerevisiae wild-type strain X2180 in minimal medium was inhibited by the addition of higher-than-supplementary levels of alpha-aminoadipate. This inhibitory effect was reversed by the addition of arginine, asparagine, aspartate, glutamine, homoserine, methionine, or serine as single amino acid supplements. Mutants belonging to the lys2 and lys14 loci were able to grow in lysine-supplemented alpha-aminoadipate medium, although not as well as when selected amino acids were added. Growth in alpha-aminoadipate medium by all strains was accompanied by an accumulation of alpha-ketoadipate. Glutamate:keto-adipate transaminase levels were derepressed two- to fivefold in lys2 mutants using alpha-aminoadipate as a nitrogen source. Wild-type strain X2180 growing in amino acid-supplemented AA medium exhibited higher levels of alpha-aminoadipate reductase. Mutants unable to use alpha-aminoadipate without amino acid supplementation were obtained by treatment of lys2 strain MW5-64 and were shown to have glutamate: ketoadipate transaminase activity and to lack alpha-aminoadipate reductase activity. Altered cell morphologies, including increased size, multiple buds, pseudohyphae, and germ tubes, evidenced by cells grown in alpha-aminoadipate medium suggest that higher-than-supplementary levels of alpha-aminoadipate result in an impairment of cell division.     

Target of serine inhibition in Escherichia coli

L-serine has long been known to inhibit growth of Escherichia coli cells cultured in minimal medium supplemented with glucose, lactate, or another carbohydrate as the sole source of carbon. However, the target of serine inhibition was not known. The growth inhibition was released by adding isoleucine, 2-ketobutyric acid, threonine or homoserine, but not by aspartate. Thus the inhibition site must be between aspartate and homoserine in the isoleucine biosynthetic pathway. We found that homoserine dehydrogenase I was strongly inhibited by serine. We isolated serine-resistant mutants, and found that in these mutants homoserine dehydrogenase I was resistant to serine. Thus, we conclude that the target of serine inhibition in Escherichia coli is homoserine dehydrogenase I.