Literature summary for 1.5.99.B2 extracted from

Monaghan, P.J.; Leys, D.; Scrutton, N.S.
Mechanistic aspects and redox properties of hyperthermophilic L-proline dehydrogenase from Pyrococcus furiosus related to dimethylglycine dehydrogenase/oxidase (2007), FEBS J., 274, 2070-2087.

Search for more literature for 1.5.99.B2

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli Rosetta(DE3)pLysS cells	Pyrococcus furiosus

Protein Variants

Protein Variants	Comment	Organism
H225A	the mutant is unstable and precipitates from solution below pH 7.0, decreased activity compared to the wild type enzyme	Pyrococcus furiosus
H225Q	the mutant displays activity down to solution pH 6.0, increased activity compared to the wild type enzyme	Pyrococcus furiosus
Y251F	wild type pH stability, increased activity compared to the wild type enzyme	Pyrococcus furiosus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
1.95	-	L-proline	mutant enzyme Y215F, in 100 mM potassium phosphate buffer, pH 7.5, at 60°C	Pyrococcus furiosus
5.3	-	L-proline	wild type enzyme, in 100 mM potassium phosphate buffer, pH 7.5, at 50°C	Pyrococcus furiosus
5.6	-	L-proline	wild type enzyme, in 100 mM potassium phosphate buffer, pH 7.5, at 40°C	Pyrococcus furiosus
9.9	-	L-proline	wild type enzyme, in 100 mM potassium phosphate buffer, pH 7.5, at 60°C	Pyrococcus furiosus
14.46	-	L-proline	mutant enzyme H225Q, in 100 mM potassium phosphate buffer, pH 7.5, at 60°C	Pyrococcus furiosus
19.67	-	L-proline	mutant enzyme H225A, in 100 mM potassium phosphate buffer, pH 7.5, at 60°C	Pyrococcus furiosus
19.9	-	L-proline	wild type enzyme, in 100 mM potassium phosphate buffer, pH 7.5, at 70°C	Pyrococcus furiosus
30.8	-	L-proline	wild type enzyme, in 100 mM potassium phosphate buffer, pH 7.5, at 80°C	Pyrococcus furiosus
212.3	-	L-pipecolic acid	wild type enzyme, in 100 mM potassium phosphate buffer, pH 7.5, at 80°C	Pyrococcus furiosus

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
mitochondrion	-	Pyrococcus furiosus	5739	-

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
42440	-	beta subunit, MALDI-TOF mass spectrometry	Pyrococcus furiosus
42470	-	beta subunit, electrospray ionization mass spectrometry	Pyrococcus furiosus

Organism

Organism	UniProt	Comment	Textmining
Pyrococcus furiosus	-	strain DSM 3638	-

Purification (Commentary)

Purification (Comment)	Organism
DE52 anion exchange column chromatography	Pyrococcus furiosus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
L-pipecolic acid + acceptor + H2O	-	Pyrococcus furiosus	? + reduced acceptor	-	?
L-proline + acceptor + H2O	preferred substrate	Pyrococcus furiosus	(S)-1-pyrroline-5-carboxylate + reduced acceptor	-	?
additional information	does not reduce NAD+	Pyrococcus furiosus	?	-	?
sarcosine + acceptor + H2O	-	Pyrococcus furiosus	? + reduced acceptor	-	?

Subunits

Subunits	Comment	Organism
heterooctamer	x-ray crystallography	Pyrococcus furiosus

Synonyms

Synonyms	Comment	Organism
L-proline dehydrogenase	-	Pyrococcus furiosus
PRODH	-	Pyrococcus furiosus

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
100	-	no loss of activity being evident up to 100°C, above this temperature, thermal denaturation of PRODH is apparent, with complete loss of activity after 10 min of incubation in glycerol buffer at temperatures above 115°C	Pyrococcus furiosus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
0.4	-	L-pipecolic acid	wild type enzyme in 100 mM potassium phosphate buffer, pH 7.5, at 80°C	Pyrococcus furiosus
0.4	-	L-proline	wild type enzyme, in 100 mM potassium phosphate buffer, pH 7.5, at 40°C	Pyrococcus furiosus
1.3	-	L-proline	wild type enzyme, in 100 mM potassium phosphate buffer, pH 7.5, at 50°C	Pyrococcus furiosus
1.4	-	L-proline	mutant enzyme H225A, in 100 mM potassium phosphate buffer, pH 7.5, at 60°C	Pyrococcus furiosus
4	-	L-proline	wild type enzyme, in 100 mM potassium phosphate buffer, pH 7.5, at 60°C	Pyrococcus furiosus
8.97	-	L-proline	mutant enzyme H225Q, in 100 mM potassium phosphate buffer, pH 7.5, at 60°C	Pyrococcus furiosus
10.4	-	L-proline	wild type enzyme, in 100 mM potassium phosphate buffer, pH 7.5, at 70°C	Pyrococcus furiosus
18.01	-	L-proline	wild type enzyme, in 100 mM potassium phosphate buffer, pH 7.5, at 80°C	Pyrococcus furiosus
37.17	-	L-proline	mutant enzyme Y215F, in 100 mM potassium phosphate buffer, pH 7.5, at 60°C	Pyrococcus furiosus

pH Range

pH Minimum	pH Maximum	Comment	Organism
5	10	-	Pyrococcus furiosus

pH Stability

pH Stability	pH Stability Maximum	Comment	Organism
5	10	-	Pyrococcus furiosus

Cofactor

Cofactor	Comment	Organism	Structure
ATP	the holoenzyme contains one ATP per alphabeta complex	Pyrococcus furiosus
FAD	flavoprotein amine dehydrogenase, the holoenzyme contains one FAD per alphabeta complex	Pyrococcus furiosus
FMN	the holoenzyme contains one FMN per alphabeta complex	Pyrococcus furiosus

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Release 2023.1 (January 2023)