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F159W
the mutant shows reduced activity and stability compared to the wild type enzyme
F200W
the mutant shows reduced activity and stability compared to the wild type enzyme
L22E/H104R
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mutation of human toward bovine purine nucleoside phosphorylase. Mutant is similar to human purine nucleoside phosphorylase in steady-state kinetic properties. Inhibitor DADMe-immucillin-G is an excellent mimic of the transition states for both human purine nucleoside phosphorylase and the mutant with a preference for the mutant. Thermodynamic parameters establish the mutant to be catalytically more efficient than the parent enzyme and reveal differences in the entropic component of catalysis
N123L/R210Q
mutation of bovine enzyme toward human enzyme. Steady-state kinetic studies indicate unchanged catalytic activity, the mutant enzyme has higher affinity for inhibitors that are mimics of a late dissociative transition state
V39T/N123L/R210Q
mutation of bovine enzyme toward human enzyme. Steady-state kinetic studies indicate unchanged catalytic activity, while presteady-state studies indicate that the chemical step is slower in the triple mutant. The mutant enzyme has higher affinity for inhibitors that are mimics of a late dissociative transition state. Mutant displays a highly dissociative SN1 transition state with low bond order to the leaving group, a transition state different from the native enzyme, with significant nucleophilic participation at the transition state
D204A
0.1% of wild-type activity towards inosine, adenosine and guanosine
D204A/R217A
about 0.2% of wild-type activity towards inosine, adenosine and guanosine
F159A
site-directed mutagenesis, the PNPF159A-FA complexes show a weak association of formycin A to the mutant's active center
F159Y
site-directed mutagenesis, a prominent quenching of the PNPF159Y emission indicates a complex formation, with the strongest association in the phosphate buffer, pH 7.0, relative to the wild-type enzyme. On the other hand, results testify to a deterioration of the interactions in the wild-type PNP/PNPF159Y mutant and formycin A complexes in the presence of the phosphate, pH 8.3
N239D
mutant enzyme shows no activity with the wild-type substrates inosine, xanthosine and guanosine. Unlike the wild-type enzyme, the mutant enzyme shows activity with adenosine
R117E/K121E/D139R
the mutant shows greater stability compared with the wild type dimer and changes in its structures compared with the wild type dimer in a hexamer
R117E/K121E/D139R/F120D
the mutant shows greater stability compared with the wild type dimer and changes in its structures compared with the wild type dimer in a hexamer
R117E/K121E/D139R/F120D/I128S/F131G
the mutant shows greater stability compared with the wild type dimer and changes in its structures compared with the wild type dimer in a hexamer
R117E/K121E/D139R/Y173S
the mutant shows greater stability compared with the wild type dimer and changes in its structures compared with the wild type dimer in a hexamer
R217A
about 0.5% of wild-type activity towards inosine and guanosine, 10.8% of activity towards adenosine
Y191L
mutant enzyme shows no activity with the wild-type substrate xanthosine. The ratio of Vmax to Km for inosine as substrate is 1.7fold lower than the ratio for wild-type enzyme
A117T
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mutation leading to purine-nucleoside phosphorylase deficiency, occuring in two sisters
F159G
mutant displays a strong increase in KM and modest decrease in kcat
F159Y
site-directed mutagenesis, pre-steadystate chemistry is reduced 32fold in mutant F159Y PNP. Pre-steady-state chemistry compares heavy and light molecular weight wild-type and mutant F159Y PNPs and finds a normal heavy-enzyme isotope effect of 1.31 for wild-type PNP and an inverse effect of 0.75 for F159Y mutant PNP. Increased isotopic mass in F159Y PNP causes more efficient transition state formation. Most heavy enzymes demonstrate normal heavy-enzyme isotope effects, and F159Y PNP is a rare example of an inverse effect
F200G
mutant displays a strong increase in KM and modest decrease in kcat
G51S
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the mutant possesses wild type phosphorylase activity levels toward inosine and ribavirin
H257D
kcat/Km for inosine is 1460fold lower than wild-type value, greatly decreased affinity for Immucillin-H
H257F
kcat/Km for inosine is 68fold lower than wild-type value, greatly decreased affinity for Immucillin-H
H257G
kcat/Km for inosine is 610fold lower than wild-type value, greatly decreased affinity for Immucillin-H
H86A
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10-25fold reduction in catalytic activity
L22E/H104R
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mutation of human toward bovine purine nucleoside phosphorylase. Mutant is similar to human purine nucleoside phosphorylase in steady-state kinetic properties. Inhibitor DADMe-immucillin-G is an excellent mimic of the transition states for both human purine nucleoside phosphorylase and the mutant with a preference for the mutant. Thermodynamic parameters establish the mutant to be catalytically more efficient than the parent enzyme and reveal differences in the entropic component of catalysis
N243A
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100fold decrease in turnover-number compared to wild-type enzyme
Q89A
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10-25fold reduction in catalytic activity
Y88A
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88C
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88D
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88E
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88F
engineering of human enzyme to accept 2',3'-dideoxyinosine as substrate. Mutant displays 9-fold improvement in kcat and greater than 2-fold reduction in KM of 2',3'-dideoxyinosine
Y88H
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88I
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88K
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88L
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88M
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88N
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88Q
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88R
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88S
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88T
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88V
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
Y88W
mutation introduced to render enzyme susceptible to substrate 2',3'-dideoxyinosine
N256D
site-directed mutagenesis, mutant KlacPNPN256D accepts both 6-oxopurines and 6-aminopurines as substrates, it shows the highest activity with adenosine
N256E
site-directed mutagenesis, the mutant is only active with inosine, no activity with guanosine, xanthosine, and adenosine
N256D
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site-directed mutagenesis, mutant KlacPNPN256D accepts both 6-oxopurines and 6-aminopurines as substrates, it shows the highest activity with adenosine
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N256E
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site-directed mutagenesis, the mutant is only active with inosine, no activity with guanosine, xanthosine, and adenosine
-
N256D
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site-directed mutagenesis, mutant KlacPNPN256D accepts both 6-oxopurines and 6-aminopurines as substrates, it shows the highest activity with adenosine
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N256E
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site-directed mutagenesis, the mutant is only active with inosine, no activity with guanosine, xanthosine, and adenosine
-
N256D
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site-directed mutagenesis, mutant KlacPNPN256D accepts both 6-oxopurines and 6-aminopurines as substrates, it shows the highest activity with adenosine
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N256E
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site-directed mutagenesis, the mutant is only active with inosine, no activity with guanosine, xanthosine, and adenosine
-
N256D
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site-directed mutagenesis, mutant KlacPNPN256D accepts both 6-oxopurines and 6-aminopurines as substrates, it shows the highest activity with adenosine
-
N256E
-
site-directed mutagenesis, the mutant is only active with inosine, no activity with guanosine, xanthosine, and adenosine
-
N256D
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site-directed mutagenesis, mutant KlacPNPN256D accepts both 6-oxopurines and 6-aminopurines as substrates, it shows the highest activity with adenosine
-
N256E
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site-directed mutagenesis, the mutant is only active with inosine, no activity with guanosine, xanthosine, and adenosine
-
N256D
-
site-directed mutagenesis, mutant KlacPNPN256D accepts both 6-oxopurines and 6-aminopurines as substrates, it shows the highest activity with adenosine
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N256E
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site-directed mutagenesis, the mutant is only active with inosine, no activity with guanosine, xanthosine, and adenosine
-
K244Q
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ration of turnover-numer/Km is 83% of that for wild-type enzyme, no activity with adenosine
N243D
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substitution results in an 8fold increase in Km-value for inosine and a 100fold decrease in ratio of turnover-number/Km. Catalyzes phosphorolysis of adenosine with a Km-value of 0.045 mM and ratio of turnover-number/Km 8fold that with inosine, wild-type enzyme shows no activity with adenosine
N243D/K244Q
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14fold increase in Km-value for inosine and 7fold decrease in the ratio of turnover-number/Km as compared to wild-type enzyme. Phosphorolysis of adenosine with a Km-value of 0.042 mM and a ratio of turnover-number/Km twice that of the single D243D substitution
N243T
-
mutant enzyme shows no activity with adenosine
V66I
the mutant shows wild type activity
V66I/V73I
the mutant shows increased activity compared to the wild type enzyme
V66I/Y160F
the mutant shows increased activity compared to the wild type enzyme
V73I
the mutant shows wild type activity
V73I/Y160F
the mutant shows wild type activity
Y160F
the mutant shows reduced activity compared to the wild type enzyme
Y160F/V66I/V73I
the mutant has a 83fold decrease in turnover number for 5'-methylthioinosine with 2fold increase in Km value compared to the wild type enzyme
T156A
about 5fold decrease in kcat/Km
T156S
increase in kcat/Km
T90A
dramatic decrase in kcat/Km
T90A/T156A
500fold reduction in catalytic activity when compared with wild-type
T90R/T156S
1000fold enhancements in kcat/Km for inosine phosphorolysis
T90S
about 5fold decrease in kcat/Km
V206I
kcat/Km similar to wild-type
T156A
-
about 5fold decrease in kcat/Km
-
T90A
-
dramatic decrase in kcat/Km
-
T90A/T156A
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500fold reduction in catalytic activity when compared with wild-type
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T90R/T156S
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1000fold enhancements in kcat/Km for inosine phosphorolysis
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T90S
-
about 5fold decrease in kcat/Km
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A196E/D238N
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the mutations in the trimeric enzyme form change substrate specificity from guanosine to adenosine
N204D
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the mutation in the hexameric enzyme form changes substrate specificity from adenosine to guanosine
N243D
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site-directed mutagenesis, the mutation in trimeric PNP changes the substrate specificity, making 6-aminopurine nucleosides good substrates
A196E/D238N
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the mutations in the trimeric enzyme form change substrate specificity from guanosine to adenosine
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N204D
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the mutation in the hexameric enzyme form changes substrate specificity from adenosine to guanosine
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N243D
site-directed mutagenesis, the mutant exhibits altered substrate specificity compared to wild-type
N243D
site-directed mutagenesis, the mutation in trimeric PNP changes the substrate specificity, making 6-aminopurine nucleosides good substrates
D204N
less than about 4% of wild-type activity towards inosine, adenosine and guanosine
D204N
site-directed mutagenesis, the enzymatic ribosylation of 1,N6-etheno-isoguanine using Escherichia coli PNP wild-type and D204N mutant enzymes gives different products, which are identified on the basis of NMR analysis and comparison with the product of the isoguanosine reaction with chloroacetic aldehyde, which gives an essentially single compound, identified unequivocally as N9-riboside
D204N
site-directed mutagenesis, the mutant exhibits altered substrate specificity compared to wild-type
M64V
-
mutant enzyme is able to cleave numerous 5-modified nucleoside analogs with much greater efficiency than the wild-type enzyme
M64V
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naturally occuring mutant, the mutant enzyme is able to cleave numerous 5-modified nucleoside analogs with much greater efficiency than the wild-type enzyme
M64V
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the mutant is able to cleave numerous 5'-modified nucleoside analogs with much greater efficiency than the wild type enzyme but no activity with adenosine or inosine
R24A
-
the mutant is almost inactive
R24A
absence of a conformational change upon binding of phosphate, about 0.2 to 0.6% of wild-type activity towards inosine, adenosine and guanosine
E201Q/N243D
crystallization data
E201Q/N243D
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unlike wild-type, mutant is able to cleave prodrugs 2-fluoro-2'-deoxyadenosine, Cladribine, and 2-fluoroadenosine
K22E/H104R
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the transition state of the mutant is fully dissociative, N7-protonated hypoxanthine with C1'-N9 distance above 3.0 A, with partial participation of phosphate, C1-Ophosphate distance is 2.26 A, 2'-C-exo-ribosyl ring pucker and the O5'-C5'-C4'-O4' dihedral angle near 60°. The transition state of the mutant is altered from the fully dissociative DN*AN character for the human wild-type purine nucleoside phosphorylase to a late phosphate-associative character
K22E/H104R
site-directed mutagenesis, mutation on the enzyme protein surface, the mutation alters the enthalpy-entropy balance with little effect on the catalytic rates
N243D
-
the mutation has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates and clearly favoring adenosine over inosine and guanosine
N243D
site-directed mutagenesis, the mutation in trimeric PNP changes the substrate specificity, making 6-aminopurine nucleosides good substrates
C254S/C256S
reduced thermodynamic and kinetic stability of the mutant with respect to the wild-type PfPNP
C254S/C256S
Tm-value is 8°C lower than Tm-value of wild-type enzyme
C254S/C256S
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after denaturation using 6 M guadinium HCl, mutant achieves a recovery of catalytic activity of 46%
additional information
a one-pot, two-enzyme mode of transglycosylation reaction is successfully constructed by combining pyrimidine nucleoside phosphorylase (BbPyNP, EC 2.4.2.2) derived from Brevibacillus borstelensis strain LK01 and Aneurinibacillus migulanus strain AM007 AmPNP for the production of purine nucleoside analogues. Conversions of 2,6-diaminopurine ribonucleoside, 2-amino-6-chloropurine ribonucleoside, and 6-thioguanine ribonucleoside synthesized still reaches over 90% on the higher concentrations of substrates (pentofuranosyl donor: purine base; 20:10 mM) with a low enzyme ratio of BbPyNP:AmPNP (2:20 microg/ml). Thus, the trimeric AmPNP is a promising biocatalyst for industrial production of purine nucleoside analogues. Method optimization and evaluation, overview
additional information
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a one-pot, two-enzyme mode of transglycosylation reaction is successfully constructed by combining pyrimidine nucleoside phosphorylase (BbPyNP, EC 2.4.2.2) derived from Brevibacillus borstelensis strain LK01 and Aneurinibacillus migulanus strain AM007 AmPNP for the production of purine nucleoside analogues. Conversions of 2,6-diaminopurine ribonucleoside, 2-amino-6-chloropurine ribonucleoside, and 6-thioguanine ribonucleoside synthesized still reaches over 90% on the higher concentrations of substrates (pentofuranosyl donor: purine base; 20:10 mM) with a low enzyme ratio of BbPyNP:AmPNP (2:20 microg/ml). Thus, the trimeric AmPNP is a promising biocatalyst for industrial production of purine nucleoside analogues. Method optimization and evaluation, overview
additional information
-
a one-pot, two-enzyme mode of transglycosylation reaction is successfully constructed by combining pyrimidine nucleoside phosphorylase (BbPyNP, EC 2.4.2.2) derived from Brevibacillus borstelensis strain LK01 and Aneurinibacillus migulanus strain AM007 AmPNP for the production of purine nucleoside analogues. Conversions of 2,6-diaminopurine ribonucleoside, 2-amino-6-chloropurine ribonucleoside, and 6-thioguanine ribonucleoside synthesized still reaches over 90% on the higher concentrations of substrates (pentofuranosyl donor: purine base; 20:10 mM) with a low enzyme ratio of BbPyNP:AmPNP (2:20 microg/ml). Thus, the trimeric AmPNP is a promising biocatalyst for industrial production of purine nucleoside analogues. Method optimization and evaluation, overview
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additional information
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the C-terminal KH/S1 domain-truncated mutant binds and cleaves RNA less efficiently with an eightfold reduced binding affinity. The mutant forms a less stable trimer than the full-length PNPase. The crystal structure of DeltaKH/S1 is more expanded, containing a slightly wider central channel than that of the wild-type PNPase, suggesting that the KH/S1 domain helps PNPase to assemble into a more compact trimer, and it regulates the channel size allosterically
additional information
mutations remote from, but linked by dynamic motion to, the catalytic site of hPNP alter at least one promoting vibration and the catalytic properties of the enzyme
additional information
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mutations remote from, but linked by dynamic motion to, the catalytic site of hPNP alter at least one promoting vibration and the catalytic properties of the enzyme
additional information
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construction of lentiviral vectors containing the human purine-nucleoside phosphorylase gene
additional information
targeted gene disruption results in loss of enzyme activity in lysate, with normal activity of adenosine deaminase. Mutant parasites show a greater requirement for exogenous purines and a severe growth defect at physiological concentrations of hypoxanthine. The mutant parasites are more sensitive to purine nucelotide phosphorylase inhibitors that bind human purine nucelotide phosphorylase tighter and less sensitive to inhibitor 5'-methylthio-immucillin-H
additional information
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targeted gene disruption results in loss of enzyme activity in lysate, with normal activity of adenosine deaminase. Mutant parasites show a greater requirement for exogenous purines and a severe growth defect at physiological concentrations of hypoxanthine. The mutant parasites are more sensitive to purine nucelotide phosphorylase inhibitors that bind human purine nucelotide phosphorylase tighter and less sensitive to inhibitor 5'-methylthio-immucillin-H
additional information
generation of a salT-deficient mutant strain