General control of amino acid biosynthesis in mutants of Candida spec. EH 15/D

The general control of amino acid biosynthesis was investigated in Candida spec. EH 15/D, using single and double mutant auxotrophic strains and prototrophic revertants starved for their required amino acids. These experiments show that starvation for lysine, histidine, arginine, leucine, threonine, proline, serine, methionine, homoserine, asparagine, glutamic acid or aspartic acid can result in derepression of enzymes. A correlation was found between the degree of derepression, growth of strains, and concentration of required amino acids. The amino acids pool pattern of mutants and revertants is different from that in the wild type strain.     

Effect of methionine and vitamin B-12 on the activities of methionine biosynthetic enzymes in metJ mutants of Escherichia coli K12

The effects of supplementation of growth medium with high concentrations of methionine (5 mm) and/or vitamin B₁₂ (10 nm) on the activities of five enzymes of the methionine regulon were measured in wild-type Escherichia coli K12, a metJ prototrophic and three metJ methionine auxotrophic derivatives. Growth on vitamin B₁₂ causes lowering of the activities of the non-B₁₂ methyltransferase while growth on methionine causes elevation of its activity in all four metJ mutants. The previous observation that this enzyme is not repressed by vitamin B₁₂ addition in metH mutants together with our observation that vitamin B₁₂ causes repression in mutants (metF) unable to synthesize the donor for homocysteine methylation supports the model of Kung et al. (10) that the holo-B₁₂-methyltransferase functions as a repressor of synthesis of the non-B₁₂-methyltransferase. Growth on methionine causes lowering of cystathionase activity, and growth on vitamin B₁₂ results in elevation of cystathionase activity in a metJ prototroph and one metJ auxotroph. The metJmetA strain (RG326) has a higher than normal level of cystathionase while the metJmetF strain (RG191) has lower than normal cystathionase activity. These results indicate the existence of a metJ independent system that modulates the activity of cystathionase possibly in response to changes in concentration of unidentified metabolite(s).     

Synthesis of cephalosporin C by a methionine analogue resistant mutant of Cephalosporium acremonium

Summary DL-seleno-methionine resistant mutants of Cephalosporium acremonium were isolated which have an enhanced capacity to utilized sulfate for the synthesis of cephalosporin C. Of these mutants, one designated as SMR-I3 produced three-fold more cephalosporin C from sulfate than its parent CW19. Mutant SMR-I3 required less dl-methionine for maximal synthesis of cephalosporin C, but an excess of dl-methionine inhibited the synthesis of the antibiotic. Furthermore, the mutant accumulated excessive methionine in the amino acid pool and possessed superior activity for sulfate uptake. These observations indicate that in the mutant SMR-I3, the biosynthesis of methionine from sulfate is very active and excess methionine becomes available for the synthesis of cephalosporin C.     

Two Feedback-Insensitive Enzymes of the Aspartate Pathway in Nicotiana sylvestris

Lysine and threonine overproducer mutants in Nicotiana sylvestris, characterized by an altered regulation of, respectively, dihydrodipicolinate synthase and aspartate kinase activities, were crossed to assess the effects of the simultaneous presence of these genes on the biosynthesis of aspartate-derived amino acids. The monogenic dominant behavior of both resistance traits was confirmed, and their loci were found to be unlinked. Study of the inhibition properties of dihydrodipicolinate synthase and aspartate kinase activities in RAEC-1 × RLT 70 confirmed the heterozygote state of both mutations, because only half of their lysine-sensitive activity could still be inhibited by this negative effector. Analysis of the free amino acid pool during the growth of the double mutant revealed a major free lysine overproduction reaching up to 50% of the total pool, whereas the other aspartate-derived amino acids remained equally or even less abundant than in the wild type. An abnormal phenotype was clearly associated with such high levels of lysine accumulation, which points out the possible role of this amino acid in the developmental features of the plant. Comparison of the amino acid content, free and total (free + protein-bound), between the wild type, the two mutants, and the double mutant obtained by crossing them brings new insights on the regulation of the aspartate pathway, and on its implications in relationship to plant nutritional value improvement.     

Genetic bases of methionine dependence in <Emphasis Type="Italic">Yersinia pestis</Emphasis> strains of main and non-main subspecies

Structural and functional organization of genes responsible for biosynthesis of amino acid methionine, which plays a leading role in cellular metabolism of bacteria, was studied in 24 natural Yersinia pestis strains of the major and non-main subspecies from various natural plague foci located in the territory of Russian Federation and neighbouring foreign countries, and also in Y. pestis and Y. pseudotuberculosis strains recorded in the files of NCBI GenBank database. Conservatism of genes metA, metC, metE, and metH as well as regulatory genes metR and metJ involved in biosynthesis of this amino acid was established. Sequencing of the variable locus of gene metB in natural Y. pestis strains of major and non-main subspecies revealed that the reason for the methionine dependence of strains belonging to the main subspecies is a deletion of a single nucleotide (−G) in the 988 position from the beginning of the gene, whereas this dependence in strains belonging to subspecies hissarica results from the appearance of a single nucleotide (+G) insertion in the 989 position of gene metB. These mutations are absent in strains of the caucasica, altaica, and ulegeica subspecies of the plague agent and in strains of pseudotuberculosis microbe, which correlates with their capacity for methionine biosynthesis.     

Cloning of the yeast methionyl-tRNA synthetase gene

A pool of random wild type yeast DNA fragments obtained by partial Sau IIIA restriction enzyme digestion and inserted in the Bam HI site of the hybrid yeast Escherichia coli plasmid ((pFL1) has been used to transform to prototrophy a methionyl-tRNA synthetase-impaired mutant requiring methionine. In the numerous prototroph strains recovered at least two independent clones have been obtained which show nonchromosomic inheritance character and an approximately 30-fold increase in methionyl-tRNA synthetase activity as compared to the wild type. Measurement of the Km for methionine in the transformed yeast cells indicates that the activity has been restored by decreasing the Km for methionine to the same level as found for the wild type methionyl-tRNA synthetase. Southern blotting experiments show that the yeast DNA's fragments inserted in the two independent plasmids share a common sequence which must correspond at least partly to the structural gene for methionyl-tRNA synthetase. They also suggest that the methionyl-tRNA synthetase gene is differently orientated in the two plasmids     

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.     

Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of α-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.     

The involvement of methionine regulatory mutants in the suppresion of b12-dependent homocysteine transmethylase (metH) mutants of Salmonella typhimurium

Summary Certain metH mutants (which lack the B₁₂-dependent homocysteine transmethylase) give rise to revertants resistant to the methionine analogue, ethionine. The revertants retain the original metH mutation and its suppression is due to two mutations, supI and supII. The supI mutation, which confers ethionine resistance, appears to be a mutation in the methionine regulatory gene, metJ, but the location and nature of supII have not been determined. It is possible that suppression results from a direct association between the metH and metJ gene products or by the introduction of an alternative pathway of homocysteine methylation.     

The synthesis of S-adenosylmethionine by mutants with defects in S-adenosylmethionine synthetase

Summary Some metK mutants of Salmonella typhimurium with constitutive methionine biosynthesis have no detectable S-adenosylmethionine (SAM) synthetase, the enzyme which converts methionine to SAM, the postulated corepressor of the methionine pathway. However these mutants are not auxotrophic for SAM, an essential compound for many reactions. Here it is shown that these mutants have normal functioning of pathways involving SAM and do in fact produce SAM at as high levels as wild-type. Also, SAM synthetase is shown to be dispensible for growth but not for methionine regulation. These results indicate that there is another route of SAM synthesis independent of SAM synthetase. This route probably also uses methionine as substrate as metK mutants are shown to convert methionine to SAM as efficiently as analogous non-metK strains. The existence of a second route of SAM synthesis makes it necessary to postulate a compartmentalization of SAM made via the SAM synthetase reaction from SAM made in any other way to explain the reduced ability of metK mutants to repress methionine biosynthesis.     

Free tryptophan pool and tryptophan biosynthetic enzymes in Saccharomyces cerevisiae

The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase) have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties.The tryptophan pool of wild type cells growing in minimal medium is 0.07 mole per g dry weight. Addition of anthranilate, indole or tryptophan to the medium produces a fifteen- to forty-fold increase in tryptophan pool, but causes no repression of the biosynthetic enzymes. Inclusion of 5-methyltryptophan in the growth medium causes a reduction in growth rate and a derepression of the biosynthetic enzymes, and this is shown here not to be correlated with a decrease in the free tryptophan pool.Mutants with an altered anthranilate synthase showing decreased sensitivity to inhibition by l-tryptophan or by the analogue dl-5-methyltryptophan have a tryptophan pool far higher than the wild type strain, but no repression of indoleglycerolphosphate synthase was observed. Mutants with an anthranilate synthase more sensitive to tryptophan inhibition show a slightly reduced tryptophan pool, but no derepression of indoleglycerolphosphate synthase was found.A mutant with constitutively derepressed levels of the biosynthetic enzymes shows a considerably increased tryptophan pool. Addition of 5-methyltryptophan to the growth medium of non-derepressible mutants causes a decrease in growth rate accompanied by a decrease in the tryptophan pool.Abbreviations CDRP 1-(o-carboxyphenylamino)-1-deoxyribulosephosphate - paba paraaminobenzoic acid - PRA N-(5-phosphoribosyl)-anthranilate - tRNA transfer ribonucleic acid; trp1 to trp5 refer to the structural genes for corresponding tryptophan biosynthetic enzymes     

Isolation and Characterization of a Xanthophyll Aberrant Mutant of the Green Alga Nannochloropsis oculata

Novel mutants (xan1 and xan2) of the unicellular green alga Nannochloropsis oculata are impaired in xanthophyll biosynthesis, thereby producing aberrant levels of xanthophylls. High-performance liquid chromatography (HPLC) analysis revealed that the xan1 and xan2 mutants have double the violaxanthin (V) content, but have significantly decreased lutein content in their cells compared to the wild type. Furthermore, these mutants contain two to three times more zeaxanthin than the wild type under low light (LL) growth conditions. However, this xanthophyll aberration in N. oculata did not affect the normal growth and the major cellular chemical composition of the xan1 strain. The xanthophyll pool size of the LL-grown mutant was 1.8-fold greater than that of the wild type. Under high light (HL) growth conditions, V content was substantially decreased in both the mutant and wild types because of the epoxidation state of the xanthophylls. Under LL growth conditions, the deepoxidation states of the xanthophyll pool sizes were 0.1 and 1.2 in the wild type and the mutant, respectively. However, the deepoxidation states of the xanthophyll pool sizes were 0.78 in the wild type and 0.87 in the mutant under HL growth conditions. We observed that the level of one of the commercially important xanthophylls, zeaxanthin, was higher in the mutant than in the wild type under all culture conditions. This mutant is discussed in terms of its commercial value and potential utilization by the algal biotechnology industry for the production of zeaxanthin.     

Effects of Regulatory Mutations upon Methionine Biosynthesis in Saccharomyces cerevisiae: Loci eth2-eth3-eth10

The effects of mutations occurring at three independent loci, eth2, eth3, and eth10, were studied on the basis of several criteria: level of resistance towards two methionine analogues (ethionine and selenomethionine), pool sizes of free methionine and S-adenosyl methionine (SAM) under different growth conditions, and susceptibility towards methionine-mediated repression and SAM-mediated repression of some enzymes involved in methionine biosynthesis (met group I enzymes). It was shown that: (i) the level of resistance towards both methionine analogues roughly correlates with the amount of methionine accumulated in the pool; (ii) the repressibility of met group I enzymes by exogenous methionine is either abolished or greatly lowered, depending upon the mutation studied; (iii) the repressibility of the same enzymes by exogenous SAM remains, in at least three mutants studied, close to that observed in a wild-type strain; (iv) the accumulation of SAM does not occur in the most extreme mutants either from endogenously overproduced or from exogenously supplied methionine: (v) the two methionine-activating enzymes, methionyl-transfer ribonucleic acid (tRNA) synthetase and methionine adenosyl transferase, do not seem modified in any of the mutants presented here; and (vi) the amount of tRNA^met and its level of charging are alike in all strains. Thus, the three recessive mutations presented here affect methionine-mediated repression, both at the level of overall methionine biosynthesis which results in its accumulation in the pool, and at the level of the synthesis of met group I enzymes. The implications of these findings are discussed.     

Storage protein characteristics of proline-requiring mutants of Zea mays (L.)

Summary Protein and amino acid composition of mature karnels from three allelic proline-requiring mutants in maize, pro _1-1, pro _1-2, and pro _1-3 were analyzed and compared to kernels of the stock A 188 containing the wild type allele. The amount of free proline was specifically reduced in the embryos of all three mutants, while in the endosperm such a reduction was only found for pro _1-2 and pro _1-3 Accumulation of the proline-rich zeins was strongly reduced in the mutants, but in contrast to opaque-2 the reduction affected all major zein polypeptides to the same extent, possibly as a consequence of the defective proline metabolism. Albumins and globulins as well as free amino acids were more abundant in the endosperms of the mutants than in the wild type. Analysis of the albumins and globulins by SDS-PAGE revealed specific increases as well as reductions of certain polypeptides in the endosperms and embryos of the mutants.     

Amino Acid Metabolism of Lemna minor L. : II. Responses to Chlorsulfuron

Chlorsulfuron, an inhibitor of acetolactate synthase (EC 4.1.3.18) (TB Ray 1984 Plant Physiol 75: 827-831), markedly inhibited the growth of Lemna minor at concentrations of 10⁻⁸ molar and above, but had no inhibitory effects on growth at 10⁻⁹ molar. At growth inhibitory concentrations, chlorsulfuron caused a pronounced increase in total free amino acid levels within 24 hours. Valine, leucine, and isoleucine, however, became smaller percentages of the total free amino acid pool as the concentration of chlorsulfuron was increased. At concentrations of chlorsulfuron of 10⁻⁸ molar and above, a new amino acid was accumulated in the free pool. This amino acid was identified as α-amino-n-butyrate by chemical ionization and electron impact gas chromatography-mass spectrometry. The amount of α-amino-n-butyrate increased from undetectable levels in untreated plants, to as high as 840 nanomoles per gram fresh weight (2.44% of the total free pool) in plants treated with 10⁻⁴ molar chlorsulfuron for 24 hours. The accumulation of this amino acid was completely inhibited by methionine sulfoximine. Chlorsulfuron did not inhibit the methionine sulfoximine induced accumulations of valine, leucine, and isoleucine, supporting the idea that the accumulation of the branched-chain amino acids in methionine sulfoximine treated plants is the result of protein turnover rather than enhanced synthesis. Protein turnover may be primarily responsible for the failure to achieve complete depletion of valine, leucine, and isoleucine even at concentrations of chlorsulfuron some 10⁴ times greater than that required to inhibit growth. Tracer studies with ¹⁵N demonstrate that chlorsulfuron inhibits the incorporation of ¹⁵N into valine, leucine, and isoleucine. The α-amino-n-butyrate accumulated in the presence of chlorsulfuron and [¹⁵N]H₄⁺ was heavily labeled with ¹⁵N at early time points and appeared to be derived by transamination from a rapidly labeled amino acid such as glutamate or alanine. We propose that chlorsulfuron inhibition of acetolactate synthase may lead to accumulation of 2-oxobutyrate in the isoleucine branch of the pathway, and transamination of 2-oxobutyrate to α-amino-n-butyrate by a constitutive transaminase utilizing either glutamate or alanine as α-amino-N donors.     

Cellular compartmentation of aromatic amino acids in Neurospora crassa. I. Occupation of a protein synthesis pool by phenylalanine in Tyr-1 mutants

The p-fluorophenylalanine (FPA) resistance of acc ^phe, which has previously been shown (Brooks et al., 1972) to be a try-1 mutant, has been further investigated. When incubated in the absence of tyrosine, acc ^phe and also tyr-1 auxotrophs show a gradual increase in free phenylalanine in the cell but a sharp decrease in FPA incorporation into protein. The decrease in FPA incorporation is apparently due to the excess phenylalanine in the mutants, since the normal endogenous pool component in wild type and also in the mutants incubated on tyrosine does not appear to compete with FPA for incorporation. The rate of FPA incorporation into protein in acc ^phe remains at 10–15% of the wild-type rate even when the ratio of free FPA to excess phenylalanine in the cell is high as 8:1. If wild type is supplied with exogenous phenylalanine and FPA simultaneously, phenylalanine is preferentially incorporated into protein but, in contrast to the mutant, the rate of FPA incorporation increases as the ratio of free FPA to phenylalanine increases. On the basis of differences in competition with FPA and in susceptibilities to mild extraction procedures, it is proposed that phenylalanine can be located in at least three compartments in Neurospora: a small constant-size endogenous pool always seen in wild type; an expandable exogenous pool; and a protein synthesis pool which is preferentially populated by endogenous phenylalanine but can be entered by exogenous molecules when biosynthesis is regulated. In acc ^phe, where phenylalanine biosynthesis is not regulated, the excess phenylalanine is located primarily in the protein synthesis pool where it only has to compete with a small FPA component and is thereby preferentially incorporated into protein in this mutant.This work was supported, in part, by an Atomic Energy Commission grant to the Institute of Molecular Biophysics, The Florida State University, and by the Genetics Training Grant, funded by the National Institutes of Health. It contains, in part, data from the doctoral thesis of the senior author, who was supported by a Florida State University Nuclear Fellowship and by a Public Health Service Fellowship.     

Enzyme-induced covalent modification of methionyl-tRNA synthetase from Bacillus stearothermophilus by methionyl-adenylate: identification of the labeled amino acid residues by matrix-assisted laser desorption-ionization mass spectrometry

Methionyl-tRNA synthetase (MetRS) from Bacillus stearothermophilus was shown to undergo covalent methionylation by a donor methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by the enzyme itself. Covalent reaction of methionyl-adenylate with the synthetase or other proteins proceeds through the formation of an isopeptide bond between the carboxylate of the amino acid and the -NH₂ group of lysyl residues. The stoichiometries of labeling, as followed by TCA precipitation, were 2.2 ± 0.1 and 4.3 ± 0.1 mol of [¹⁴C]Met incorporated by 1 mol of the monomeric MS534 and the native dimeric species of B. stearo methionyl-tRNA synthetase, respectively. Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-261, -295, -301 and -528 (or -534) of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine. By analogy with the 3D structure of the monomeric M547 species of E. coli methionyl-tRNA synthetase, lysines-261, -295, and -301 would be located in the catalytic crevice of the thermostable enzyme where methionine activation and transfer take place. It is proposed that, once activated by ATP, most of the methionine molecules react with the closest reactive lysyl residues.     

Determination of 35S-aminoacyl-transfer ribonucleic acid specific radioactivity in small tissue samples

Rate determination of protein synthesis utilizing tracer amino acid incorporation requires accurate assessment of the specific radioactivity of the labeled precursor aminoacyl-tRNA pool. Previously published methods presumably useful for the measurement of any aminoacyl-tRNA were unsuccessful when applied to [³⁵S]methionine, due to the unique chemical properties of this amino acid. Herein we describe modifications of these methods necessary for the measurement of ³⁵S-aminoacyl-tRNA specific radioactivity from small tissue samples incubated in the presence of [³⁵S]methionine. The use of [³⁵S]methionine of high specific radioactivity enables analysis of the methionyl-tRNA from less than 100 mg of tissue. Conditions for optimal recovery of ³⁵S-labeled dansyl-amino acid derivatives are presented and possible applications of this method are discussed.