FKBP12 physically and functionally interacts with aspartokinase in Saccharomyces cerevisiae.

The peptidyl-prolyl isomerase FKBP12 was originally identified as the intracellular receptor for the immunosuppressive drugs FK506 (tacrolimus) and rapamycin (sirolimus). Although peptidyl-prolyl isomerases have been implicated in catalyzing protein folding, the cellular functions of FKBP12 in Saccharomyces cerevisiae and other organisms are largely unknown. Using the yeast two-hybrid system, we identified aspartokinase, an enzyme that catalyzes an intermediate step in threonine and methionine biosynthesis, as an in vivo binding target of FKBP12. Aspartokinase also binds FKBP12 in vitro, and drugs that bind the FKBP12 active site, or mutations in FKBP12 surface and active site residues, disrupt the FKBP12-aspartokinase complex in vivo and in vitro.fpr1 mutants lacking FKBP12 are viable, are not threonine or methionine auxotrophs, and express wild-type levels of aspartokinase protein and activity; thus, FKBP12 is not essential for aspartokinase activity. The activity of aspartokinase is regulated by feedback inhibition by product, and genetic analyses reveal that FKBP12 is important for this feedback inhibition, possibly by catalyzing aspartokinase conformational changes in response to product binding.     

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.     

Genetic and biochemical study of threonine-overproducing mutants of Saccharomyces cerevisiae.

Three threonine-overproducing mutants were obtained as prototrophic revertants of a hom3 mutant strain of Saccharomyces cerevisiae. The gene HOM3 codes for aspartokinase (aspartate kinase; EC 2.7.2.4), the first enzyme of the threonine-methionine biosynthetic route, which is subjected to feedback inhibition by threonine. Enzymatic studies indicated that aspartokinase from the revertants has lost the feedback inhibition, resulting in overproduction of threonine. These revertants also bore one or two additional mutations, named tex1-1 and tex2-1, which alone or jointly made possible the excretion of the threonine accumulated. The effect of these two genes on excretion is potentiated by excess inositol in the medium.     

Transductional construction of a threonine-hyperproducing strain of Serratia marcescens: lack of feedback controls of three aspartokinases and two homoserine dehydrogenases

To construct a threonine-hyperproducing strain of Serratia marcescens Sr41, the six regulatory mutations for three aspartokinases and two homoserine dehydrogenases were combined in a single strain by three transductional crosses. The constructed strain, T-1026, carried the lysC1 mutation leading to lack of feedback inhibition and repression of aspartokinase III, the thrA1(1) mutation desensitizing aspartokinase I to feedback inhibition, the thrA2(1) mutation releasing feedback inhibition of homoserine dehydrogenase I, the two hnr mutations derepressing aspartokinase I and homoserine dehydrogenase I, and the etr-1 mutation derepressing aspartokinase II and homoserine dehydrogenase II. The strain produced ca. 40 mg of threonine per ml of medium containing sucrose and urea. Furthermore, the productivity of strain T-1026 was compared with those of strains devoid of more than one of the six regulatory mutations.     

Transductional construction of a threonine-hyperproducing strain of Serratia marcescens: lack of feedback controls of three aspartokinases and two homoserine dehydrogenases.

To construct a threonine-hyperproducing strain of Serratia marcescens Sr41, the six regulatory mutations for three aspartokinases and two homoserine dehydrogenases were combined in a single strain by three transductional crosses. The constructed strain, T-1026, carried the lysC1 mutation leading to lack of feedback inhibition and repression of aspartokinase III, the thrA1(1) mutation desensitizing aspartokinase I to feedback inhibition, the thrA2(1) mutation releasing feedback inhibition of homoserine dehydrogenase I, the two hnr mutations derepressing aspartokinase I and homoserine dehydrogenase I, and the etr-1 mutation derepressing aspartokinase II and homoserine dehydrogenase II. The strain produced ca. 40 mg of threonine per ml of medium containing sucrose and urea. Furthermore, the productivity of strain T-1026 was compared with those of strains devoid of more than one of the six regulatory mutations.     

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.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. Kinetic and spectroscopic effects upon binding of serine and threonine.

The two threonine-sensitive activities aspartokinase and homoserine dehydrogenase are inhibited by L-serine. The inhibition of the aspartokinase by L-serine displays homotropic cooperative effects and is competitive versus aspartate. The inhibition by L-serine of the homoserine dehydrogenase displays Michaelis-Menten kinetics which are of a competitive nature versus homoserine. Characteristic effects of L-serine on the protein include a perturbation of its absorption and fluorescence spectra, with an increase in the fluorescence of the protein-NADPH complex. L-serine shifts the allosteric equilibrium of the protein to a "T-like" conformation to which L-threonine binds noncooperatively. L-Serine, a threonine analog, is not capable, as the physiological effector, of inducing a complete R to T transition of the enzyme; the aspartokinase globules show a cooperative conformation change upon serine binding, but this conformation change is not found in the homoserine dehydrogenase globules.     

Homoserine and threonine pools of borrelidin resistant Saccharomyces cerevisiae mutants with an altered aspartokinase

Borrelidin is a specific inhibitor of the threonyl-tRNA-synthethase. A class of dominant borrelidin resistant mutants (BOR1) of Saccharomyces cerevisiae was biochemically characterized. The mutants possess an altered aspartokinase which is insensitive to threonine inhibition. The threonine and homoserine pools in these mutants are 22 times larger than in the wild type. By feeding aspartate under a variety of conditions the homoserine pool is increased even 57 times whilst the threonine pool is reduced.     

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.     

Dissociation properties of aspartokinase I-homoserine dehydrogenase I extracted from a missense mutant of E. coli K12

The threonine sensitive aspartokinase-homoserine dehydrogenase devoid of aspartokinase activity has been extracted from a missense mutant of $\text{E. coli}$ K12 and some of its properties have been investigated. The genetic localization of the corresponding mutation indicated that the amino acid replacement lies in the kinase region of the molecule. The cooperativity of threonine inhibition of the homoserine dehydrogenase activity is lowered. The measurement of the molecular weight of the enzyme in presence or absence of threonine indicates that the molecule dissociates more easily than the wild type enzyme. These results are discussed in view of the recent structural model proposed for aspartokinase I-homoserine dehydrogenase I.     

Hmo1p, a high mobility group 1/2 homolog, genetically and physically interacts with the yeast FKBP12 prolyl isomerase

The immunosuppressive drugs FK506 and rapamycin bind to the cellular protein FKBP12, and the resulting FKBP12-drug complexes inhibit signal transduction. FKBP12 is a ubiquitous, highly conserved, abundant enzyme that catalyzes a rate-limiting step in protein folding: peptidyl-prolyl cis-trans isomerization. However, FKBP12 is dispensible for viability in both yeast and mice, and therefore does not play an essential role in protein folding. The functions of FKBP12 may involve interactions with a number of partner proteins, and a few proteins that interact with FKBP12 in the absence of FK506 or rapamycin have been identified, including the ryanodine receptor, aspartokinase, and the type II TGF-beta receptor; however, none of these are conserved from yeast to humans. To identify other targets and functions of FKBP12, we have screened for mutations that are synthetically lethal with an FKBP12 mutation in yeast. We find that mutations in HMO1, which encodes a high mobility group 1/2 homolog, are synthetically lethal with mutations in the yeast FPR1 gene encoding FKBP12. Deltahmo1 and Deltafpr1 mutants share two phenotypes: an increased rate of plasmid loss and slow growth. In addition, Hmo1p and FKBP12 physically interact in FKBP12 affinity chromatography experiments, and two-hybrid experiments suggest that FKBP12 regulates Hmo1p-Hmo1p or Hmo1p-DNA interactions. Because HMG1/2 proteins are conserved from yeast to humans, our findings suggest that FKBP12-HMG1/2 interactions could represent the first conserved function of FKBP12 other than mediating FK506 and rapamycin actions.     

Enhancement of isoleucine hydroxamate-mediated growth inhibition and improvement of isoleucine-producing strains of Serratia marcescens.

Growth inhibition by isoleucine hydroxamate in Serratia marcescens was significantly enhanced by adding valine plus leucine and by using glycerol as the carbon source. Isoleucine hydroxamate-resistant mutants were isolated under conditions in which growth inhibition was enhanced. One of the mutants, strain GIHVLr2179, lacked both feedback inhibition and repression of threonine deaminase. An alpha-aminobutyric acid-resistant mutant derived from strain GIHVLr2179, strain GIHVLAr2795, produced 12 mg of isoleucine per ml in the medium containing glucose and urea as carbon and nitrogen sources (a twofold increase over prior reports). This strain had increased activities of threonine deaminase, acetohydroxy acid synthase, aspartokinase, and homoserine dehydrogenase.     

Effect of gene amplification on threonine production by yeast

In this work, we have studied the effect of amplifying different alleles involved in the threonine biosynthesis on the amino acid production by Saccharomyces cerevisiae. The genes used were wild-type HOM3, HOM2, HOM6, THR1, and THR4, and two mutant alleles of HOM3 (namely HOM3-R2 and HOM3-R6), that code for feedback-insensitive aspartate kinases. The results show that only the amplification of the HOM3 alleles leads to threonine and, in some instances, to homoserine overproduction. In terms of the regulation of the pathway, the data indicate that the main control is exerted by inhibition of the aspartate kinase and that, probably, a second and less important regulation takes place at the level of the homoserine kinase, the THR1 gene product. However, amplification of THR1 in two related Hom3-R2 strains does not increase the amount of threonine but, in one of them, it does induce accumulation of more homoserine. This result probably reflects differences between these strains in some undetermined genetic factor/s related with threonine metabolism. In general, the data indicate that the common laboratory yeast strains are genetically rather heterogeneous and, thus, extrapolation of conclusions must be done carefully. (c) 1996 John Wiley & Sons, Inc.     

Enhancement of isoleucine hydroxamate-mediated growth inhibition and improvement of isoleucine-producing strains of Serratia marcescens.

Growth inhibition by isoleucine hydroxamate in Serratia marcescens was significantly enhanced by adding valine plus leucine and by using glycerol as the carbon source. Isoleucine hydroxamate-resistant mutants were isolated under conditions in which growth inhibition was enhanced. One of the mutants, strain GIHVLr2179, lacked both feedback inhibition and repression of threonine deaminase. An alpha-aminobutyric acid-resistant mutant derived from strain GIHVLr2179, strain GIHVLAr2795, produced 12 mg of isoleucine per ml in the medium containing glucose and urea as carbon and nitrogen sources (a twofold increase over prior reports). This strain had increased activities of threonine deaminase, acetohydroxy acid synthase, aspartokinase, and homoserine dehydrogenase.     

Transductional construction of a threonine-producing strain of Serratia marcescens.

A threonine-producing strain of Serratia marcescens Sr41 was constructed according to the following process. Thr- strain E-60 was derived from strain HNr59 having constitutive levels of threonine-sensitive aspartokinase and homoserine dehydrogenase. Thr+ transductant T-570 was constructed from strain E-60 and phage grown on strain HNr21 having feedback-resistant threonine-sensitive aspartokinase and homoserine dehydrogenase. This transductant lacked both feedback inhibition and repression for the two enzymes. Thr- strain N-11 was derived from strain AECr174 lacking feedback inhibition and repression of lysine-sensitive aspartokinase. Subsequently, the threonine region of strain T-570 was transduced into strain N-11. One of the THR+ transductants, strain T-693, produced markedly high levels of the two aspartokinases and homoserine dehydrogenase, which were insensitive to feedback inhibition. This strain produced about 25 mg of threonine per ml in the medium containing sucrose and urea.     

Regulatory Structure of the Biosynthetic Pathway for the Aspartate Family of Amino Acids in Lemna paucicostata Hegelm. 6746, with Special Reference to the Role of Aspartokinase

Comprehensive studies were made with Lemna paucicostata Hegelm. 6746 of the effects of combinations of lysine, methionine, and threonine on growth rates, soluble amino acid contents, aspartokinase activities, and fluxes of 4-carbon moieties from aspartate through the aspartokinase step into the amino acids of the aspartate family. These studies show that flux in vitro through the aspartokinase step is insensitive to inhibition by lysine or threonine, and confirm previous in vitro data in establishing that aspartokinase in vivo is present in two orders of magnitude excess of its requirements. No evidence of channeling of the products of the lysine- and threonine-sensitive aspartokinases was obtained, either form of the enzyme alone being more than adequate for the combined in vivo flux through the aspartokinase step. The marked insensitivity of flux through the aspartokinase step to inhibition by lysine or threonine strongly suggests that inhibition of aspartokinase by these amino acids is not normally a major factor in regulation of entry of 4-carbon units into the aspartate family of amino acids. Direct measurement of fluxes of 4-carbon units demonstrated that: (a) Lysine strongly feedback regulates its own synthesis, probably at the step catalyzed by dihydrodipicolinate synthase. (b) Threonine alone does not regulate its own synthesis in vivo, thereby confirming previous studies of the metabolism of [¹⁴C]threonine and [¹⁴C]homoserine in Lemna. This finding excludes not only aspartokinases as an important regulatory determinant of threonine synthesis, but also two other enzymes (homoserine dehydrogenase and threonine synthase) suggested to fulfill this role. Complete inhibition of threonine synthesis was observed only in the combined presence of accumulated threonine and lysine. The physiological significance of this single example of apparent regulation of flux at the aspartokinase step, albeit under unusually stringent conditions of aspartokinase inhibition, remains to be determined. (c) Isoleucine strongly inhibits its own synthesis, probably at threonine dehydratase, without causing compensatory reduction in threonine synthesis. A fundamentally changed scheme for regulation of synthesis of the aspartate family of amino acids is presented that has important implications for improvement of the nutritional contents of these amino acids in plants.     

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.