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H180A
the mutation completely inactivates the enzyme
H180Q
the mutation completely inactivates the enzyme
H260A
the mutation completely inactivates the enzyme
H260Q
the mutation completely inactivates the enzyme
K270A
the mutation completely inactivates the enzyme
K270R
the mutation completely inactivates the enzyme
N182A
the mutation completely inactivates the enzyme
N182Q
the mutation completely inactivates the enzyme
R278A
the mutation completely inactivates the enzyme
R278H
the mutation reduces the enzyme activity to approximately 26%
S272A
the mutation reduces the enzyme activity by 83%
D405
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inactivation of catalytic site in PHY-2 subunit, 8% of wild-type PHY-1/PHY-2/(PDI-2)2 P4H tetramer activity
D407N
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inactivation of catalytic site in PHY-1 subunit, 5% of wild-type PHY-1/PHY-2/(PDI-2)2 P4H tetramer activity
L484A
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lysine of PHY-2 that binds the C-5 carboxyl group of 2-oxoglutarate, 27% of wild-type PHY-1/PHY-2/(PDI-2)2 P4H tetramer activity
L486A
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lysine of PHY-1 that binds the C-5 carboxyl group of 2-oxoglutarate, 25% of wild-type PHY-1/PHY-2/(PDI-2)2 P4H tetramer activity
D149A
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a mutation at the tip of the betaII-betaIII loop, the mutant shows reduced activity and narrower substrate specificity due to altered substrate binding compared to the wild-type enzyme
D149N
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a mutation at the tip of the betaII-betaIII loop, the mutant shows reduced activity and narrower substrate specificity due to altered substrate binding compared to the wild-type enzyme
D81A
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a mutation at the tip of the beta3-beta4 loop, the mutant shows reduced activity and narrower substrate specificity due to altered substrate binding compared to the wild-type enzyme
E127A
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very low catalytic activity
G85A
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a mutation at the tip of the beta3-beta4 loop, the mutant shows reduced activity and narrower substrate specificity due to altered substrate binding compared to the wild-type enzyme
H245A
-
no catalytic activity
N152A
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a mutation at the tip of the betaII-betaIII loop, the mutant shows reduced activity and narrower substrate specificity due to altered substrate binding compared to the wild-type enzyme
Q130A
-
almost 2-fold increase in Km for poly(L-Pro)
R161A
-
no catalytic activity
R93A
-
no catalytic activity
S78T
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a mutation at the entrance and exit of the beta3-beta4 loop, the mutant shows slightly reduced activity compared to the wild-type enzyme
S78T/S87L
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a mutation at the entrance and exit of the beta3-beta4 loop, the mutant shows slightly reduced activity compared to the wild-type enzyme
S84A
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a mutation at the tip of the beta3-beta4 loop, the mutant shows reduced activity and narrower substrate specificity due to altered substrate binding compared to the wild-type enzyme
S87L
-
a mutation at the entrance and exit of the beta3-beta4 loop, the mutant shows slightly reduced activity compared to the wild-type enzyme
S95A
-
no catalytic activity
W243A
-
Km values similar to wild-type, 10-fold decrease in kcat
W99A
-
2.5-fold increase in Km for poly(L-Pro)
Y134F
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6-fold increase in Km for 2-oxoglutarate, decrease in kcat
Y140A
-
no catalytic activity
Y140F
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a mutation at the betaI-betaII loop, the mutant shows reduced activity and narrower substrate specificity due to altered substrate binding compared to the wild-type enzyme
Y168F
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3-4-fold increase in Km for 2-oxoglutarate, decrease in kcat
R490H
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the mutation reduces the percentage of uncoupled decarboxylation
R490S
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the mutation increases the Km for 2-oxoglutarate, reduces the reaction velocity and increases the percentage of uncoupled decarboxylation
C150S
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the mutation has no major effect on tetramer assembly, but the amount of tetramer is slightly reduced, being about 80% of that of the wild-type enzyme
C486S
-
the mutation totally prevents tetramer assembly
C511S
-
the mutation totally prevents tetramer assembly
D414A
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site-directed mutagenesis, the mutation mimics the active site of a halogenase. The substitutions does not convert P4H into a halogenase, but the hydroxylase activity of D414A P4H cannot be rescued with small molecule, the mutant is inactive
D414G
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site-directed mutagenesis, the mutation mimics the active site of a halogenase. The substitutions does not convert P4H into a halogenase, but the hydroxylase activity of D414A P4H cannot be rescued with small molecule, the mutant is inactive
D414H
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site-directed mutagenesis, the mutation mimics the active site of a cysteine dioxygenase, the mutant is inactive
D414H/H483D
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site-directed mutagenesis, the mutant is inactive
H131A/D315A
-
inactive mutant
H141S
-
the mutation has no effect on enzyme activity and does not inhibit tetramer assembly
H165S
-
the mutation produces a reduction of about 60% in enzyme activity per unit extractable cell protein relative to that obtained with the wild-type alpha subunit, the amount of tetramer is reduced by about 20-25%, the Km values for Fe2+, 2-oxoglutarate, ascorbate and the peptide substrate with the mutant are identical to those with the wild-type enzyme
H221S
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the mutation produces a reduction of about 30% in enzyme activity per unit extractable cell protein relative to that obtained with the wild-type alpha subunit, the amount of tetramer is reduced by about 20-25%, the Km values for Fe2+, 2-oxoglutarate, ascorbate and the peptide substrate with the mutant are identical to those with the wild-type enzyme
H296S
-
the mutation has no effect on enzyme activity and does not inhibit tetramer assembly
H324S
-
the mutation totally prevents tetramer assembly
H412D/D414H
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site-directed mutagenesis, the mutant is inactive
H412S
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the mutation causes a complete inactivation of the enzyme with no effect on tetramer assembly or binding of the tetramer to poly(L-proline), role in the binding of Fe2+ to a catalytic site
H483S
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the mutation causes a complete inactivation of the enzyme with no effect on tetramer assembly or binding of the tetramer to poly(L-proline), role in the binding of Fe2+ to a catalytic site
H501S
-
the mutation reduces the enzyme activity to about 4% with no effect on tetramer assembly or binding of the tetramer to poly(L-proline), role in the binding of Fe2+ to a catalytic site, the Km values for Fe2+, ascorbate and the peptide substrate with the mutant are identical to those with the wild-type enzyme, but the Km for 2-oxoglutarate is about 2.5fold higher. The main difference is that the Vmax determined from kinetic plots is consistently less than about 5% of that of the wild-type enzyme
H63S
-
the mutation has no effect on enzyme activity and does not inhibit tetramer assembly
N96Q/N242Q
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the amount of enzyme activity observed with the double mutant alpha subunit is identical to that of the wild-type enzyme, the size of the double mutant alpha subunit is distinctly smaller than that of either the diglycosylated or monoglycosylated alpha subunit present in the wild-type enzyme, the difference being consistent with loss of all the carbohydrate
P191A
-
loss of hypoxic inducibility
P317R
-
naturally occuring mutation, near the Fe2+ binding site, causing erythrocytosis
R371H
-
naturally occuring mutation causing erythrocytosis
W243F
site-directed mutagenesis, mutant structure analysis compared to the wild-type enzyme, the substrate reaches equilibrium within 10 ns. Replacement of Trp243 by Phe increases the accessibility to the oxidant
W243G
site-directed mutagenesis, mutant structure analysis compared to the wild-type enzyme, the substrate equilibrium is not reached after 10 ns
Y140F
site-directed mutagenesis, mutant structure analysis compared to the wild-type enzyme, the substrate reaches equilibrium within 10 ns, inactive mutant
Y140G
site-directed mutagenesis, mutant structure analysis compared to the wild-type enzyme, the substrate reaches equilibrium within 10 ns
Y140G/W243G
site-directed mutagenesis, mutant structure analysis compared to the wild-type enzyme, the substrate equilibrium is not reached after 10 ns. The Y140G/W243G double mutant shows the accumulative effect of both the Y140G and W243G mutations
additional information
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co-expression of PHY-4.1, product of alpha subunit-like gene Y43F8B.4, and beta-subunit protein-disulfide isomerase PDI2 results in active (PHY-4.1)2(PDI2)2 tetramers and in PHY-4.1/PDI2 dimers. Contrary to alpha subunits PHY-1, PHY-2, PHY-3, tetramers containing PHY-4.1 are able to hydroxylate poly(L-Pro) and other proline-rich peptides
additional information
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suppression by RNAi leads to a defective cell wall consisting of a loose network of fibrils resembling the inner and outer W1 and W7-layers of the wild-type cell wall. The nine remaining P4H-like polypeptides of Chlamydomonas cannot fully compensate for the lack of isoform Cr-P4H-1 activity
additional information
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P4H1 null mutant cells differentiate the basic pre-stalk and pre-spore cell types but exhibit a selectively increased O2 requirement for culmination, from 12% to near or above ambient levels. Overexpression of P4H1 reduces the O2 requirement to below 5%. P4H1-expressing cells accumulate at the anterior end, suggesting that P4H1 enables transcellular signaling by the tip
additional information
generation of chimaeric constructs by swapping the regions of isoforms EGLN1 and EGLN3
additional information
generation of chimaeric constructs by swapping the regions of isoforms EGLN1 and EGLN3
additional information
generation of chimaeric constructs by swapping the regions of isoforms EGLN1 and EGLN3
additional information
generation of chimaeric constructs by swapping the regions of isoforms EGLN1 and EGLN3. A minimal sequence of residues 236-252 of EGLN1 is involved in substrate discrimination
additional information
generation of chimaeric constructs by swapping the regions of isoforms EGLN1 and EGLN3. A minimal sequence of residues 236-252 of EGLN1 is involved in substrate discrimination
additional information
generation of chimaeric constructs by swapping the regions of isoforms EGLN1 and EGLN3. A minimal sequence of residues 236-252 of EGLN1 is involved in substrate discrimination
additional information
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overexpression in cultured neuroblastoma cells reduces levels of hypoxia-induced factor alpha oxygen-dependent degradation domain reporter constructs, whereas depletion by siRNA increases hypoxia-induced factor alpha protein level. Recombinant enzyme hydroxylates the two critical prolines in hypoxia-induced factor alpha oxygen-dependent degradation domain in vitro, but not prolines in recombinant type I procollagen chains
additional information
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construction of point mutants
additional information
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stable knockdown of PHDs in A375 cells does not affect cell proliferation, overview. Sections from PHD3 knockdown xenografts show little increase in cleaved caspase-3 staining after treatment with 2-oxoglutarate
additional information
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transient transfection of HeLa and A549 cells with siRNA-targeting PHD2 or PHD3, transient downregulation of PHD3 in HeLa cells or transfection of the control siRNA does not affect the phosphorylation of Cof-1, downregulation of PHD2 leads to an increase of phosphorylated Cof-1. Induction of a PHD2 knockdown in tetracycline-inducible HeLa PHD2 knockdown cells, 1B6 and 3B7 cells from cell line 2.1.1-16, results in increased F-actin formation as detected by phalloidin staining. PHD2 knockdown impairs cell migration. Wild-type PHD2 reverses the increased p-Cof1 levels in 3B7 cells, whereas the mutant PHD2 has no similar effect
additional information
P4halpha1 gene silencing by specific siRNA expression in human aortic smooth muscle cells
additional information
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P4halpha1 gene silencing by specific siRNA expression in human aortic smooth muscle cells
additional information
recombinant expression of sh-P4HA2-1 and sh-P4HA2-2 in breast cancer cell lines HMT-3522 T4-2, MDA-MB-231, ZR-75-1, and MDA-MB-157 for gene silencing, phenotypes, detailed overview
additional information
wild-type and mutant enzymes structure-function analysis by quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) study, QM/MM optimized hydrogen atom abstraction structures for wild-type and mutants at the C4 positions of the substrate, overview
additional information
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wild-type and mutant enzymes structure-function analysis by quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) study, QM/MM optimized hydrogen atom abstraction structures for wild-type and mutants at the C4 positions of the substrate, overview
additional information
recombinant lentiviral-based overexpression of prolyl-4-hydroxylase-alpha1 in mice, the plaques show increased collgane accumulation but no difference of lipid accumulation. The protein expressions of TGF-beta1 are higher in lenti-P4Ha1 mice than in mock or lenti-EGFP mice. Overexpression of P4Ha1 reduced macrophage infiltration
additional information
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recombinant lentiviral-based overexpression of prolyl-4-hydroxylase-alpha1 in mice, the plaques show increased collgane accumulation but no difference of lipid accumulation. The protein expressions of TGF-beta1 are higher in lenti-P4Ha1 mice than in mock or lenti-EGFP mice. Overexpression of P4Ha1 reduced macrophage infiltration
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additional information
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a GFP fusion protein is expressed in BY-2 cells containing mutant versions of NtP4H1.1 ((a) truncated version including transmembrane region in whcih the GFP is fused just after the first linker sequence and same mutant as (a) which in addition lacks three basic amino acids in the cytosolic tail). Mutant proteins are still found to be localized in the Golgi, indicating that NtP4H1.1 contains multiple targeting information in different regions