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Information on EC 1.1.99.31 - (S)-mandelate dehydrogenase and Organism(s) Pseudomonas putida and UniProt Accession P20932
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1.1.99.31 (S)-mandelate dehydrogenaseIUBMB Comments
This enzyme is a member of the FMN-dependent alpha-hydroxy-acid oxidase/dehydrogenase family . While all enzymes of this family oxidize the (S)-enantiomer of an alpha-hydroxy acid to an alpha-oxo acid, the ultimate oxidant (oxygen, intramolecular heme or some other acceptor) depends on the particular enzyme. This enzyme transfers the electron pair from FMNH2 to a component of the electron transport chain, most probably ubiquinone [1,2]. It is part of a metabolic pathway in Pseudomonads that allows these organisms to utilize mandelic acid, derivatized from the common soil metabolite amygdalin, as the sole source of carbon and energy . The enzyme has a large active-site pocket and preferentially binds substrates with longer sidechains, e.g. 2-hydroxyoctanoate rather than 2-hydroxybutyrate . It also prefers substrates that, like (S)-mandelate, have beta unsaturation, e.g. (indol-3-yl)glycolate compared with (indol-3-yl)lactate . Esters of mandelate, such as methyl (S)-mandelate, are also substrates .
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Word Map
- 1.1.99.31
- s-mandelate
- putida
- benzoylformate
-
fmn-dependent
- benzaldehyde
- calcoaceticus
- fmn
- mitra
-
alpha-hydroxy
- l-lactate
- phenylglyoxylate
-
carbanion
-
flavocytochrome
-
membrane-binding
-
flavoenzyme
-
alpha-protons
-
half-reaction
-
rhodotorula
- graminis
-
electron-rich
The taxonomic range for the selected organisms is: Pseudomonas putida
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Synonyms
(s)-mandelate dehydrogenase, l-mandelate dehydrogenase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
PATHWAY SOURCE
PATHWAYS
-
-
SYSTEMATIC NAME
IUBMB Comments
(S)-2-hydroxy-2-phenylacetate:acceptor 2-oxidoreductase
This enzyme is a member of the FMN-dependent alpha-hydroxy-acid oxidase/dehydrogenase family [1]. While all enzymes of this family oxidize the (S)-enantiomer of an alpha-hydroxy acid to an alpha-oxo acid, the ultimate oxidant (oxygen, intramolecular heme or some other acceptor) depends on the particular enzyme. This enzyme transfers the electron pair from FMNH2 to a component of the electron transport chain, most probably ubiquinone [1,2]. It is part of a metabolic pathway in Pseudomonads that allows these organisms to utilize mandelic acid, derivatized from the common soil metabolite amygdalin, as the sole source of carbon and energy [2]. The enzyme has a large active-site pocket and preferentially binds substrates with longer sidechains, e.g. 2-hydroxyoctanoate rather than 2-hydroxybutyrate [1]. It also prefers substrates that, like (S)-mandelate, have beta unsaturation, e.g. (indol-3-yl)glycolate compared with (indol-3-yl)lactate [1]. Esters of mandelate, such as methyl (S)-mandelate, are also substrates [3].
CAS REGISTRY NUMBER
COMMENTARY
9067-95-2
-
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
(R,S)-2-hydroxy-3-butenoic acid + acceptor
2-oxo-3-butenoic acid + reduced acceptor
very low binding affinity for MDH
-
-
?
(R,S)-2-hydroxybutyrate + acceptor
2-oxobutyrate + reduced acceptor
slow substrate
-
-
?
(R,S)-2-hydroxybutyric acid + acceptor
2-oxo-3-butynoic acid + reduced acceptor
very low binding affinity for MDH
-
-
?
(R,S)-2-hydroxyhexanoate + acceptor
2-oxohexanoate + reduced acceptor
-
-
-
?
(R,S)-2-hydroxyisocaproate + acceptor
2-oxoisocaproate + reduced acceptor
-
-
-
?
(R,S)-2-hydroxyisovalerate + acceptor
2-oxo-isovalerate + reduced acceptor
-
-
-
?
(R,S)-2-hydroxyoctanoate + acceptor
2-oxooctanoate + reduced acceptor
slow substrate
-
-
?
(R,S)-2-hydroxyvalerate + acceptor
2-oxovalerate + reduced acceptor
-
-
-
?
(R,S)-3-indolelactate + acceptor
3-(1H-indol-2-yl)-2-oxopropanoate + reduced acceptor
-
-
-
?
(R,S)-3-phenyllactate + 2,6-dichlorophenolindophenol
phenylpyruvate + reduced 2,6-dichlorophenolindophenol
slow substrate
-
-
?
(R,S)-indoleglycolate + acceptor
indol-2-yl(oxo)acetate + reduced acceptor
-
-
-
?
(R,S)-mandelate + 2,6-dichlorophenolindophenol
2-oxo-2-phenylacetate + reduced 2,6-dichlorophenolindophenol
-
-
-
?
(R,S)-mandelate + ferricyanide
2-oxo-2-phenylacetate + ferrocyanide
-
-
-
?
(S)-mandelate + 2 ferricyanide
2-oxo-2-phenylacetate + 2 ferrocyanide + 2 H+
-
-
-
?
(S)-mandelate + acceptor
phenylglyoxylate + reduced acceptor
-
-
-
?
2-hydroxy-3-butynoate + acceptor
2-oxo-3-butynoate + reduced acceptor
-
-
-
?
mandelate + acceptor
2-oxo-2-phenylacetate + reduced acceptor
-
-
-
?
(R,S)-2-hydroxy-3-butenoic acid + acceptor
2-oxo-3-butenoic acid + reduced acceptor
-
-
-
-
?
(R,S)-2-hydroxyisovalerate + acceptor
2-oxo-isovalerate + reduced acceptor
-
-
-
-
?
(S)-2-hydroxy-2-phenylacetate + O2
2-oxo-2-phenylacetate + H2O2
-
in absence of any other electron acceptor, oxygen is used by the wild-type enzyme for reoxidation of the reduced flavin, at a rather slow rate. Ping-pong kinetics
-
-
?
(S)-mandelate + 2,6-dichlorophenolindophenol
2-oxo-2-phenylacetate + reduced 2,6-dichlorophenolindophenol
-
-
-
-
?
2-hydroxy-3-butynoate + acceptor
2-oxo-3-butynoate + reduced acceptor
-
-
-
-
?
2-hydroxybutyrate + acceptor
2-oxobutyrate + reduced acceptor
-
-
-
-
?
2-hydroxyhexanoate + acceptor
2-oxohexanoate + reduced acceptor
-
-
-
-
?
2-hydroxyisocaproate + acceptor
2-oxoisocaproate + reduced acceptor
-
-
-
-
?
2-hydroxyoctanoate + acceptor
2-oxooctanoate + reduced acceptor
-
-
-
-
?
2-hydroxyvalerate + acceptor
2-oxovalerate + reduced acceptor
-
-
-
-
?
3-indolelactate + acceptor
3-(1H-indol-2-yl)-2-oxopropanoate + reduced acceptor
-
-
-
-
?
3-phenyllactate + acceptor
2-oxo-3-phenylpropanoate + reduced acceptor
-
-
-
-
?
4-chloromandelate + acceptor
2-oxo-2-(4-chlorophenyl)acetate + reduced acceptor
-
-
-
-
?
indoleglycolate + acceptor
indol-2-yl(oxo)acetate + reduced acceptor
-
-
-
-
?
mandelate + acceptor
2-oxo-2-phenylacetate + reduced acceptor
-
-
-
-
?
(S)-mandelate + acceptor
2-oxo-2-phenylacetate + reduced acceptor
-
-
-
-
?
(S)-mandelate + acceptor
2-oxo-2-phenylacetate + reduced acceptor
-
-
-
-
r
(S)-mandelate + acceptor
2-oxo-2-phenylacetate + reduced acceptor
-
acceptor: 2,6-dichloroindophenol plus phenazine methosulfate. The MDH reaction has two rate-limiting step of similar activation energies: the formation and breakdown of a distinct intermediate, with the latter step being slightly more rate limiting. MDH is capable of catalyzing the reverse reaction, the reoxidation of reduced MDH by the product ketoacid, benzoylformate. The transient intermediate is observed during the reverse reaction as well
-
-
r
(S)-mandelate + acceptor
2-oxo-2-phenylacetate + reduced acceptor
-
phenazine methosulfate and 2,6-dichloroindophenol as acceptor. In absence of any other electron acceptor, oxygen is used by the wild-type enzyme for reoxidation of the reduced flavin, at a rather slow rate
-
-
?
(S)-mandelate + acceptor
2-oxo-2-phenylacetate + reduced acceptor
-
reduction of 2,6-dichloroindophenol in presence of N-methylphenazonium methosulfate
-
-
?
additional information
?
-
(S)-mandelamide, (S)-1-phenyl-2-propen-1-ol, (S)-1-phenyl-1,2-ethanediol, (S)-1-phenyl-2-propyn-1-ol, (R,S)-pantoyllactone, and (S)-1-phenyl-2,2,2-trifluoroethanol are no substrates
-
-
?
additional information
?
-
-
(S)-mandelamide, (S)-1-phenyl-2-propen-1-ol, (S)-1-phenyl-1,2-ethanediol, (S)-1-phenyl-2-propyn-1-ol, (R,S)-pantoyllactone, and (S)-1-phenyl-2,2,2-trifluoroethanol are no 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
(S)-mandelate + acceptor
phenylglyoxylate + reduced acceptor
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
FMN
-
dependent on. In the reductive half-reaction (S)-mandelate is oxidized to 2-oxo-2-phenylacetate, resulting in the reduction of FMN
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
22
(R,S)-2-hydroxy-3-butynoic acid
in 0.1 M potassium phosphate buffer, pH 7.5 at 20°C
32
(R,S)-2-hydroxybutyrate
in 0.1 M potassium phosphate buffer, pH 7.5 at 20°C
4.9
(R,S)-2-hydroxyhexanoate
in 0.1 M potassium phosphate buffer, pH 7.5 at 20°C
4.3
(R,S)-2-hydroxyisocaproate
in 0.1 M potassium phosphate buffer, pH 7.5 at 20°C
9.5
(R,S)-2-hydroxyisovalerate
in 0.1 M potassium phosphate buffer, pH 7.5 at 20°C
0.75
(R,S)-2-hydroxyoctanoate
in 0.1 M potassium phosphate buffer, pH 7.5 at 20°C
15.3
(R,S)-2-hydroxyvalerate
in 0.1 M potassium phosphate buffer, pH 7.5 at 20°C
0.9
(R,S)-3-indolelactate
in 0.1 M potassium phosphate buffer, pH 7.5 at 20°C
0.4
(R,S)-indoleglycolate
in 0.1 M potassium phosphate buffer, pH 7.5 at 20°C
0.33
(R,S)-mandelate
in 0.1 M potassium phosphate buffer, pH 7.5 at 20°C
2
DL-3-phenyllactate
in 0.1 M potassium phosphate buffer, pH 7.5 at 20°C
10.3
2-Hydroxy-3-butenoate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
2.6
2-Hydroxyisovalerate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.12
(S)-Mandelate
wild type enzyme, at 20°C, in 0.1 M potassium phosphate, pH 7.5
5.6
(S)-Mandelate
mutant enzyme R277K, at 20°C, in 0.1 M potassium phosphate, pH 7.5
17
(S)-Mandelate
mutant enzyme R277G, at 20°C, in 0.1 M potassium phosphate, pH 7.5
2.7
ethyl-(S)-mandelate
mutant enzyme R277K, at 20°C, in 0.1 M potassium phosphate, pH 7.5
2.9
ethyl-(S)-mandelate
wild type enzyme, at 20°C, in 0.1 M potassium phosphate, pH 7.5
3.7
ethyl-(S)-mandelate
mutant enzyme R277G, at 20°C, in 0.1 M potassium phosphate, pH 7.5
7.4
methyl-(S)-mandelate
mutant enzyme R277G, at 20°C, in 0.1 M potassium phosphate, pH 7.5
10.3
methyl-(S)-mandelate
mutant enzyme R277K, at 20°C, in 0.1 M potassium phosphate, pH 7.5
11.3
methyl-(S)-mandelate
wild type enzyme, at 20°C, in 0.1 M potassium phosphate, pH 7.5
0.04
(S)-Mandelate
-
pH 7.5, 20°C, mutant enzyme, G81A/MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach and a G81A mutation in MDH
0.085
(S)-Mandelate
-
pH 7.5, 20°C, cosubstrate: 2,6-dichloroindophenol, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.09
(S)-Mandelate
-
pH 7.5, 20°C, mutant enzyme MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.158
(S)-Mandelate
-
pH 7.5, (S)-mandelate dehydrogenase with 17 residues deleted at the carboxy terminus
0.18
(S)-Mandelate
-
pH 7.5, 20°C, cosubstrate: 2,6-dichloroindophenol, wild-type enzyme
0.225
(S)-Mandelate
-
pH 7.5, (S)-mandelate dehydrogenase with residues 2-4 deleted
0.229
(S)-Mandelate
-
pH 7.5, chimeric mutant of (S)-mandelate dehydrogenase with membrane anchoring loop replaced by a portion of glycolate oxidase from spinach
0.78
(S)-phenyllactate
-
pH 7.5, chimeric mutant of (S)-mandelate dehydrogenase with membrane anchoring loop replaced by a portion of glycolate oxidase from spinach
17.2
2-Hydroxy-3-butynoate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
13.2
2-Hydroxybutyrate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.89
2-hydroxyhexanoate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.49
2-hydroxyisocaproate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.29
2-Hydroxyoctanoate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
3.2
2-hydroxyvalerate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.64
3-indolelactate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
1.3
3-Phenyllactate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.24
indoleglycolate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.14
Mandelate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.49
O2
-
pH 7.5, 20°C, mutant enzyme, G81A/MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach and a G81A mutation in MDH
0.68
O2
-
pH 7.5, 20°C, mutant enzyme MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with 205 residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.03
(R,S)-p-chloromandelate
-
mutant enzyme H274G, in presence of 20 mM imidazole
0.5
2-Hydroxy-3-butenoate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.03
2-Hydroxyisovalerate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.23
(S)-Mandelate
mutant enzyme R277G, at 20°C, in 0.1 M potassium phosphate, pH 7.5
66
(S)-Mandelate
mutant enzyme R277K, at 20°C, in 0.1 M potassium phosphate, pH 7.5
360
(S)-Mandelate
wild type enzyme, at 20°C, in 0.1 M potassium phosphate, pH 7.5
0.3
ethyl-(S)-mandelate
mutant enzyme R277G, at 20°C, in 0.1 M potassium phosphate, pH 7.5
2.6
ethyl-(S)-mandelate
mutant enzyme R277K, at 20°C, in 0.1 M potassium phosphate, pH 7.5
204
ethyl-(S)-mandelate
wild type enzyme, at 20°C, in 0.1 M potassium phosphate, pH 7.5
2.3
methyl-(S)-mandelate
mutant enzyme R277K, at 20°C, in 0.1 M potassium phosphate, pH 7.5
2.5
methyl-(S)-mandelate
mutant enzyme R277G, at 20°C, in 0.1 M potassium phosphate, pH 7.5
151
methyl-(S)-mandelate
wild type enzyme, at 20°C, in 0.1 M potassium phosphate, pH 7.5
1.6
(S)-Mandelate
-
pH 7.5, chimeric mutant of (S)-mandelate dehydrogenase with membrane anchoring loop replaced by a portion of glycolate oxidase from spinach
2.3
(S)-Mandelate
-
pH 7.5, 20°C, mutant enzyme, G81A/MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach and a G81A mutation in MDH
9
(S)-Mandelate
-
pH 7.5, (S)-mandelate dehydrogenase with residues 2-4 deleted
24
(S)-Mandelate
-
pH 7.5, (S)-mandelate dehydrogenase with 17 residues deleted at the carboxy terminus
205
(S)-Mandelate
-
pH 7.5, 20°C, cosubstrate: 2,6-dichloroindophenol, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
205
(S)-Mandelate
-
pH 7.5, 20°C, mutant enzyme MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with 205 residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
290
(S)-Mandelate
-
pH 7.5, 20°C, cosubstrate: 2,6-dichloroindophenol, wild-type enzyme
6.6
2-Hydroxy-3-butynoate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.1
2-Hydroxybutyrate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.48
2-hydroxyhexanoate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
1.28
2-hydroxyisocaproate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.96
2-Hydroxyoctanoate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.5
2-hydroxyvalerate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
3.13
3-indolelactate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
3.85
3-indolelactate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.87
3-Phenyllactate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
38.5
indoleglycolate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
195
Mandelate
-
pH 7.5, 20°C, MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.03
O2
-
pH 7.5, 20°C, mutant enzyme MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with 205 residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
0.22
O2
-
pH 7.5, 20°C, mutant enzyme, G81A/MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach and a G81A mutation in MDH
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
36
(R,S)-2-hydroxy-3-butynoic acid
in 0.1 M potassium phosphate buffer, pH 7.5 at 20°C
53.8
(S)-1-phenyl-1,2-ethanediol
wild type enzyme, at 20°C, in 0.1 M potassium phosphate, pH 7.5
2.6
(S)-1-phenyl-2,2,2-trifluoroethanol
wild type enzyme, at 20°C, in 0.1 M potassium phosphate, pH 7.5
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapor-diffusion method. Crystal structure of MDH-GOX2, a chimeric mutant of MDH with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
mutant G81A of the catalytically similar, soluble chimera MDH-GOX2 (mandelate dehydrogenase/glycolate oxidase)
1.35 A resolution structure of oxidized form and of the substrate-reduced form of MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
G81A
R277G
reduced activity compared to the wild type enzyme, the mutation at Arg277 has no influence on the binding affinity of ester substrates
R277K
reduced activity compared to the wild type enzyme, the mutation at Arg277 has no influence on the binding affinity of ester substrates
G81A
-
higher specificity for small substrates, compared to that of wild-type enzyme, the affinity for (S)-mandelate is relatively unchanged. The rate of the first half-reaction is slower than the wild-type rate. 2fold increase in oxidative half-reaction. Affinity for oxygen increases 10-15fold
G81D
-
extreme low activity, reduction in activity is due to the decrease in electrophilicity of the FMN
G81S
-
the rate of the first half-reaction is slower than the wild-type rate. The rate of the first half-reaction is slower than the wild-type rate. Affinity for O2 increases 10-15fold
G81V
-
extreme low activity, reduction in activity is due to the decrease in electrophilicity of the FMN
H274A
-
inactive mutant, activity can partially be restored by the addition of exogenous imidazole
H274G
-
inactive mutant, activity can be restored by the addition of exogenous imidazole
R165E
-
mutant is less stable than wild-type enzyme, mutant enzyme is only partially reduced by (S)-mandelate. 81.8fold decrease in kcat for (S)-mandelate, 199fold increase in KM-value for (S)-mandelate compared to wild-type enzyme
R165G
-
mutant is less stable than wild-type enzyme, mutant enzyme is only partially reduced by (S)-mandelate. 27.3fold decrease in kcat for (S)-mandelate, 48.3fold increase in KM-value for (S)-mandelate compared to wild-type enzyme
R165G/R277G
-
no activity. Double mutant is less stable than single Arg165 mutant in term of long-term storage
R165G/R277K
-
double mutant is less stable than single Arg165 mutant in term of long-term storage. 9000fold decrease in kcat for (S)-mandelate, 261.7fold increase in KM-value for (S)-mandelate compared to wild-type enzyme
R165K
-
mutant is less stable than wild-type enzyme, mutant enzyme is fully reduced on addition of (S)-mandelate. 3fold decrease in kcat for (S)-mandelate, 12.5fold increase in KM-value for (S)-mandelate compared to wild-type enzyme
R165K/R277G
-
no activity. Double mutant is less stable than single Arg165 mutant in term of long-term storage
R165K/R277K
-
double mutant is less stable than single Arg165 mutant in term of long-term storage. 327fold decrease in kcat for (S)-mandelate, 126.7fold increase in KM-value for (S)-mandelate compared to wild-type enzyme
R165M
-
mutant is less stable than wild-type enzyme, mutant enzyme is fully reduced on addition of (S)-mandelate. 19.6fold decrease in kcat for (S)-mandelate, 36.7fold increase in KM-value for (S)-mandelate compared to wild-type enzyme
R277G
R277H
-
kcat for (S)-mandelate is 1000fold lower than wild-type value, KM-value for (S)-mandelate is 608fold higher than wild-type value
R277K
R277L
-
kcat for (S)-mandelate is 421fold lower fold than wild-type value, KM-value for (S)-mandelate is 392fold higher than wild-type value
additional information
G81A
mutation in the catalytically similar chimera MDH-GOX2 (mandelate dehydrogenase/glycolate oxidase), mutant has lower reactivity with substrate
R277G
-
1565fold decrease in kcat for (S)-mandelate, 141fold increase in KM-value for (S)-mandelate compared to wild-type enzyme
R277G
-
kcat for (S)-mandelate is 178fold lower than wild-type value, KM-value for (S)-mandelate is 160fold higher than wild-type value, KM-value for {(S)-3-phenyllactate is 1.6fold lower than wild-type value
R277K
-
5.5 fold decrease in kcat for (S)-mandelate, 46.7fold increase in KM-value for (S)-mandelate compared to wild-type enzyme
R277K
-
kcat for (S)-mandelate is 3.7fold lower than wild-type value, KM-value for (S)-mandelate is 36.7fold higher than wild-type value
additional information
-
chimeric mutant of (S)-mandelate dehydrogenase with membrane anchoring loop replaced by a portion of glycolate oxidase from spinach is soluble and retains partial catalytic activity (about 1%) using (S)-mandelate as substrate. The activities of the soluble mutant enzymes (S)-mandelate dehydrogenase with residues 2-4 deleted and (S)-mandelate dehydrogenase with 17 residues deleted at the carboxy terminus are nearly the same when (S)-phenyllactate is used as substrate
additional information
-
MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach. G81A/MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach and a G81A mutation in MDH
additional information
-
MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach. This mutant is very similar to the wild-type membrane-bound enzyme in its spectroscopic properties, substrate specificity, catalytic activity, kinetic mechanism, and lack of reactivity toeards oxygen. It should prove to be a highly useful model for structural studies of MDH
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, wild-type enzyme can be stored frozen in 20% ethanediol for weeks without loss of activity
-
-70°C, wild-type enzyme retains activity for more than 1 year. The single Arg165 mutant lose activity after 2-3 months, double mutants R165K/R277K and R165G/R277K are inactivated after 2-3 weeks in storage
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
His6-tagged enzyme, Ni-resin column chromatography and Sephadex G-25 gel filtration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Mitra, B.; Gerlt, J.A.; Babbitt, P.C.; Koo, C.W.; Kenyon, G.L.; Joseph, D.; Petsko, G.A.
A novel structural basis for membrane association of a protein: construction of a chimeric soluble mutant of (S)-mandelate dehydrogenase from Pseudomonas putida
Biochemistry
32
12959-12967
1993
Pseudomonas putida
Xu, Y.; Mitra, B.
A highly active, soluble mutant of the membrane-associated (S)-mandelate dehydrogenase from Pseudomonas putida
Biochemistry
38
12367-12376
1999
Pseudomonas putida
Lehoux, I.E.; Mitra, B.
(S)-Mandelate dehydrogenase from Pseudomonas putida: mutations of the catalytic base histidine-274 and chemical rescue of activity
Biochemistry
38
9948-9955
1999
Pseudomonas putida
Lehoux, I.E.; Mitra, B.
Role of arginine 277 in (S)-mandelate dehydrogenase from Pseudomonas putida in substrate binding and transition state stabilization
Biochemistry
39
10055-10065
2000
Pseudomonas putida
Sukumar, N.; Xu, Y.; Gatti, D.L.; Mitra, B.; Mathews, F.S.
Structure of an active soluble mutant of the membrane-associated (S)-mandelate dehydrogenase
Biochemistry
40
9870-9878
2001
Pseudomonas putida (P20932), Pseudomonas putida
Xu, Y.; Dewanti, A.R.; Mitra, B.
Arginine 165/arginine 277 pair in (S)-mandelate dehydrogenase from Pseudomonas putida: role in catalysis and substrate binding
Biochemistry
41
12313-12319
2002
Pseudomonas putida
Dewanti, A.R.; Mitra, B.
A transient intermediate in the reaction catalyzed by (S)-mandelate dehydrogenase from Pseudomonas putida
Biochemistry
42
12893-12901
2003
Pseudomonas putida
Dewanti, A.R.; Xu, Y.; Mitra, B.
Role of glycine 81 in (S)-mandelate dehydrogenase from Pseudomonas putida in substrate specificity and oxidase activity
Biochemistry
43
10692-10700
2004
Pseudomonas putida
Sukumar, N.; Dewanti, A.R.; Mitra, B.; Mathews, F.S.
High resolution structures of an oxidized and reduced flavoprotein. The water switch in a soluble form of (S)-mandelate dehydrogenase
J. Biol. Chem.
279
3749-3757
2004
Pseudomonas putida
Lehoux, I.E.; Mitra, B.
(S)-Mandelate dehydrogenase from Pseudomonas putida: mechanistic studies with alternate substrates and pH and kinetic isotope effects
Biochemistry
38
5836-5848
1999
Pseudomonas putida (P20932), Pseudomonas putida
Dewanti, A.R.; Xu, Y.; Mitra, B.
Esters of mandelic acid as substrates for (S)-mandelate dehydrogenase from Pseudomonas putida: implications for the reaction mechanism
Biochemistry
43
1883-1890
2004
Pseudomonas putida (P20932), Pseudomonas putida
Sukumar, N.; Dewanti, A.; Merli, A.; Rossi, G.L.; Mitra, B.; Mathews, F.S.
Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates
Acta Crystallogr. Sect. D
65
543-552
2009
Pseudomonas putida (P20932), Pseudomonas putida
Mazurenko, I.; Ghach, W.; Kohring, G.W.; Despas, C.; Walcarius, A.; Etienne, M.
Immobilization of membrane-bounded (S)-mandelate dehydrogenase in sol-gel matrix for electroenzymatic synthesis
Bioelectrochemistry
104
65-70
2015
Pseudomonas putida, Pseudomonas putida DSM 291
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