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A173S/Q536E
cell line V79 compared to wild-type CHO-K1 cells, also with silent polymorphisms at codons 94 and 187
A155H
drastic increases in Km values
A155W
13fold increase in Km value for dihydropteroate
D154A
mutation in residue specific for dihydropteroate binding, 200fold increase in Km value for substrate 5,6,7,8-tetrahydropteroyl-gamma-(L-Glu)2
E146Q
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completely inactive, no ATP binding is observed with this mutant
T122H
drastic increases in Km values
T122W
drastic increases in Km values
A22G
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a naturally occuring mutation, rs10760502 , phenotype, overview. The genotypes of the A22G polymorphism may be risk factors for acute lymphoblastic leukemia (ALL) and may play a role in the survival of patients with ALL
A382T
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94% of the wild type enzyme activity
A447V
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does not affect enzyme activity
C209R
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8% of the wild type enzyme activity
C346A
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activity comparable with that of wild type enzyme
C388F
MTXR5 resistant leukemia cells established by repeated cycles of 24 h-pulse exposures to methotrexate (MTX), harbor a C388F substitution resulting in a 91% loss of their cellular FPGS activity. Kinetic analysis reveals that the mutant protein retains parental affinity toward MTX, while the Km toward L-glutamate increases by 23fold. 3D-modeling analysis suggests that C388 is located at the entrance to the active site of this enzyme
D376A
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only slightly lower activity than the wild type enzyme
D378A
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only slightly lower activity than the wild type enzyme
G569C
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13% of the wild type enzyme activity
K384A
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only slightly lower activity than the wild type enzyme
R377A
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with no significant activity
R377A D335A
site-directed mutagenesis, the substitution results in a 1500fold increase in the Km for glutamate, and a 20fold decrease in the Kcat
R424C
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reduced efficacy with substrates compared to the wild type enzyme
S457F
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reduced efficacy with substrates compared to the wild type enzyme
D151A
mutation in residue specific for dihydropteroate binding, mutant is defective in using tetrahydrofolate as substrate
D313A
mutation in the C-terminal of the enzyme
D345C
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more than 85% of the wild type activity, with 5,10-methylene-5,6,7,8-tetrahydrofolic acid as substrate
E81A
mutation in the omega-loop of the enzyme
F121A
50fold increase in Km for 5,10-methylene-5,6,7,8-tetrahydrofolic acid
F75A
increase in Km value for 5,10-methylene-5,6,7,8-tetrahydrofolic acid, decreases in Km values for ATP and L-Glu
G411A
mutation in the C-terminal of the enzyme
G51S/S52T
no FGPS activity
H316A
mutation in the C-terminal of the enzyme
K172C
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more than 85% of the wild type activity, with 5,10-methylene-5,6,7,8-tetrahydrofolic acid as substrate
K185A
mutation in the MgATP2- binding site of the enzyme
P74A
mutation in the omega-loop of the enzyme
R15E
increases in Km values
R82A
mutation in the omega-loop of the enzyme
S152W
increases in Km values
S412A
mutation in the C-terminal of the enzyme
T119W
100fold increase in Km for 5,10-methylene-5,6,7,8-tetrahydrofolic acid
Y414A
decrease in Km values for ATP, increase in Km for L-Glu
C346F
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MTXR5 antifolates-resistant cell subline
C346F
MTXR5 resistant leukemia cells, established by repeated cycles of 24 h-pulse exposures to methotrexate (MTX), harbor a C346F substitution resulting in a 91% loss of their cellular FPGS activity. Kinetic analysis reveals that the mutant protein retains parental affinity toward MTX, while the Km toward L-glutamate increases by 23fold. Substitution of active site residues D335, H338 and R377 results in an over 500fold decrease in the Vmax of the polyglutamylation reaction when compared to the wild-type hcFPGS protein
D335A
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with no significant activity
D335A
site-directed mutagenesis, the substitution leads to a catalytically dead FPGS with diminished binding of glutamate, ATP and the antifolate
H338A
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only 0.3% activity of the wild type enzyme
H338A
site-directed mutagenesis, the mutation increases the Kmfor glutamic acid by 600fold foldwith negligible residual catalytic activity
E143A
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almost inactive
E143A
mutation in the MgATP2- binding site of the enzyme
E143D
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almost inactive
E143D
mutation in the MgATP2- binding site of the enzyme
E143Q
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almost inactive
E143Q
mutation in the MgATP2- binding site of the enzyme
S73A
mutation in the omega-loop of the enzyme
S73A
mutant retains some FGPS activity
S67F
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L51 cells
additional information
construction of a fpgs1 knockout mutant, transcripts of a number of genes in the C1 and lignin pathways had altered expression in fpgs1 mutants, microarray analysis and quantitative real-time RT-PCR. Total lignin is low in fpgs1 mutant plants leading to higher saccharification efficiency of the mutant. The decrease in total lignin in fpgs1 is mainly due to lower guaiacyl lignin levels. Glycome profiling reveals subtle alterations in the cell walls of fpgs1, the degree of methylation of 4-O-methyl glucuronoxylan in hemicellulosic polysaccharides is reduced in the fpgs1 mutant, NMR analysis
additional information
generation of a T-DNA insertion mutant (atdfb-3) of the plastidial folylpolyglutamate synthetase gene (AtDFB). The mutant is defective in seed reserves and skotomorphogenesis. Lower carbon and higher nitrogen content in the mutant seeds compared to wild-type are indicative of an altered C and N partitioning capacity. Higher levels of organic acids and sugars are detected in the mutant seeds compared with the wild-type. Mutant atdfb-3 seeds contain less total amino acids and individual Asn and Glu as well as NO3-. The mutant exhibts significant changes in seed storage. Defects in hypocotyl elongation are observed in atdfb-3 in darkness under sufficient nitrate conditions, and further enhances under nitrate limited conditions. Mutant sensitivity to limited nitrate during a narrow developmental window. Exogenous 5-formyl-tetrahydrofolate completely restores the hypocotyl length in atdfb-3 seedlings with nitrate as the sole N source. Further study demonstrated that folate profiling and N metabolism are perturbed in atdfb-3 etiolated seedlings. The activity of enzymes involved in N reduction and assimilation is altered in atdfb-3. Exogenous 5-fluoro-tetrahydrofolate restores the wild-type hypocotyl phenotype in etiolated atdfb-3 mutant seedlings
additional information
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construction of a fpgs1 knockout mutant, transcripts of a number of genes in the C1 and lignin pathways had altered expression in fpgs1 mutants, microarray analysis and quantitative real-time RT-PCR. Total lignin is low in fpgs1 mutant plants leading to higher saccharification efficiency of the mutant. The decrease in total lignin in fpgs1 is mainly due to lower guaiacyl lignin levels. Glycome profiling reveals subtle alterations in the cell walls of fpgs1, the degree of methylation of 4-O-methyl glucuronoxylan in hemicellulosic polysaccharides is reduced in the fpgs1 mutant, NMR analysis
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additional information
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C to T transition at nucleotide 1294 generating a premature stop at codon 432 in AUXB1 mutant cell line. The M7.2 cell line has superphysiologic levels of FPGS protein in both the mitochondria and cytosol
additional information
C to T transition at nucleotide 1294 generating a premature stop at codon 432 in AUXB1 mutant cell line. The M7.2 cell line has superphysiologic levels of FPGS protein in both the mitochondria and cytosol
additional information
generation of a FPGS-null Chinese hamster ovary (CHO) cell line AUXB1. The cells are isolated as auxotrophs for glycine, adenosine and thymidine. AUXB1 cells are hemizygous for the FPGS gene and harbor a nonsense mutation at position 432 which renders the enzyme catalytically dead. AUXB1 cells are transfected with human FPGS cDNA presenting high FPGS activity
additional information
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the enzyme cloned in a pUc19 vector is mutagenized by progressive deletion from both the 5'-end and the 3ïend and by TAB linker insertion. The specific activities of the extracts of the mutant enzyme are 4-16% that of the wild type enzyme. Non of the carboxy terminal or linker insertion mutants has a specific activity greater than 0.5% that of the wild type enzyme
additional information
expression of the amino-terminal domain with residues 1-287. The domain binds tetrahydrofolate and dihydropteroate with the same affinity as the intact enzyme. Construction of domain-swap chimera proteins between the Escherichia coli and the Lactobacillus casei enzymes. Chimera possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Mutants with swapped omega loops, containing a folate binding site, retain the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating Lactobacillus casei folypolyglutamate synthetase to contain an Escherichia coli folypolyglutamate synthetase dihydropteroate binding loop does not alter its substrate specificity to using dihydropteroate as a substrate
additional information
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expression of the amino-terminal domain with residues 1-287. The domain binds tetrahydrofolate and dihydropteroate with the same affinity as the intact enzyme. Construction of domain-swap chimera proteins between the Escherichia coli and the Lactobacillus casei enzymes. Chimera possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Mutants with swapped omega loops, containing a folate binding site, retain the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating Lactobacillus casei folypolyglutamate synthetase to contain an Escherichia coli folypolyglutamate synthetase dihydropteroate binding loop does not alter its substrate specificity to using dihydropteroate as a substrate
additional information
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siRNA transfected into DLD-1 cells to downregulate folylpolyglutamate synthase reduces the basal level of reduced folate, the folate level after leucovorin treatment, and the enhancement of 5-fluoro-2'-deoxyuridine-induced cytotoxicity elicited by leucovorin. Folylpolyglutamate synthase and gamma-glutamyl hydrolase expression levels in tumors are determinants of the efficacy of leucovorin in enhancing the antitumor activity of 5-fluorouracil
additional information
aberrant FPGS preRNA splicing, FPGS preRNA splicing, five sublines, isolated by 24 h-pulse exposure of CCRF-CEM cells to MTX or 7OH-MTX, as well as ALL patients' specimens display aberrant splicing of the FPGS mRNA, comprised of both intron retention and exon skipping. FPGS mutations in antifolate selected tumor cell lines are identified in antifolate-resistant sublines of the human T-ALL cell line CCRF-CEM. The resistant sublines, MTXR5, CEM-p, MTAR1.5, ZD1694 C-9 andMTA C-3, all harboring point mutations in the FPGS open reading frame, are selected with different polyglutamatable (i.e. classical) antifolates using diverse selection methods. These drug-resistant sublines are all crossresistant to classical antifolates (e.g. raltitrexed/ZD1694/tomudex) while retaining sensitivity to non-polyglutamatable antifolates (e.g. plevitrexed/ZD9331/vamidex), compared to their parental counterparts, hence exhibiting a major loss of FPGS activity
additional information
aberrant FPGS preRNA splicing, FPGS preRNA splicing, five sublines, isolated by 24 h-pulse exposure of CCRF-CEM cells to MTX or 7OH-MTX, as well as ALL patients' specimens display aberrant splicing of the FPGS mRNA, comprised of both intron retention and exon skipping. FPGS mutations in antifolate selected tumor cell lines are identified in antifolate-resistant sublines of the human T-ALL cell line CCRF-CEM. The resistant sublines, MTXR5, CEM-p, MTAR1.5, ZD1694 C-9 andMTA C-3, all harboring point mutations in the FPGS open reading frame, are selected with different polyglutamatable (i.e. classical) antifolates using diverse selection methods. These drug-resistant sublines are all crossresistant to classical antifolates (e.g. raltitrexed/ZD1694/tomudex) while retaining sensitivity to non-polyglutamatable antifolates (e.g. plevitrexed/ZD9331/vamidex), compared to their parental counterparts, hence exhibiting a major loss of FPGS activity
additional information
aberrant FPGS preRNA splicing, FPGS preRNA splicing, five sublines, isolated by 24 h-pulse exposure of CCRF-CEM cells to MTX or 7OH-MTX, as well as ALL patients' specimens display aberrant splicing of the FPGS mRNA, comprised of both intron retention and exon skipping. FPGS mutations in antifolate selected tumor cell lines are identified in antifolate-resistant sublines of the human T-ALL cell line CCRF-CEM. The resistant sublines, MTXR5, CEM-p, MTAR1.5, ZD1694 C-9 andMTA C-3, all harboring point mutations in the FPGS open reading frame, are selected with different polyglutamatable (i.e. classical) antifolates using diverse selection methods. These drug-resistant sublines are all crossresistant to classical antifolates (e.g. raltitrexed/ZD1694/tomudex) while retaining sensitivity to non-polyglutamatable antifolates (e.g. plevitrexed/ZD9331/vamidex), compared to their parental counterparts, hence exhibiting a majorloss of FPGS activity
additional information
aberrant FPGS preRNA splicing, FPGS preRNA splicing, five sublines, isolated by 24 h-pulse exposure of CCRF-CEM cells to MTX or 7OH-MTX, as well as ALL patients' specimens display aberrant splicing of the FPGS mRNA, comprised of both intron retention and exon skipping. FPGS mutations in antifolate selected tumor cell lines are identified in antifolate-resistant sublines of the human T-ALL cell line CCRF-CEM. The resistant sublines, MTXR5, CEM-p, MTAR1.5, ZD1694 C-9 andMTA C-3, all harboring point mutations in the FPGS open reading frame, are selected with different polyglutamatable (i.e. classical) antifolates using diverse selection methods. These drug-resistant sublines are all crossresistant to classical antifolates (e.g. raltitrexed/ZD1694/tomudex) while retaining sensitivity to non-polyglutamatable antifolates (e.g. plevitrexed/ZD9331/vamidex), compared to their parental counterparts, hence exhibiting a majorloss of FPGS activity
additional information
enzyme genotyping, 3 genotypes for 3 SNPs of FPGS, the methotrexate polyglutamates MTXPG3-5/1-2 ratio shows significant differences among these 3 genotypes for 3 different SNPs of FPGS
additional information
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enzyme genotyping, 3 genotypes for 3 SNPs of FPGS, the methotrexate polyglutamates MTXPG3-5/1-2 ratio shows significant differences among these 3 genotypes for 3 different SNPs of FPGS
additional information
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FPGS overexpression and suppression in colon and breast cancers cells, HCT-116 and MDA-MB-435 cell lines, FPGS inhibition is developed by transfecting HCT-116 cells and MDA-MB-435 cells with the antisense FPGS cDNA and the FPGS-targeted small interfering RNA (siRNA), respectively. FPGS overexpression is associated with significantly lower (by 18%) global DNA methylation than controls in HCT-116 cells, but is associated with significantly higher (by 13%) global DNA methylation than controls in MDA-MB-435 cells. FPGS suppression is associated with significantly higher (by 12%) global DNA methylation than controls in HCT-116 cells, but has no effect in MDA-MB-435 cells. In MDA-MB-435 cells, 2239 differentially methylated genes (1161 hypermethylated and 1078 hypomethylated) occur in response to FPGS overexpression and 2024 differentially methylated genes (1150 hypermethylated and 874 hypomethylated) occur in response to FPGS suppression. Differentially methylated genes are involved in molecular transport and cell death in both the FPGS-overexpressed and silenced MDA-MB-435 cells
additional information
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genotyping of FPGS rs1544105 polymorphism in 57 ALL patients and 31 age and sex-matched children, overall survival is analyzed by Kaplan-Meier method. No differences are observed between patients and controls regarding the distribution frequency of genotype and alleles of rs1544105. Patients carrying AA genotype have a significantly higher plasma concentration of methotrexate after 24 h than those carrying GG or GA (P<0.05) and no differences are found after 44 h. Kaplan-Meier survival analysis shows a longer median survival time in patients with AA than other genotypes with significant difference in overall survival
additional information
construction of domain-swap chimera proteins between the Escherichia coli and the Lactobacillus casei enzymes. Chimera possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Mutants with swapped omega loops, containing a folate binding site, retain the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating Lactobacillus casei folypolyglutamate synthetase to contain an Escherichia coli folypolyglutamate synthetase dihydropteroate binding loop does not alter its substrate specificity to using dihydropteroate as a substrate
additional information
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construction of domain-swap chimera proteins between the Escherichia coli and the Lactobacillus casei enzymes. Chimera possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Mutants with swapped omega loops, containing a folate binding site, retain the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating Lactobacillus casei folypolyglutamate synthetase to contain an Escherichia coli folypolyglutamate synthetase dihydropteroate binding loop does not alter its substrate specificity to using dihydropteroate as a substrate
additional information
construction of homologous recombination vectors for conditional and complete deletion of the fpgs P1 promoter and the associated exons, conversion of the dual-promoter mouse folylpolyglutamate synthetase gene to the human single-promoter structure. Deletion of the mouse P1 promoter through homologous recombination to study the dual-promoter mouse system and to create a mouse with a humanized fpgs gene structure reveals that despite the loss of the predominant fpgs mRNA species in liver and kidney, P1-knockout mice develop and reproduce normally. The survival of the mutant mice is explained by increased P2 transcription due to relief of transcriptional interference, by a 3fold more efficient translation of P2-derived than P1-derived transcripts, and by 2fold higher stability of P2-derived FPGS. In combination, all 3 effects reinstate FPGS function, even in liver. Eliminating mouse P1 gives a mouse model that mimicks the human housekeeping pattern of fpgs gene expression. Generation of embryonic stem (ES) cells with P1 conditional- or complete-KO fpgs allele, and generation of homozygous P1-knockout mice, Recombinant expression of enzyme constructs in the enzyme-deficient AUXB1 cell from Cricetulus griseus
additional information
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construction of homologous recombination vectors for conditional and complete deletion of the fpgs P1 promoter and the associated exons, conversion of the dual-promoter mouse folylpolyglutamate synthetase gene to the human single-promoter structure. Deletion of the mouse P1 promoter through homologous recombination to study the dual-promoter mouse system and to create a mouse with a humanized fpgs gene structure reveals that despite the loss of the predominant fpgs mRNA species in liver and kidney, P1-knockout mice develop and reproduce normally. The survival of the mutant mice is explained by increased P2 transcription due to relief of transcriptional interference, by a 3fold more efficient translation of P2-derived than P1-derived transcripts, and by 2fold higher stability of P2-derived FPGS. In combination, all 3 effects reinstate FPGS function, even in liver. Eliminating mouse P1 gives a mouse model that mimicks the human housekeeping pattern of fpgs gene expression. Generation of embryonic stem (ES) cells with P1 conditional- or complete-KO fpgs allele, and generation of homozygous P1-knockout mice, Recombinant expression of enzyme constructs in the enzyme-deficient AUXB1 cell from Cricetulus griseus
additional information
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removal of the first 16 or 40 residues of the protein abolishes the enzyme activity in the yeast cell host, Saccharomyces cerevisiae but not in Escherichia coli
additional information
FPGS promoter methylation leads to reduced transcription in rats following three months of alcohol consumption. This reduction in FPGS activity results in decreased levels of total as well as polyglutamated folates in different tissues
additional information
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generation of methionine auxotrophs mutants by disruption of met7 gene, which encodes the enzyme
additional information
at least four of 10 Mu-induced bm4 mutant alleles contain a Mu insertion in the FPGS GRMZM2G393334 gene, PCR-based sequencing of Mu-tagging alleles, the enzyme expression is reduced in the brown midrib4 (bm4) mutant, bm4 encodes a functional FPGS, sequence analysis and quantitative RT-PCR expression analysis of bm4-Mu alleles
additional information
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at least four of 10 Mu-induced bm4 mutant alleles contain a Mu insertion in the FPGS GRMZM2G393334 gene, PCR-based sequencing of Mu-tagging alleles, the enzyme expression is reduced in the brown midrib4 (bm4) mutant, bm4 encodes a functional FPGS, sequence analysis and quantitative RT-PCR expression analysis of bm4-Mu alleles