Literature summary for 1.20.1.1 extracted from

McLachlan, M.J.; Johannes, T.W.; Zhao, H.
Further improvement of phosphite dehydrogenase thermostability by saturation mutagenesis (2008), Biotechnol. Bioeng., 99, 268-274.

Application

Application	Comment	Organism
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	Pseudomonas stutzeri

Cloned(Commentary)

Cloned (Comment)	Organism
mutants cloned into pET15b as a N-terminal His-tagged construct	Pseudomonas stutzeri

Protein Variants

Protein Variants	Comment	Organism
A146S	increases the half-life of thermal inactivation at 45°C from around 1 min to 8 min	Pseudomonas stutzeri
A319E	increases the half-life of thermal inactivation at 45°C from around 1 min to 2 min	Pseudomonas stutzeri
A319E/T101A	increases the half-life of thermal inactivation at 45°C from around 1 min to 5 min	Pseudomonas stutzeri
A325V	thermostability almost identical to that of the wild-type enzyme	Pseudomonas stutzeri
C336D	increases solubility and activity, thermostability almost identical to that of the wild-type enzyme	Pseudomonas stutzeri
D13E	increases solubility and activity, thermostability almost identical to that of the wild-type enzyme	Pseudomonas stutzeri
E130K	increases the half-life of thermal inactivation at 45°C from around 1 min to 12.5 min	Pseudomonas stutzeri
E130Q	increases the half-life of thermal inactivation at 45°C from around 1 min to 7 min	Pseudomonas stutzeri
E130R	increases the half-life of thermal inactivation at 45°C from around 1 min to 9 min	Pseudomonas stutzeri
E175A	increases solubility and activity, thermostability almost identical to that of the wild-type enzyme	Pseudomonas stutzeri
E332N	increases solubility and activity, thermostability almost identical to that of the wild-type enzyme	Pseudomonas stutzeri
F198I	leads to low activity	Pseudomonas stutzeri
F198M	increases the half-life of thermal inactivation at 45°C from around 1 min to 2 min	Pseudomonas stutzeri
I150F	increases the half-life of thermal inactivation at 45°C from around 1 min to 7 min	Pseudomonas stutzeri
I313L	thermostability almost identical to that of the wild-type enzyme	Pseudomonas stutzeri
L276C	increases the half-life of thermal inactivation at 45°C from around 1 min to 12 min	Pseudomonas stutzeri
L276H	increases the half-life of thermal inactivation at 45°C from around 1 min to 2 min	Pseudomonas stutzeri
L276Q	increases the half-life of thermal inactivation at 45°C from around 1 min to 3.5 min	Pseudomonas stutzeri
L276R	increases the half-life of thermal inactivation at 45°C from around 1 min to 8 min	Pseudomonas stutzeri
L276S	increases the half-life of thermal inactivation at 45°C from around 1 min to 3 min	Pseudomonas stutzeri
M26I	increases solubility and activity, thermostability almost identical to that of the wild-type enzyme	Pseudomonas stutzeri
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	Pseudomonas stutzeri
Q132K	increases the half-life of thermal inactivation at 45°C from around 1 min to 3 min	Pseudomonas stutzeri
Q132R	increases the half-life of thermal inactivation at 45°C from around 1 min to 2 min	Pseudomonas stutzeri
Q137H	increases the half-life of thermal inactivation at 45°C from around 1 min to 4.5 min	Pseudomonas stutzeri
Q137R	increases the half-life of thermal inactivation at 45°C from around 1 min to 4 min	Pseudomonas stutzeri
Q215L	increases the half-life of thermal inactivation at 45°C from around 1 min to 9 min	Pseudomonas stutzeri
Q215M	increases the half-life of thermal inactivation at 45°C from around 1 min to 2.5 min	Pseudomonas stutzeri
R275L	increases the half-life of thermal inactivation at 45°C from around 1 min to 9 min	Pseudomonas stutzeri
R275Q	increases the half-life of thermal inactivation at 45°C from around 1 min to 4.5 min	Pseudomonas stutzeri
T101A	increases the half-life of thermal inactivation at 45°C from around 1 min to 4.5 min	Pseudomonas stutzeri
V315A	thermostability almost identical to that of the wild-type enzyme	Pseudomonas stutzeri
V71Ia	thermostability almost identical to that of the wild-type enzyme	Pseudomonas stutzeri

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.04	-	NAD+	12 x PTDH mutant	Pseudomonas stutzeri
0.046	-	phosphite	12 x PTDH mutant	Pseudomonas stutzeri
0.05	-	NAD+	Opt12 variant	Pseudomonas stutzeri
0.054	-	NAD+	Opt13 variant	Pseudomonas stutzeri
0.057	-	phosphite	parent enzyme	Pseudomonas stutzeri
0.066	-	NAD+	parent enzyme	Pseudomonas stutzeri
0.079	-	phosphite	Opt12 variant	Pseudomonas stutzeri
0.09	-	phosphite	Opt13 variant	Pseudomonas stutzeri
0.105	-	NAD+	Opt14 variant	Pseudomonas stutzeri
0.142	-	phosphite	Opt14 variant	Pseudomonas stutzeri

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas stutzeri	O69054	-	-

Purification (Commentary)

Purification (Comment)	Organism
by small-scale protein purification	Pseudomonas stutzeri

Storage Stability

Storage Stability	Organism
-80°C, 20% glycerol	Pseudomonas stutzeri

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
phosphite + H2O + NAD+	-	Pseudomonas stutzeri	phosphate + NADH + H+	-	?

Synonyms

Synonyms	Comment	Organism
phosphite dehydrogenase	-	Pseudomonas stutzeri
PTDH	-	Pseudomonas stutzeri

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
3.25	-	phosphite	12 x PTDH mutant	Pseudomonas stutzeri
3.55	-	phosphite	Opt12 and Opt13 variants	Pseudomonas stutzeri
3.65	-	phosphite	Opt14 variant	Pseudomonas stutzeri
4.367	-	phosphite	parent enzyme	Pseudomonas stutzeri

Cofactor

Cofactor	Comment	Organism	Structure
NAD+	-	Pseudomonas stutzeri