Selenalysine and protein synthesis.

Selenalysine is a lysine analog having the gamma-methylene group substituted by a selenium atom. It has been demonstrated that selenalysine is activated and transferred to tRNAlys by either Escherichia coli or rat liver aminoacyl-tRNA synthetases, and inhibits lysine incorporation into polypeptides in protein-synthesizing systems from E. coli, rat liver or rabbit reticulocytes. All tests were performed in comparison with thialysine, a lysine analog having the gamma-methylene group substituted by a sulfur atom. In all the reactions studied, both thialysine and selenalysine act as competitive inhibitors of lysine. With respect to thialysine, selenalysine act as competitive inhibitors of lysine. With respect to thialysine, selenalysine shows a slightly lower activity as lysine inhibitor.     

Aspartokinase III repression and lysine analogs utilization for protein synthesis

The extents of thialysine and selenalysine incorporation into cell proteins were compared in E. coli KL16 and in a mutant able to grow equally well in the presence or in the absence of both lysine analogs. The mutant differs from the parental strain in the repression of aspartokinase III (AKIII), the first enzyme of the lysine biosynthetic pathway. No analog incorporation into proteins was observed in mutant cells grown in the presence of either analog, whereas a marked analog incorporation was observed in the parental strain, where up to 17% and 12% of protein lysine can be substituted by thialysine and selenalysine respectively. In the parental strain grown in media containing either analog at different concentration the extent of analog incorporation into proteins is related to the extent of AKIII repression.     

Thialysine and selenalysine utilization for protein synthesis by E. coli in comparison with lysine

Utilization of thialysine and selenalysine for protein synthesis by a lysine requiring E. coli mutant was studied. Incorporation into proteins of thialysine or selenalysine, added to culture medium together with lysine, becomes evident when the amount of available lysine in the medium is highly reduced, that is the mutant utilizes the isologs only after all the available natural aminoacid has been utilized. Compared to selenalysine, thialysine is better utilized; when both isologs are present in the medium at equal concentrations, up to 46% of protein lysine is substituted by thialysine and only 12% by selenalysine.     

Thialysine- and selenalysine-resistance in a E. coli mutant

A thialysine-resistant mutant of E. coli strain KL16 also shows a lower sensitivity to selenalysine, the lysine analog containing selenium. No difference between the mutant and the parental strain has been shown regarding the affinities of the transport systems and the lysyl-tRNA synthetase for selenalysine, thialysine and lysine as well as the inhibitory effects of these three aminoacids on the activity of the lysine biosynthetic pathway. A marked difference between the two strains has been evidenced in the AK III repression: in the mutant the repression by selenalysine, thialysine and lysine is much lower than in the parental strain.     

Utilization of lysine analogs by a lysine-requiring E. coli mutant

Thialysine and selenalysine cannot substitute lysine as a growth factor for a lysine-requiring E. coli mutant, but can nevertheless be utilized for protein synthesis in the presence of lysine. In order to have information about the effects of lysine on the utilization of the two analogs, the extent of the incorporation of the three aminoacids into newly synthesized proteins has been determined. The analog starts to be utilized by cells growing in a medium containing either analog and lysine when lysine concentration becomes very low. Of the two analogs, thialysine is more easily utilized. In fact thialysine can be utilized when the lysine/thialysine ratio in the medium is 1/25. Selenalysine starts to be utilized when the lysine/selenalysine ratio is 1/200.     

Degradation of thialysine- or selenalysine-containing abnormal proteins in CHO cells

CHO cells can incorporate thialysine and selenalysine in their proteins in substitution of lysine. Data are reported in the present paper showing that proteins containing either thialysine or selenalysine are unstable and quite rapidly degraded. The degradation rate is strictly related to the extent of protein lysine substitution. At similar extent of substitution, selenalysine-containing proteins are more unstable that thialysine-containing ones.     

Biochemical characterization of a thialysine-resistant clone of CHO cells

The intracellular transport and the activation of lysine, thialysine and selenalysine have been investigated in a thialysine-resistant CHO cell mutant strain in comparison with the parental strain. The cationic amino acid transport system responsible for the transport of these 3 amino acids shows no differences between the 2 strains as regards its affinity for each of these amino acids. On the other hand the Vmax of the transport system in the mutant is about double that in the parental strain. The lysyl-tRNA synthetase, assayed both as ATP = PPi exchange reaction and lysyl-tRNA synthesis, shows a lower affinity for thialysine and selenalysine than for lysine in both strains; in the mutant, however, the difference is even greater. Thus the thialysine resistance of the mutant is mainly due to the properties of its lysyl-tRNA synthetase, which shows a greater difference of the affinities for lysine and thialysine with respect to the parental strain.     

Repression of lysine transport in E. coli KL16

Data reported in this paper show that both lysine transport systems in E. coli KL16 can be repressed by lysine and its isologs, thialysine and selenalysine, whereas they are not repressed by ornithine. The repression is specific on lysine transport systems; it is evident with 0.01 mM lysine or isolog concentration and reaches a maximum with 0.1 mM concentration. By comparing the extent of repression by lysine and its isologs, lysine gives the highest and selenalysine the lowest degree of repression. The shift from the repressed to the depressed state is rather immediate once the amino acid is removed from the culture medium.     

Thialysine utilization by E. coli and its effects on cell growth

Summary Thialysine and selenalysine, two lysine isologs having the -methylene group substituted by a sulfur or a selenium atom, respectively, inhibit E. coli lysine-sensitive aspartokinase. The inhibition is specific, reversible and non-competitive. Compared to lysine, the two isologs have a less marked inhibitory effect, but show a similar homotropic cooperativity with a Hill's coefficient of about 2. The inhibition by each isolog is additive to that by lysine. Both compounds protect the enzyme against thermal inactivation. Overall, the data reported indicate that thialysine and selenalysine bind to the same allosteric site of lysine, the physiological modulator of the enzyme.     

Intracellular transport of thialysine and selenalysine in CHO cells

The intracellular transport of thialysine and selenalysine in CHO cells has been studied. Data have been obtained indicating that the two lysine analogs can be transported by both the cationic aminoacid transport system and by the L transport system. The affinity of the cationic aminoacid transport system is similar for the two lysine analogs but lower than that for lysine and the affinity of the L transport system for the two lysine analogs is lower than that for leucine.     

Selenalysine utilization for growth and protein synthesis by a lysine requiring E. coli mutant

Summary Selenalysine can be utilized in substitution of lysine by a lysine requiring E. coli mutant. The presence of some lysine in the culture medium is necessary to allow selenalysine utilization for growth; in the presence of an excess of lysine, selenalysine is not utilized. When utilized, selenalysine gives rise to an increase of final growth. However, it shows some toxic effects as demonstrated by the decrease of both growth rate and cell viability. Selenalysine is incorporated into proteins in substitution of lysine. Up to a maximum of 50% of total protein lysine can be substituted. The decrease of cell viability is correlated with the extent of lysine substitution.This paper is dedicated to Professor A. E. Braunstein on his 80th birthday.     

Isolation and properties of a thialysine-resistant clone of CHO cells

A variant clone of Chinese hamster ovary (CHO) cells resistant to thialysine has been isolated. It maintains the phenotypic properties even after 250 generations in medium without thialysine. Growth rate, cell viability and protein synthesis rate of the variant are much less affected by thialysine than the parental strain. In both the parental strain and the variant, thialysine acts in competition with lysine as indicated by the fact that all thialysine effects can be completely reversed by lysine.     

Transport systems for lysine, thialysine and selenalysine in E. coli KL16

Two lysine transport systems have been identified in E. coli KL16. They differ in their affinity for lysine, one showing a KM of 0.36 microM and the other a KM of 4.7 microM. Different compounds with chemical similarities to lysine were tested for their capacity to interfere with lysine transport. Among these only thialysine and selenalysine competitively inhibit lysine transport. The inhibition is on both transport systems. Thialysine shows a KI of 4 microM for the low affinity system and a KI of 8 microM for the high affinity system. Selenalysine shows values of 6 microM and 12 microM respectively.     

Thialysine utilization for protein synthesis by an exponentially growing E. coli culture

The extent of protein lysine substitution by thialysine in E. coli cells grown in media containing the analog depends on the time interval the cells are grown in the presence of analog and on the analog concentration in the medium. By calculating the percent of lysine substitution in newly synthesized proteins it was shown that this reaches, after one cell doubling in the presence of analog, a maximum which is 17% in the cells grown with 0.1 or 0.2 mM thialysine and 8% in cells grown with 0.05 mM thialysine. Proteins synthesized in the presence of analog in the concentration range 0.05-0.2 mM show similar stability to those synthesized in the absence of analog. The extent of analog incorporation into newly synthesized proteins, as regards both the time course and the dependence on analog concentration in the medium, is strictly related to the extent of the repression of AK III, the first enzyme of lysine biosynthetic pathway.     

Thialysine utilization by a lysine-requiring Escherichia coli mutant

Summary Thialysine cannot completely substitute lysine as growth factor for a lysine-requiring E. coli mutant. However it can be utilized for growth in the presence of limiting amounts of lysine, in substitution of, and in competition with this latter. The effects of thialysine on growth rate, protein synthesis rate and cell viability, and its incorporation into proteins were studied in function of lysine and thialysine concentration in the culture media. Up to 60% of protein lysine substitution by thialysine is observed, without appreciable effects on cell viability.     

Selenalysine utilization by CHO cells

CHO cells allowed to grow in a medium containing selenalysine can utilize it for protein synthesis. Selenalysine is incorporated into cell proteins in substitution of lysine: a maximum of 5% of protein lysine can be substituted. Protein lysine substitution by selenalysine can be correlated to the reduced viability of cells grown in its presence.     

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.     

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 caused 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 enzymes synthesis as well as inhibition of activity.     

Degradation of thialysine- or selenalysine-containing abnormal proteins in E. coli

Thialysine and selenalysine can be utilized for protein synthesis by lysine-requiring E. coli cells even in the absence of lysine. Protein synthesis has been determined as labeled leucine incorporation into acid-insoluble material, as increase of cell proteins and as protein-lysine substitution by the analog. Either analog can be incorporated into proteins, in the absence of lysine, for a limited time interval after which cells stop to duplicate. Proteins synthesized during this period contain most of their lysine residues substituted by the analog. Moreover, it has been shown that the analog-containing proteins are unstable and rapidly degraded. Their instability would account for the inability of lysine-requiring E. coli cells to utilize the analog as growth factor.     

Lysine overproduction mutations in the yeast Saccharomyces cerevisiae and its transfection into industrial Yeast strains ]

Yeast mutants resistant to a toxic lysine analog, thialysine were obtained by a method described in the literature. A strain excreting the maximum amount of lysine (0.45 g/l) was selected from these mutants. The intracellular content of lysine was also increased by 30%. The genetic nature of lysine overproduction was studied in this strain. An increase in the amount of excreted lysine was shown to be determined by at least two genes, one of which carries a mutation of thialysine resistance manifesting the pleiotropic effect of lysine overproduction (Th1R) and the other is involved in the regulation of lysine production (PRL). Linkage groups of these genes were determined: the first gene was mapped to the IV chromosome and the second, to the XV chromosome. Both genetic characters were introduced into industrial baker's yeast strains via a series of backcrosses. The stabilization of the genome in the newly derived strains was confirmed by electrokaryotyping.