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E108A
-
in contrast to wild-type mutant existed as monomer even at pH 7
K263A
the mutant shows strongly reduced activity compared to the wild type enzyme
A143A
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
A144V
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
A374V
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
D317A
site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
D386A
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
E299A
site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
E302A
site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
E307A
site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
E345A
site-directed mutagenesis, mutation of a residue in the serine binding site, the mutant shows only slightly altered kinetics and activity compared to the wild-type enzyme
E360A
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
E387A
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
G145V
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
G336V/G337V
changing glycine residues 336 and 337 to valine affect the sensitivity of the enzyme to inhibition by L-serine but not the extent of inhibition. The decrease in sensitivity is caused primarily by a decrease in the affinity of the enzyme for L-serine. The mutations also affect the domain rotation of the subunits in response to L-serine binding
G337V
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
G349V
site-directed mutagenesis, mutation of a residue in the serine binding site, the mutant shows only slightly altered kinetics and activity compared to the wild-type enzyme
G362V
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
H335A
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
H344A/N364A
-
no inhibition by L-serine
K141A
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
K141R
kcat/KM for 2-oxoglutarate is 4.8fold lower than the value for the wild-type enzyme, kcat/KM for 3-phosphooxypyruvate is 2.6fold higher than the value for the wild-type enzyme
K311A
site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
N190A
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, no protein expression
N303A
site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
N303A/K311A
site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
N346A/H344A
-
no inhibition by L-serine
N346A/N364A
-
no inhibition by L-serine
P348A
site-directed mutagenesis, mutation of a residue in the serine binding site, the mutant shows only slightly altered kinetics and activity compared to the wild-type enzyme
Q298A
site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
Q301A
site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
Q361A
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
Q375A
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
R338A
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
R347A
site-directed mutagenesis, mutation of a residue in the serine binding site, the mutant shows only slightly altered kinetics and activity compared to the wild-type enzyme
S107A
site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
S107A/S111A
site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
S111A
site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
S111A/K311A
site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
S296A
site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
S316A
site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
S323A
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
S373A
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
T297A
site-directed mutagenesis, mutation of a residue in the polypeptide connecting the substrate binding domain and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
W139F/E360W
exhibits catalytic activity comparable to that of the native enzyme and is effectively inhibited by L-serine. The only fluorescence signal of the mutant is due to the single tryptophan at position 360. Pre-steady state analysis of binding of inhibitor serine shows that each serine binding interface produces an integrated fluorescent signal
W139F/E360W/G294V
-
placement of a tryptophanyl residue near the serine binding site (W139F/E360W) allows serine binding to be monitored by fluorescence quenching analysis. Pre-steady state analysis demonstrate that serine binds to two forms of the free enzyme, E and E*. Conversion of Gly-336 to valine has its main effect on the Kd of serine binding to one form of the free enzyme (E) while maintaining the cooperativity of binding observed in the native enzyme
W139F/E360W/G336V
-
placement of a tryptophanyl residue near the serine binding site (W139F/E360W) allows serine binding to be monitored by fluorescence quenching analysis. Pre-steady state analysis demonstrate that serine binds to two forms of the free enzyme, E and E*. Conversion of Gly-294 to valine eliminates a rate limiting conformational change that follows serine binding to E. The conformational change between the two forms of free enzyme is maintained, but the Hill coefficient for cooperativity is significantly lowered
K141R
-
kcat/KM for 2-oxoglutarate is 4.8fold lower than the value for the wild-type enzyme, kcat/KM for 3-phosphooxypyruvate is 2.6fold higher than the value for the wild-type enzyme
-
A373T
naturally occuring mutation located in the nucleotide binding and regulatory domains of 3-PGDH, the mutation does not affect steady-state expression, protein stability, and protein degradation rates, the mutant is almost catalytically inactive
G377S
naturally occuring mutation located in the nucleotide binding and regulatory domains of 3-PGDH, the mutation does not affect steady-state expression, protein stability, and protein degradation rates, the mutant is almost catalytically inactive
R135W
naturally occuring mutation located in the nucleotide binding and regulatory domains of 3-PGDH, the mutation does not affect steady-state expression, protein stability, and protein degradation rates, the mutant is almost catalytically inactive
V261M
naturally occuring mutation located in the nucleotide binding and regulatory domains of 3-PGDH, the mutation does not affect steady-state expression, protein stability, and protein degradation rates, the mutant is almost catalytically inactive
V425M
naturally occuring mutation in the carboxy-terminal regulatory domain, leads to 3-PGDH deficiency, the mutant is almost catalytically inactive
V490M
naturally occuring mutation in the carboxy-terminal regulatory domain, leads to 3-PGDH deficiency, the mutant is almost catalytically inactive
up
-
Phgdh is transiently upregulated in the brain by kainic acid-treatment, causing selective lesions in the hippocampal region, kainic acid is injected into the bregma, overview
D463A
site-directed mutagenesis, a very large reduction in the sensitivity of the mutant enzyme to L-serine
G316V
site-directed mutagenesis, the mutant shows slightly reduced activity and decreased sensitivity to L-serine compared to the wild-type
G316V/G317V
site-directed mutagenesis, the mutant shows reduced activity and decreased sensitivity to L-serine compared to the wild-type
G316V/G317V/G318V
site-directed mutagenesis, the mutant is not producable
G316V/G318V
site-directed mutagenesis, the mutant shows reduced activity and decreased sensitivity to L-serine compared to the wild-type
G317V
site-directed mutagenesis, the mutant shows slightly reduced activity and decreased sensitivity to L-serine compared to the wild-type
G317V/G318V
site-directed mutagenesis, the mutant shows reduced activity and decreased sensitivity to L-serine compared to the wild-type
G318V
site-directed mutagenesis, the mutant shows slightly reduced activity and decreased sensitivity to L-serine compared to the wild-type
K439A/R451A/R501A
site-directed mutagenesis, the mutation eliminates substrate inhibition and pH-dependent depression in activity
N481A
site-directed mutagenesis, a very large reduction in the sensitivity of the mutant enzyme to L-serine. Mutant N481A co-elutes with native PGDH in gel filtration, it shows loss of cooperativity, which cannot be explained by a change in the quaternary structure of the enzyme from tetramer to dimer or monomer
R132K
mutation decreases the Km-value for 3-phosphooxypyruvate by approximately 10fold
R451A/R501A/K439A
site-directed mutagenesis, the mutation eliminates substrate inhibition and pH-dependent depression in activity
R501A/R451A/K439A
anion binding site mutant: Km (mM) (3-phosphohydroxypyruvate): 0.243, kcat: 1558, Ki (mM) (3-phosphohydroxypyruvate): 7.218, mutant displays only little uncompetitive substrate inhibition, no dual pH optima compared to wild-type
R72A
mutant enzyme utilize 2-oxoglutarate as substrate
R72L
mutant enzyme utilize 2-oxoglutarate as substrate
W130F
site-directed mutagenesis, catalytically inactive mutant
W29F
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
W327F
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
Y461A
site-directed mutagenesis, a very large reduction in the sensitivity of the mutant enzyme to L-serine
D463A
-
site-directed mutagenesis, a very large reduction in the sensitivity of the mutant enzyme to L-serine
-
G316V
-
site-directed mutagenesis, the mutant shows slightly reduced activity and decreased sensitivity to L-serine compared to the wild-type
-
G316V/G317V/G318V
-
site-directed mutagenesis, the mutant is not producable
-
N481A
-
site-directed mutagenesis, a very large reduction in the sensitivity of the mutant enzyme to L-serine. Mutant N481A co-elutes with native PGDH in gel filtration, it shows loss of cooperativity, which cannot be explained by a change in the quaternary structure of the enzyme from tetramer to dimer or monomer
-
R132K
-
mutation decreases the Km-value for 3-phosphooxypyruvate by approximately 10fold
-
R72A
-
mutant enzyme utilize 2-oxoglutarate as substrate
-
R72L
-
mutant enzyme utilize 2-oxoglutarate as substrate
-
Y461A
-
site-directed mutagenesis, a very large reduction in the sensitivity of the mutant enzyme to L-serine
-
G336V
site-directed mutagenesis, mutation of a residue in the Trp-139-loop and the regulatory domain, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
G336V
changing glycine residues 336 and/or 337 to valine affect the sensitivity of the enzyme to inhibition by L-serine but not the extent of inhibition. The decrease in sensitivity is caused primarily by a decrease in the affinity of the enzyme for L-serine. The mutations also affect the domain rotation of the subunits in response to L-serine binding. Crystal structure of G336V demonstrates that the minimal effect of L-serine binding leading to inhibition of enzyme activity requires a domain rotation of approximately only 6° in just two of the four subunits of the enzyme that are oriented diagonally across from each other in the tetramer
H344A
-
50% inhibition at 0.8 mM L-serine
H344A
site-directed mutagenesis, mutation of a residue in the serine binding site, the mutant shows only slightly altered kinetics and activity compared to the wild-type enzyme
N346A
-
50% inhibition at 6 mM L-serine
N346A
site-directed mutagenesis, mutation of a residue in the serine binding site, the mutant shows only slightly altered kinetics and activity compared to the wild-type enzyme
N364A
-
50% inhibition at 48 mM L-serine
N364A
site-directed mutagenesis, mutation of a residue in the serine binding site, the mutant shows only slightly altered kinetics and activity compared to the wild-type enzyme
H447A
anion binding site mutant: Km (mM) (3-phosphohydroxypyruvate): 0.16, kcat: 1446, Ki (mM) (3-phosphohydroxypyruvate): 0.87, mutant displays complete uncompetitive substrate inhibition, no dual pH optima compared to wild-type
H447A
site-directed mutagenesis, the mutant shows altered L-serine binding, kinetics for NADH, and activity compared to the wild-type enzyme
K439A
anion binding site mutant: Km (mM) (3-phosphohydroxypyruvate): 0.075, kcat: 368, Ki (mM) (3-phosphohydroxypyruvate): 0.054, mutant displays partial uncompetitive substrate inhibition, mutant retains dual pH optima
K439A
site-directed mutagenesis, the mutant shows altered L-serine binding, kinetics for NADH, and activity compared to the wild-type enzyme
R446A
anion binding site mutant: Km (mM) (3-phosphohydroxypyruvate): 0.123 kcat: 467, Ki (mM) (3-phosphohydroxypyruvate): 0.289, mutant displays partial uncompetitive substrate inhibition, no dual pH optima compared to wild-type
R446A
site-directed mutagenesis, the mutant shows altered L-serine binding, kinetics for NADH, and activity compared to the wild-type enzyme
R451A
anion binding site mutant: Km (mM) (3-phosphohydroxypyruvate): 0.19, kcat: 1881, Ki (mM) (3-phosphohydroxypyruvate): 0.95, mutant displays complete uncompetitive substrate inhibition, no dual pH optima compared to wild-type
R451A
site-directed mutagenesis, the mutant shows altered L-serine binding, kinetics for NADH, and activity compared to the wild-type enzyme
R501A
anion binding site mutant: Km (mM) (3-phosphohydroxypyruvate): 0.18, kcat: 1989, Ki (mM) (3-phosphohydroxypyruvate): 1.02, mutant displays complete uncompetitive substrate inhibition, no dual pH optima compared to wild-type
R501A
site-directed mutagenesis, the mutant shows altered L-serine binding, kinetics for NADH, and activity compared to the wild-type enzyme
H447A
-
anion binding site mutant: Km (mM) (3-phosphohydroxypyruvate): 0.16, kcat: 1446, Ki (mM) (3-phosphohydroxypyruvate): 0.87, mutant displays complete uncompetitive substrate inhibition, no dual pH optima compared to wild-type
-
H447A
-
site-directed mutagenesis, the mutant shows altered L-serine binding, kinetics for NADH, and activity compared to the wild-type enzyme
-
K439A
-
anion binding site mutant: Km (mM) (3-phosphohydroxypyruvate): 0.075, kcat: 368, Ki (mM) (3-phosphohydroxypyruvate): 0.054, mutant displays partial uncompetitive substrate inhibition, mutant retains dual pH optima
-
K439A
-
site-directed mutagenesis, the mutant shows altered L-serine binding, kinetics for NADH, and activity compared to the wild-type enzyme
-
R446A
-
anion binding site mutant: Km (mM) (3-phosphohydroxypyruvate): 0.123 kcat: 467, Ki (mM) (3-phosphohydroxypyruvate): 0.289, mutant displays partial uncompetitive substrate inhibition, no dual pH optima compared to wild-type
-
R446A
-
site-directed mutagenesis, the mutant shows altered L-serine binding, kinetics for NADH, and activity compared to the wild-type enzyme
-
R451A
-
anion binding site mutant: Km (mM) (3-phosphohydroxypyruvate): 0.19, kcat: 1881, Ki (mM) (3-phosphohydroxypyruvate): 0.95, mutant displays complete uncompetitive substrate inhibition, no dual pH optima compared to wild-type
-
R451A
-
site-directed mutagenesis, the mutant shows altered L-serine binding, kinetics for NADH, and activity compared to the wild-type enzyme
-
R501A
-
anion binding site mutant: Km (mM) (3-phosphohydroxypyruvate): 0.18, kcat: 1989, Ki (mM) (3-phosphohydroxypyruvate): 1.02, mutant displays complete uncompetitive substrate inhibition, no dual pH optima compared to wild-type
-
R501A
-
site-directed mutagenesis, the mutant shows altered L-serine binding, kinetics for NADH, and activity compared to the wild-type enzyme
-
additional information
-
expression of the Aphanothece PGDH gene in Escherichia coli causes an increase in levels of betaine as well as glycine and serine. Expression of the Aphanothece PGDH gene in Arabidopsis thaliana, in which the betaine synthetic pathway is introduced via glycine methylation, further increases betaine levels and improves the stress tolerance
additional information
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expression of GFP-mutant enzyme in Arabidopsis to investigate the tissue distribution, complementation of Escherichia coli serA- mutant
additional information
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C-terminally truncated SerA-mutant 197 shows phosphoglycerate dehydrogenase activity of 1.3 U/mg protein, the activity is no longer sensitive to L-Ser. The mutant enzyme exists as a dimer
additional information
-
construction of an L-serine-insensitive mutant serADELAT197
additional information
-
diverse mutants with different interaction between the 3 binding domains of each of 4 subunits and modified kinetics
additional information
-
diverse mutants with modified active siteand increased Km values
additional information
-
sequence analysis of the PHGDH promoter region exhibits several putative transcription factor binding sites for NF-Y, Sp1, GATA-1, p53, AP2, and AP1, with no TATA-box motif at an appropriate position. Transfection of a series of deletion constructs of the promoter region into HeLa cells reveals that the core positive promoter activity resides in the -276 to +1, which contains two GC-motifs for binding Sp1 and one CCAAT-motif for NF-Y. Mutational analysis and electrophoretic mobility shift assay indicates that both the proximal GC-motif and CCAAT-motif are crucial for full induction of the promoter activity
additional information
p.G238fsX is a naturally occuring frameshift mutation, the mutant is almost catalytically inactive with a 4fold increased Km for 3-phosphohydroxypyruvate
additional information
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p.G238fsX is a naturally occuring frameshift mutation, the mutant is almost catalytically inactive with a 4fold increased Km for 3-phosphohydroxypyruvate
additional information
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generation of Phgdh heterozygous mice, Phgdh+/-, using E14 embryonic stem cells 129P2/OlaHsd background