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Information on EC 1.20.1.1 - phosphonate dehydrogenase and Organism(s) Pseudomonas stutzeri and UniProt Accession O69054
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1.20.1.1 phosphonate dehydrogenaseIUBMB Comments
NADP+ is a poor substitute for NAD+ in the enzyme from Pseudomonas stutzeri WM88.
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Word Map
- 1.20.1.1
- phosphorus
- stutzeri
-
hydride
-
baeyer-villiger
- biotechnology
-
bvmos
- phenylacetone
-
d-hydroxy
- synthesis
The taxonomic range for the selected organisms is: Pseudomonas stutzeri
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
phosphite dehydrogenase, ptdh, nad-dependent phosphite dehydrogenase, nad:phosphite oxidoreductase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
NAD-dependent phosphite dehydrogenase
-
-
-
-
NAD:phosphite oxidoreductase
-
-
-
-
phosphite dehydrogenase
phosphite dehydrogenase
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
phosphonate + NAD+ + H2O = phosphate + NADH + H+
different reaction mechanism discussed
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
phosphonate:NAD+ oxidoreductase
NADP+ is a poor substitute for NAD+ in the enzyme from Pseudomonas stutzeri WM88.
CAS REGISTRY NUMBER
COMMENTARY
9031-35-0
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
hydroxypyruvate + NAD+
pyruvate + NADH
very poor substrate
-
?
phosphite + H2O + NADP+
phosphate + NADPH + H+
NADP poorly substitutes for NAD+
-
?
phosphonate + NAD+ + H2O
phosphate + NADH + H+
-
-
-
?
phosphite + H2O + NAD+
phosphate + NADH + H+
use of phosphite as energy source, regeneration of NADH
-
ir
phosphite + H2O + NAD+
phosphate + NADH + H+
-
Asp79 is important in orienting Arg237 for proper interaction with phosphite
-
-
?
phosphite + H2O + NADP+
phosphate + NADPH + H+
-
NADP poorly substitutes for NAD+
-
?
phosphonate + H2O + NAD+
phosphate + NADH
-
-
-
-
ir
phosphonate + H2O + NAD+
phosphate + NADH
-
enables growth on phosphonate as sole phosphorous source
-
-
ir
phosphonate + H2O + NAD+
phosphate + NADH
-
NADH regeneration system
-
-
ir
phosphonate + H2O + NADP+
phosphate + NADPH
-
100-fold lower activity than with NAD+
-
-
ir
phosphonate + H2O + NADP+
phosphate + NADPH
-
NADP+ is a very poor hydride acceptor with the wild type enzyme
-
-
ir
additional information
?
-
-
enzyme does not accept any alternative substrates
-
-
?
additional information
?
-
-
enzyme does not accept any alternative substrates
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
phosphite + H2O + NAD+
phosphate + NADH + H+
use of phosphite as energy source, regeneration of NADH
-
ir
phosphonate + H2O + NAD+
phosphate + NADH
-
enables growth on phosphonate as sole phosphorous source
-
-
ir
phosphonate + H2O + NAD+
phosphate + NADH
-
NADH regeneration system
-
-
ir
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
slow steps after hydride transfer do not significantly limit the rate of reaction for the wild-type enzyme, the active site mutants, or the thermostable mutant
-
sulfite
-
strong inhibitor, competitive with respect to phosphonate, uncompetitive with respect to NAD+
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.04
NAD+
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R/V71I/E130K/I313L/A325V, pH 7.3, 25°C
0.045
NAD+
-
mutant D13E/M26I/E175A/T181S/A308T/E332N/C336D, pH 7.3, 25°C
0.05
NAD+
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/A325V, pH 7.3, 25°C
0.057
NAD+
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R, pH 7.3, 25°C
0.063
NAD+
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/V71I, pH 7.3, 25°C
0.066
NAD+
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A, pH 7.3, 25°C
0.071
NAD+
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/I313L, pH 7.3, 25°C
0.075
NAD+
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/E130K, pH 7.3, 25°C
0.056
NADP+
-
mutant D13E/M26I/E175A/T181S/A308T/E332N/C336D, pH 7.3, 25°C
0.027
phosphite
-
cosubstrate NAD+, mutant M26I/E332N/C336D, pH 7.3, 25°C
0.03
phosphite
-
cosubstrate NAD+, mutant D13E/M26I/E332N/C336D, pH 7.3, 25°C
0.046
phosphite
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R/V71I/E130K/I313L/A325V, pH 7.3, 25°C
0.048
phosphite
-
cosubstrate NAD+, mutant D13E/M26I/E175A/T181S/A308T/E332N/C336D, pH 7.3, 25°C
0.062
phosphite
-
cosubstrate NADP+, mutant D13E/M26I/E175A/T181S/E332N/C336D, pH 7.3, 25°C
0.07
phosphite
-
cosubstrate NADP+, mutant D13E/M26I/E175A/T181S/A308T/E332N/C336D, pH 7.3, 25°C
0.082
phosphite
-
cosubstrate NAD+, mutant D13E/M26I/E175A/E332N/C336D, pH 7.3, 25°C
0.084
phosphite
-
cosubstrate NAD+, mutant D13E/M26I/E175A/T181S/E332N/C336D, pH 7.3, 25°C
0.087
phosphite
-
cosubstrate NADP+, mutant D13E/M26I/E175A/E332N/C336D, pH 7.3, 25°C
0.096
phosphite
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A, pH 7.3, 25°C
0.105
phosphite
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/V71I, pH 7.3, 25°C
0.123
phosphite
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/E130K, pH 7.3, 25°C
0.135
phosphite
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/A325V, pH 7.3, 25°C
0.155
phosphite
-
mutant Q137R/I150F/Q215L/R275Q/D162N/V315A, pH 7.3, 25°C
0.156
phosphite
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/I313L, pH 7.3, 25°C
0.169
phosphite
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R, pH 7.3, 25°C
1.9
phosphite
-
cosubstrate NADP+, mutant D13E/M26I/E332N/C336D, pH 7.3, 25°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2.9
NAD+
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A, pH 7.3, 25°C
3.2
NAD+
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R, pH 7.3, 25°C
3.2
NAD+
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R/V71I/E130K/I313L/A325V, pH 7.3, 25°C
3.5
NAD+
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/A325V, pH 7.3, 25°C
3.5
NAD+
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/E130K, pH 7.3, 25°C
3.5
NAD+
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/I313L, pH 7.3, 25°C
3.5
NAD+
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/V71I, pH 7.3, 25°C
1.9
NADP+
-
mutant D13E/M26I/E175A/T181S/A308T/E332N/C336D, pH 7.3, 25°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6.2
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
36400
-
x * 38500, SDS-PAGE, His-tag fusion protein, x * 36400, SDS-PAGE, native protein
38500
38500
-
x * 38500, SDS-PAGE, His-tag fusion protein, x * 36400, SDS-PAGE, native protein
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
-
x * 38500, SDS-PAGE, His-tag fusion protein, x * 36400, SDS-PAGE, native protein
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
mutant D13E/M26I/V71I/E130K/Q132R/Q137R/I150F/E175A/Q215L/R275Q/L276Q/I313L/V315A/A319E/A325V/E332N/C336D, to 2.3 A resolution. The TS-PTDH monomer may be divided into a large and a small domain, separated by a flexible hinge region. The NAD+ cofactor is housed at the junction between the two domains, where residues from the large subunit engage in interactions with the ligand. In the cocrystal structure with NAD+ and inhibitor sulfite, the sulfite anion is situated proximal to the nicotinamide of the cofactor, where it is engaged through interactions with the side chains of Arg237, His292, and the backbone amides of Lys76 and Gly77
mutant enzymes R301K and R301A, hanging drop vapor diffusion method, using 0.1 M NaCl, 0.1 M sodium cacodylate pH 5.8, 16% PEG 6000 (w/v) (for mutant R301K) or 0.1 M ammonium acetate, 0.1 M Bis Tris pH 5.5, 17% PEG 10000 (w/v) (for mutant R301A)
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A146S
increases the half-life of thermal inactivation at 45°C from around 1 min to 8 min
A319E
increases the half-life of thermal inactivation at 45°C from around 1 min to 2 min
A319E/T101A
increases the half-life of thermal inactivation at 45°C from around 1 min to 5 min
A325V
thermostability almost identical to that of the wild-type enzyme
C336D
increases solubility and activity, thermostability almost identical to that of the wild-type enzyme
D13E
increases solubility and activity, thermostability almost identical to that of the wild-type enzyme
D13E/M26I/V71I/E130K/Q132R/Q137R/I150F/E175A/Q215L/R275Q/L276Q/I313L/V315A/A319E/A325V/E332N/C336D
thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity. Mutation E175A leads to relaxation of cofactor specificity
E130K
increases the half-life of thermal inactivation at 45°C from around 1 min to 12.5 min
E130Q
increases the half-life of thermal inactivation at 45°C from around 1 min to 7 min
E130R
increases the half-life of thermal inactivation at 45°C from around 1 min to 9 min
E175A
E332N
increases solubility and activity, thermostability almost identical to that of the wild-type enzyme
F198M
increases the half-life of thermal inactivation at 45°C from around 1 min to 2 min
I150F
increases the half-life of thermal inactivation at 45°C from around 1 min to 7 min
I313L
thermostability almost identical to that of the wild-type enzyme
L276C
increases the half-life of thermal inactivation at 45°C from around 1 min to 12 min
L276H
increases the half-life of thermal inactivation at 45°C from around 1 min to 2 min
L276Q
increases the half-life of thermal inactivation at 45°C from around 1 min to 3.5 min
L276R
increases the half-life of thermal inactivation at 45°C from around 1 min to 8 min
L276S
increases the half-life of thermal inactivation at 45°C from around 1 min to 3 min
M26I
increases solubility and activity, thermostability almost identical to that of the wild-type enzyme
M53A
the KM values for phosphonate and NAD+ are little different from the parent enzyme, while kcat is reduced by a factor of 50
M53N
the KM values for phosphonate and NAD+ are little different from the parent enzyme, while kcat is reduced by a factor of 200
Q132K
increases the half-life of thermal inactivation at 45°C from around 1 min to 3 min
Q132R
increases the half-life of thermal inactivation at 45°C from around 1 min to 2 min
Q137H
increases the half-life of thermal inactivation at 45°C from around 1 min to 4.5 min
Q137R
increases the half-life of thermal inactivation at 45°C from around 1 min to 4 min
Q215L
increases the half-life of thermal inactivation at 45°C from around 1 min to 9 min
Q215M
increases the half-life of thermal inactivation at 45°C from around 1 min to 2.5 min
R275L
increases the half-life of thermal inactivation at 45°C from around 1 min to 9 min
R275Q
increases the half-life of thermal inactivation at 45°C from around 1 min to 4.5 min
R301A
R301K
S295A
mutant based on a thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity, plus mutation E175A that allows this mutant to use both NAD+ and NADP+. Mutation S295A leads to sharp decrease in activity, while kcat value is similar to wild-type
T101A
increases the half-life of thermal inactivation at 45°C from around 1 min to 4.5 min
V315A
thermostability almost identical to that of the wild-type enzyme
V71Ia
thermostability almost identical to that of the wild-type enzyme
W134A
mutant based on a thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity, plus mutation E175A that allows this mutant to use both NAD+ and NADP+. Mutation W134A leads to sharp decrease in activity, while kcat value is similar to wild-type
W134F
mutant based on a thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity, plus mutation E175A that allows this mutant to use both NAD+ and NADP+. Mutation W134F leads to sharp decrease in activity, while kcat value is similar to wild-type
Y139F
mutant based on a thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity, plus mutation E175A that allows this mutant to use both NAD+ and NADP+. Mutation Y139F leads to sharp decrease in activity, while kcat value is similar to wild-type
D13E/M26I/E175A/E332N/C336D
-
mutant obtained by directed evolution, round 4
D13E/M26I/E175A/T181S/A308T/E332N/C336D
-
mutant obtained by directed evolution, round 6, strong decrease in KM value for NADP compared to wild-type
D13E/M26I/E175A/T181S/E332N/C336D
-
mutant obtained by directed evolution, round 5
D79A
-
significant differences in its kinetic constants compared to the wild-type enzyme. 2600fold decrease in catalytic efficiency. Pre-steady-state rates are approximately the same as the steady-state rates
E175A
E175A/A176R
E266Q
H292F
H292K
H292N
I150F
-
thermostable mutant, half-life at 45°C 7.0 min compared to 1.4 min of wild-type
K76A
Q132R
-
thermostable mutant, half-life at 45°C 2.3 min compared to 1.4 min of wild-type
Q132R/Q137R/I150F/Q215L/R275Q
-
thermostable mutant, half-life at 45°C 161 min compared to 1.4 min of wild-type
Q137R
-
thermostable mutant, half-life at 45°C 3.8 min compared to 1.4 min of wild-type
Q137R/I150F/Q215L/R275Q
-
thermostable mutant, half-life at 45°C 200 min compared to 1.4 min of wild-type
Q137R/I150F/Q215L/R275Q/A319E
-
thermostable mutant, half-life at 45°C 567 min compared to 1.4 min of wild-type
Q137R/I150F/Q215L/R275Q/D162N/V315A
-
thermostable mutant, half-life at 45°C 614 min compared to 1.4 min of wild-type
Q137R/I150F/Q215L/R275Q/L276Q
-
thermostable mutant, half-life at 45°C 437 min compared to 1.4 min of wild-type
Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A
-
thermostable mutant, half-life at 45°C 1421 min compared to 1.4 min of wild-type
Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/A325V
-
thermostable mutant, half-life at 45°C 2315 min compared to 1.4 min of wild-type
Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/E130K
-
thermostable mutant, half-life at 45°C 1515 min compared to 1.4 min of wild-type
Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/I313L
-
thermostable mutant, half-life at 45°C 1765 min compared to 1.4 min of wild-type
Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R
-
thermostable mutant, half-life at 45°C 2350 min compared to 1.4 min of wild-type
Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R/V71I/E130K/I313L/A325V
-
thermostable mutant, half-life at 45°C 8440 min compared to 1.4 min of wild-type
Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R/V71I/E130K/I313L/A325V/A176R
-
highly stable and active mutant engineered for regeneration of NADPH and enzyme membrane reactors
Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/V71I
-
thermostable mutant, half-life at 45°C 2000 min compared to 1.4 min of wild-type
Q215L
-
thermostable mutant, half-life at 45°C 8.7 min compared to 1.4 min of wild-type
R237K
R275Q
-
thermostable mutant, half-life at 45°C 4.6 min compared to 1.4 min of wild-type
additional information
E175A
increases solubility and activity, thermostability almost identical to that of the wild-type enzyme
R301A
mutant based on a thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity, plus mutation E175A that allows this mutant to use both NAD+ and NADP+. Mutation R301A leads to sharp decrease in activity, while kcat value is similar to wild-type
R301A
the mutant shows about 100fold loss in kcat and a 500fold increased Km value for phosphonate. The Ki value of sulfite with the mutant is 400fold increased compared to the wild type enzyme
R301K
mutant based on a thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity, plus mutation E175A that allows this mutant to use both NAD+ and NADP+. Mutation R301K leads to sharp decrease in activity, while kcat value is similar to wild-type
R301K
the mutant exhibits a slightly higher kcat than the wild type enzyme and a 20fold increased Km value for phosphonate
E175A/A176R
-
double mutant, 3.6-fold higher efficiency with NAD+, 1000-fold higher efficiency with NADP+, 3-fold favor for NADP+ over NAD+ as cofactor
E175A/A176R
-
mutant utilizes both NAD+ and NADP+, kinetic isotope effects study, hydride transfer step is partially rate determining
E266Q
-
significant increase in Km for both substrates, increase in turnover
E266Q
-
higher activity, steady-state and pre-steady-state rates are comparable
K76A
-
pre-steady-state rates are approximately the same as the steady-state rates
R237K
-
low activity, absence of a significant burst in the pre-steady-state
additional information
12 x PTDH mutant, saturation mutagenesis performed separately on each of the following residues in the parent PTDH template: V71, E130, Q132, Q137, I150, Q215, R275, L276, I313, V315, A319, and A325. The most thermostabilizing mutation discovered for each particular site is incorporated into the 12 x PTDH mutant, performed for K132, H137, L275, and C276, forming an optimized thermally stable phosphite dehydrogenase termed Opt12. Addition of A146S to Opt12 leads to the Opt13 variant, and the further addition of F198M leads to Opt14
additional information
mutant 17x-PTDH is based on a thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity, plus mutation E175A that allows this mutant to use both NAD+ and NADP+
additional information
-
mutant 17x-PTDH is based on a thermostable mutant TS-PTDH which contains 12 mutations that result in considerably increased thermostability with minimal change in activity, and four additional mutations that increase its activity, plus mutation E175A that allows this mutant to use both NAD+ and NADP+
additional information
-
thermostable mutant 12X-PTDH with higher solubility than the wild-type. Thermostable mutant with dual cofactor specificity NADP-12X NAD shows pre-steady-state behavior very similar to that observed with 12X-PTDH and the wild-type enzyme. Pre-steady-state traces of thermostable mutant with dual cofactor specificity NADP-12X NADP shows curvature with NADP, particularly with protiated phosphite
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
40.5
-
rapid inactivation, half life less than 10 min, 1 mM NAD+ partially protects against thermal inactivation, E175A/A176R double mutant is readily protected against inactivation by addition of 1 mM NADP+ for at least 15 min
59.3
-
mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R/V71I/E130K/I313L/A325V, melting temperature
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Ni2+-affinity column chromatography and Superdex 200 gel filtration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
His6-tagged PTDH overexpressed in Escherichia coli BL21(DE3) harboring a pET15b vector
-
wild type and mutant proteins expressed in Escherichia coli BL21(DE3) as His-tag fusion protein
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
biotechnology
synthesis
-
production of deuterium- or tritium-labeled substances, mutant enzymes could be applied as NADPH regeneration systems
additional information
biotechnology
-
application of mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R/V71I/E130K/I313L/A325V/A176R for regeneration of NADPH in xylose reductase-catalyzed xylitol synthesis and alcohol dehydrogenase-catalyzed (R)-phenylethanol synthesis. comparison of enzyme with commercial Pseudomonas sp. formate dehydrogenase. Mutant Q137R/I150F/Q215L/R275Q/L276Q/A319E/V315A/Q132R/V71I/E130K/I313L/A325V/A176R shows higher substrate conversion and higher total turnover numbers for NADP+ than formate dehydrogenase
biotechnology
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evaluation of enzyme mutants for regeneration of reduced cofactors NADH and NADPH in industrial processes and comparison with Candida boidinii formate dehydrogenase
biotechnology
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improvement of enzyme for use in regeneration of NADH and NADPH, application in bioconversion of trimethylpyruvate to L-tert-leucine as model reaction
biotechnology
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use of enzyme mutant E175A/A176R for regeneration of NADH and NADPH. Model system converting xylose into xylitol by NADP-dependent xylose reductase and comparison of regeneration of NADPH by enzyme mutant and by Pseudomonas sp. formate dehydrogenase
additional information
random mutagenesis followed by comprehensive saturation mutagenesis further improves the enzyme thermostability while maintaining its activity. Mutant has improved the half-life of thermal inactivation at 45°C by 23,000fold over the parent enzyme. The engineered phosphite dehydrogenase will be useful in NAD(P)H regeneration
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Costas, A.M.G.; White, A.K.; Metcalf, W.W.
Purification and characterization of a novel phosphorus-oxidizing enzyme from Pseudomonas stutzeri WM88
J. Biol. Chem.
276
17429-17436
2001
Pseudomonas stutzeri (O69054)
Vrtis, J.M.; White, A.K.; Metcalf, W.W.; van der Donk, W.A.
Phosphite dehydrogenase: An unusual phosphoryl transfer reaction
J. Am. Chem. Soc.
123
2672-2673
2001
Pseudomonas stutzeri
Woodyer, R.; van der Donk, W.A.; Zhao, H.
Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design
Biochemistry
42
11604-11614
2003
Pseudomonas stutzeri
Woodyer, R.; Wheatley, J.L.; Relyea, H.A.; Rimkus, S.; van der Donk, W.A.
Site-directed mutagenesis of active site residues of phosphite dehydrogenase
Biochemistry
44
4765-4774
2005
Pseudomonas stutzeri, Pseudomonas stutzeri WM 88
Relyea, H.A.; Vrtis, J.M.; Woodyer, R.; Rimkus, S.A.; van der Donk, W.A.
Inhibition and pH dependence of phosphite dehydrogenase
Biochemistry
44
6640-6649
2005
Pseudomonas stutzeri
Relyea, H.A.; van der Donk, W.A.
Mechanism and applications of phosphite dehydrogenase
Bioorg. Chem.
33
171-189
2005
Alcaligenes faecalis, Pseudomonas stutzeri, Pseudomonas stutzeri WM88
Johannes, T.W.; Woodyer, R.D.; Zhao, H.
Directed evolution of a thermostable phosphite dehydrogenase for NAD(P)H regeneration
Appl. Environ. Microbiol.
71
5728-5734
2005
Pseudomonas stutzeri
Johannes, T.W.; Woodyer, R.D.; Zhao, H.
Efficient regeneration of NADPH using an engineered phosphite dehydrogenase
Biotechnol. Bioeng.
96
18-26
2007
Pseudomonas stutzeri
Woodyer, R.; van der Donk, W.A.; Zhao, H.
Optimizing a biocatalyst for improved NAD(P)H regeneration: directed evolution of phosphite dehydrogenase
Comb. Chem. High Throughput Screen.
9
237-245
2006
Pseudomonas stutzeri
Woodyer, R.; Zhao, H.; van der Donk, W.A.
Mechanistic investigation of a highly active phosphite dehydrogenase mutant and its application for NADPH regeneration
FEBS J.
272
3816-3827
2005
Pseudomonas stutzeri
Fogle, E.J.; van der Donk, W.A.
Pre-steady-state studies of phosphite dehydrogenase demonstrate that hydride transfer is fully rate limiting
Biochemistry
46
13101-13108
2007
Pseudomonas stutzeri, Pseudomonas stutzeri WW88
McLachlan, M.J.; Johannes, T.W.; Zhao, H.
Further improvement of phosphite dehydrogenase thermostability by saturation mutagenesis
Biotechnol. Bioeng.
99
268-274
2008
Pseudomonas stutzeri (O69054)
Hung, J.E.; Fogle, E.J.; Christman, H.D.; Johannes, T.W.; Zhao, H.; Metcalf, W.W.; van der Donk, W.A.
Investigation of the role of Arg301 identified in the X-ray structure of phosphite dehydrogenase
Biochemistry
51
4254-4262
2012
Cupriavidus metallidurans, Cupriavidus metallidurans CH34, Methylorubrum extorquens, Methylorubrum extorquens ATCC 14718 / DSM 1338 / JCM 2805 / NCIMB 9133 / AM1, Nostoc punctiforme, Pseudomonas stutzeri (O69054), Pseudomonas stutzeri
Zou, Y.; Zhang, H.; Brunzelle, J.S.; Johannes, T.W.; Woodyer, R.; Hung, J.E.; Nair, N.; van der Donk, W.A.; Zhao, H.; Nair, S.K.
Crystal structures of phosphite dehydrogenase provide insights into nicotinamide cofactor regeneration
Biochemistry
51
4263-4270
2012
Pseudomonas stutzeri (O69054)
Hirota, R.; Yamane, S.T.; Fujibuchi, T.; Motomura, K.; Ishida, T.; Ikeda, T.; Kuroda, A.
Isolation and characterization of a soluble and thermostable phosphite dehydrogenase from Ralstonia sp. strain 4506
J. Biosci. Bioeng.
113
445-450
2012
Pseudomonas stutzeri, Pseudomonas stutzeri WM88, Ralstonia sp. (G4XDR8), Ralstonia sp., Ralstonia sp. 4506 (G4XDR8)
Beyer, N.; Kulig, J.K.; Bartsch, A.; Hayes, M.A.; Janssen, D.B.; Fraaije, M.W.
P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations
Appl. Microbiol. Biotechnol.
101
2319-2331
2017
Pseudomonas stutzeri, Pseudomonas stutzeri WM88
Ranaghan, K.; Hung, J.; Bartlett, G.; Mooibroek, T.; Harvey, J.; Woolfson, D.; Van Der Donk, W.; Mulholland, A.
A catalytic role for methionine revealed by a combination of computation and experiments on phosphite dehydrogenase
Chem. Sci.
5
2191-2199
2014
Pseudomonas stutzeri (O69054), Pseudomonas stutzeri WM88 (O69054)
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