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

1982 ◽  
Vol 2 (7) ◽  
pp. 731-736 ◽  
Author(s):  
M A Delgado ◽  
J Guerrero ◽  
J Conde
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutant Strain ◽  
Feedback Inhibition ◽  
Biochemical Study ◽  
Aspartate Kinase

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.

scholarly journals Genetic and biochemical study of threonine-overproducing mutants of Saccharomyces cerevisiae

1982 ◽  
Vol 2 (7) ◽  
pp. 731-736
Author(s):  
M A Delgado ◽  
J Guerrero ◽  
J Conde
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutant Strain ◽  
Feedback Inhibition ◽  
Biochemical Study ◽  
Aspartate Kinase

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.

The GCR1 gene encodes a positive transcriptional regulator of the enolase and glyceraldehyde-3-phosphate dehydrogenase gene families in Saccharomyces cerevisiae

1987 ◽  
Vol 7 (2) ◽  
pp. 813-820
Author(s):  
M J Holland ◽  
T Yokoi ◽  
J P Holland ◽  
K Myambo ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutant Strain ◽  
Gene Families ◽  
Gene Replacement ◽  
Insertion Mutation ◽  
Gene Insertion ◽  
Glycolytic Gene ◽  
Dehydrogenase Gene ◽  
Multiple Copies ◽  
State Concentration

The intracellular concentrations of the polypeptides encoded by the two enolase (ENO1 and ENO2) and three glyceraldehyde-3-phosphate dehydrogenase (TDH1, TDH2, and TDH3) genes were coordinately reduced more than 20-fold in a Saccharomyces cerevisiae strain carrying the gcr1-1 mutation. The steady-state concentration of glyceraldehyde-3-phosphate dehydrogenase mRNA was shown to be approximately 50-fold reduced in the mutant strain. Overexpression of enolase and glyceraldehyde-3-phosphate dehydrogenase in strains carrying multiple copies of either ENO1 or TDH3 was reduced more than 50-fold in strains carrying the gcr1-1 mutation. These results demonstrated that the GCR1 gene encodes a trans-acting factor which is required for efficient and coordinate expression of these glycolytic gene families. The GCR1 gene and the gcr1-1 mutant allele were cloned and sequenced. GCR1 encodes a predicted 844-amino-acid polypeptide; the gcr1-1 allele contains a 1-base-pair insertion mutation at codon 304. A null mutant carrying a deletion of 90% of the GCR1 coding sequence and a URA3 gene insertion was constructed by gene replacement. The phenotype of a strain carrying this null mutation was identical to that of the gcr1-1 mutant strain.

Cellular Amino Acid Concentrations and Regulation of Aspartate Kinase in the Thermophilic Phototrophic Prokaryote Chloroflexus aurantiacus

1990 ◽  
Vol 45 (1-2) ◽  
pp. 74-78 ◽  
Author(s):  
Jobst-Heinrich Klemme ◽  
Gisela Laakmann-Ditges ◽  
Jutta Mertschuweit
Keyword(s):  
Amino Acid ◽  
Feedback Inhibition ◽  
Gel Filtration ◽  
Break Point ◽  
Test System ◽  
Chloroflexus Aurantiacus ◽  
Aspartate Kinase ◽  
Homoserine Dehydrogenase ◽  
Molecular Weights ◽  
Amino Acid Concentrations

Aspartate kinase (AK , EC 2.7.2.4) from the thermophilic, phototrophic prokaryote, Chloroflexus aurantiacus, was partially purified and separated from homoserine dehydrogenase (HSDH, EC 1.1.1.3). The molecular weights as determined by gel filtration were 130,000 and 46,000, respectively. HSDH had a moderately high thermal stability (50% inactivation at 84 °C) and displayed its activity optimum at 72 °C. By contrast, AK had its activity optimum at 52 °C (with a break-point in the Arrhenius plot at 42 °C) and was much less thermostable (50% inactivation at 67 °C). The Km-values for aspartate and ATP (determined in a pyruvate kinase-coupled test system) were 10.5 and 0.63 mM , respectively. The enzyme was strongly inhibited by L-threonine (Ki = 10 μm) and activated by alanine, isoleucine, valine and methionine. L-Threonine acted as a mixed-type inhibitor in respect to aspartate, and non-competitively in respect to ATP. Contrary to AKs from Rhodospirillaceae, the enzyme from Chloroflexus aurantiacus was not subject to a concerted feedback inhibition by two amino acids of the aspartate family. The regulatory properties of the aspartate kinase are discussed in relation to the cellular amino acid concentrations.

Effect of valine and the herbicide sulfometuron methyl on acetolactate synthase activity in nuclear and plasmid-borne sulphometuron methyl resistant Saccharomyces cerevisiae strains

1988 ◽  
Vol 34 (5) ◽  
pp. 680-685 ◽  
Author(s):  
S. N. Maiti ◽  
M. W. Zink ◽  
G. H. Rank
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Copy Number ◽  
Specific Activity ◽  
Feedback Inhibition ◽  
Acetolactate Synthase ◽  
Wild Type ◽  
Sulfometuron Methyl ◽  
Isogenic Lines

Acetolactate synthase (ALS) specific activity was evaluated in isogenic lines of Saccharomyces cerevisiae carrying the wild-type ILV2 gene or mutations in this gene for resistance to the herbicide sulfometuron methyl (SM). Statistical comparisons were made between two nuclear alleles and among five alleles borne on a YE chimaeric plasmid transformed into a strain carrying a 1.5-kilobase deletion in the nuclear ILV2 gene. Decreased ALS activity of plasmid-borne SM-resistant mutations was shown not to be caused by copy number effects. ALS-specific activity in strains carrying the wild-type ILV2 allele exhibited strong feedback inhibition by valine and was sensitive to SM. All nuclear and plasmid-borne SM-resistance alleles resulted in ALS-specific activity highly resistant to SM and resistant to valine feedback inhibition.

scholarly journals FKBP12 Controls Aspartate Pathway Flux in Saccharomyces cerevisiae To Prevent Toxic Intermediate Accumulation

2004 ◽  
Vol 3 (5) ◽  
pp. 1287-1296 ◽  
Author(s):  
Miguel Arévalo-Rodríguez ◽  
Xuewen Pan ◽  
Jef D. Boeke ◽  
Joseph Heitman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Feedback Inhibition ◽  
Cellular Functions ◽  
Prolyl Isomerase ◽  
Homoserine Dehydrogenase ◽  
Semialdehyde Dehydrogenase ◽  
Common Precursor ◽  
Aspartate Pathway ◽  
Synthetically Lethal ◽  
High Throughput Assay

ABSTRACT FKBP12 is a conserved member of the prolyl-isomerase enzyme family and serves as the intracellular receptor for FK506 that mediates immunosuppression in mammals and antimicrobial actions in fungi. To investigate the cellular functions of FKBP12 in Saccharomyces cerevisiae, we employed a high-throughput assay to identify mutations that are synthetically lethal with a mutation in the FPR1 gene, which encodes FKBP12. This screen identified a mutation in the HOM6 gene, which encodes homoserine dehydrogenase, the enzyme catalyzing the last step in conversion of aspartic acid into homoserine, the common precursor in threonine and methionine synthesis. Lethality of fpr1 hom6 double mutants was suppressed by null mutations in HOM3 or HOM2, encoding aspartokinase and aspartate β-semialdehyde dehydrogenase, respectively, supporting the hypothesis that fpr1 hom6 double mutants are inviable because of toxic accumulation of aspartate β-semialdehyde, the substrate of homoserine dehydrogenase. Our findings also indicate that mutation or inhibition of FKBP12 dysregulates the homoserine synthetic pathway by perturbing aspartokinase feedback inhibition by threonine. Because this pathway is conserved in fungi but not in mammals, our findings suggest a facile route to synergistic antifungal drug development via concomitant inhibition of FKBP12 and Hom6.

scholarly journals FKBP12 physically and functionally interacts with aspartokinase in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (10) ◽  
pp. 5968-5975 ◽  
Author(s):  
C M Alarcón ◽  
J Heitman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Conformational Changes ◽  
Active Site ◽  
Feedback Inhibition ◽  
Immunosuppressive Drugs ◽  
Intermediate Step ◽  
Active Site Residues ◽  
Peptidyl Prolyl Isomerase

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.

Molecular basis of the inhibitor selectivity and insights into the feedback inhibition mechanism of citramalate synthase from Leptospira interrogans

2009 ◽  
Vol 421 (1) ◽  
pp. 133-143 ◽  
Author(s):  
Peng Zhang ◽  
Jun Ma ◽  
Zilong Zhang ◽  
Manwu Zha ◽  
Hai Xu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Reaction ◽  
Molecular Basis ◽  
High Selectivity ◽  
Feedback Inhibition ◽  
Biochemical Study ◽  
Inhibition Mechanism ◽  
Leptospira Interrogans ◽  
Regulatory Domain ◽  
Dimer Interface

LiCMS (Leptospira interrogans citramalate synthase) catalyses the first reaction of the isoleucine biosynthesis pathway in L. interrogans, the pathogen of leptospirosis. The catalytic reaction is regulated through feedback inhibition by its end product isoleucine. To understand the molecular basis of the high selectivity of the inhibitor and the mechanism of feedback inhibition, we determined the crystal structure of LiCMSC (C-terminal regulatory domain of LiCMS) in complex with isoleucine, and performed a biochemical study of the inhibition of LiCMS using mutagenesis and kinetic methods. LiCMSC forms a dimer of dimers in both the crystal structure and solution and the dimeric LiCMSC is the basic functional unit. LiCMSC consists of six β-strands forming two anti-parallel β-sheets and two α-helices and assumes a βαβ three-layer sandwich structure. The inhibitor isoleucine is bound in a pocket at the dimer interface and has both hydrophobic and hydrogen-bonding interactions with several conserved residues of both subunits. The high selectivity of LiCMS for isoleucine over leucine is primarily dictated by the residues, Tyr430, Leu451, Tyr454, Ile458 and Val468, that form a hydrophobic pocket to accommodate the side chain of the inhibitor. The binding of isoleucine has inhibitory effects on the binding of both the substrate, pyruvate, and coenzyme, acetyl-CoA, in a typical pattern of K-type inhibition. The structural and biochemical data from the present study together suggest that the binding of isoleucine affects the binding of the substrate and coenzyme at the active site, possibly via conformational change of the dimer interface of the regulatory domain, leading to inhibition of the catalytic reaction.

Studies on mutants affecting amidophosphoribosyltransferase activity in Saccharomyces cerevisiae

1983 ◽  
Vol 29 (6) ◽  
pp. 681-688 ◽  
Author(s):  
Daniel J. Nieto ◽  
Robin A. Woods
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzyme Activity ◽  
Ultraviolet Light ◽  
Temperature Sensitivity ◽  
De Novo ◽  
Feedback Inhibition ◽  
Temperature Sensitive ◽  
Ethylmethane Sulphonate ◽  
Interallelic Complementation ◽  
Different Levels

Mutants at the ade4 locus of yeast were isolated following mutagenesis of ade+ and ade2 with ultraviolet light (UV), ethylmethane sulphonate, and the acridine half mustard ICR-170. Tests for interallelic complementation, osmotic remediality, temperature sensitivity, and mutagen-specific reversion were carried out on 19 mutants. Six mutants showed interallelic complementation and fell into four groups, defining three complons. Three mutants were osmotic remedial and the same three were temperature sensitive. Three mutants induced by ICR-170 gave purine-excreting revertants, designated Pur6 or ade4.RCF, after exposure to UV. Activity of amidophosphoribosyltransferase (PRPPAT) was assayed in the ade4 mutants and other alleles at this locus. The ade4 mutants lacked activity of the enzyme; the alleles su-pur+, su-pur, PUR6, and Pur6, showed different levels of activity. The enzyme was subject to feedback inhibition by AMP and IMP in su-pur+ and PUR6; su-pur was hypersensitive to inhibition by AMP, whereas Pur6 was slightly resistant. Purine synthesis de novo was shown to be repressible in su-pur+ and constitutive in PUR6 and Pur6 by following the accumulation of aminoimidazole ribotide in the presence and absence of cycloheximide. These observations were confirmed by direct assay of enzyme activity.

scholarly journals The GCR1 gene encodes a positive transcriptional regulator of the enolase and glyceraldehyde-3-phosphate dehydrogenase gene families in Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (2) ◽  
pp. 813-820 ◽  
Author(s):  
M J Holland ◽  
T Yokoi ◽  
J P Holland ◽  
K Myambo ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutant Strain ◽  
Gene Families ◽  
Gene Replacement ◽  
Insertion Mutation ◽  
Gene Insertion ◽  
Glycolytic Gene ◽  
Dehydrogenase Gene ◽  
Multiple Copies ◽  
State Concentration

The intracellular concentrations of the polypeptides encoded by the two enolase (ENO1 and ENO2) and three glyceraldehyde-3-phosphate dehydrogenase (TDH1, TDH2, and TDH3) genes were coordinately reduced more than 20-fold in a Saccharomyces cerevisiae strain carrying the gcr1-1 mutation. The steady-state concentration of glyceraldehyde-3-phosphate dehydrogenase mRNA was shown to be approximately 50-fold reduced in the mutant strain. Overexpression of enolase and glyceraldehyde-3-phosphate dehydrogenase in strains carrying multiple copies of either ENO1 or TDH3 was reduced more than 50-fold in strains carrying the gcr1-1 mutation. These results demonstrated that the GCR1 gene encodes a trans-acting factor which is required for efficient and coordinate expression of these glycolytic gene families. The GCR1 gene and the gcr1-1 mutant allele were cloned and sequenced. GCR1 encodes a predicted 844-amino-acid polypeptide; the gcr1-1 allele contains a 1-base-pair insertion mutation at codon 304. A null mutant carrying a deletion of 90% of the GCR1 coding sequence and a URA3 gene insertion was constructed by gene replacement. The phenotype of a strain carrying this null mutation was identical to that of the gcr1-1 mutant strain.

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