Information on EC 1.1.2.3 - L-lactate dehydrogenase (cytochrome)

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
1.1.2.3
-
RECOMMENDED NAME
GeneOntology No.
L-lactate dehydrogenase (cytochrome)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(S)-lactate + 2 ferricytochrome c = pyruvate + 2 ferrocytochrome c + 2 H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
lactate fermentation
-
-
Metabolic pathways
-
-
methylglyoxal degradation V
-
-
Pyruvate metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
(S)-lactate:ferricytochrome-c 2-oxidoreductase
Identical with cytochrome b2; a flavohemoprotein (FMN).
CAS REGISTRY NUMBER
COMMENTARY hide
9078-32-4
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
356
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
K-105
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
IZR-106
-
-
Manually annotated by BRENDA team
IZR-42
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
-
the pyruvate produced as a result of L-lactate oxidation is utilised by the Krebs cycle, but flavocytochrome b2 also forms part of a short respiratory electron transport chain which results in one ATP molecule being produced for every L-lactate consumed
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(S)-2-hydroxybutyrate + ferricytochrome c
2-oxobutyrate + ferrocytochrome c
show the reaction diagram
-
-
-
-
?
(S)-2-hydroxyhexanoate + ferricytochrome c
2-oxohexanoate + ferrocytochrome c
show the reaction diagram
-
-
-
-
?
(S)-2-hydroxyoctanoate + ferricytochrome c
2-oxooctanoate + ferrocytochrome c
show the reaction diagram
-
-
-
-
?
(S)-2-hydroxyvalerate + ferricytochrome c
2-oxovalerate + ferrocytochrome c
show the reaction diagram
-
-
-
-
?
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
show the reaction diagram
(S)-lactate + 2 potassium ferricyanide
pyruvate + 2 reduced potassium ferricyanide + 2 H+
show the reaction diagram
-
-
-
-
?
(S)-lactate + cytochrome c
pyruvate + oxidized cytochrome c
show the reaction diagram
(S)-lactate + K3[Fe(CN)6]
?
show the reaction diagram
(S)-lactate + potassium ferricyanide
pyruvate + potassium ferrocyanide
show the reaction diagram
-
-
-
-
?
(S)-mandelate + ferricytochrome c
hydroxy(phenyl)acetate + ferrocytochrome c
show the reaction diagram
(S)-phenyllactate + ferricytochrome c
phenylpyruvate + ferrocytochrome c
show the reaction diagram
-
-
-
-
?
glycolate + ferricytochrome c
glyoxylate + ferrocytochrome c
show the reaction diagram
-
very poor substrate
-
-
?
L-lactate + ferricyanide
pyruvate + ferrocyanide + H+
show the reaction diagram
L-lactate + ferricytochrome c
pyruvate + ferrocytochrome c + H+
show the reaction diagram
L-lactate + O2
? + superoxide anion
show the reaction diagram
-
the FDH domain reacts slowly with oxygen with formation of superoxide anion when separated from its natural electron acceptor, whether isolated or included in the holoenzyme
-
-
?
L-lactate + potassium ferricyanide
pyruvate + potassium ferrocyanide + H+
show the reaction diagram
phenyllactate + ferricytochrome c
phenylpyruvate + ferrocytochrome c
show the reaction diagram
-
-
-
-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(S)-lactate + 2 ferricytochrome c
pyruvate + 2 ferrocytochrome c + 2 H+
show the reaction diagram
L-lactate + ferricytochrome c
pyruvate + ferrocytochrome c + H+
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,6-dichlorophenolindophenol
-
-
cytochrome
-
each subunit of the tetramer is composed of two domains, one binding a heme and the other an FMN prosthetic group. The cytochrome domain consists of residues 1 to 99
-
cytochrome c
FAD
-
the flavoenzyme possesses flavocytochrome b2 subunits, that each consists of an N-terminal cytochrome domain and a C-terminal flavodehydrogenase, FDH, domain
ferricytochrome c
flavocytochrome c
-
-
-
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
D-lactate
ethane nitronate
-
competitive
ferricyanide
-
-
Ferrocytochrome
-
-
Fluoropyruvate
-
-
L-lactate
L-Mandelate
-
competitive
malate
oxalate
p-mercuribenzoate
-
-
Propionate
-
competitive
pyruvate
sulfite
-
competitive
zinc-substituted cytochrome c
-
-
-
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.16 - 5
(S)-Lactate
0.02 - 131
cytochrome c
0.03 - 6
ferricyanide
0.0015 - 0.131
ferricytochrome c
0.0001 - 38
L-lactate
0.22 - 1.43
L-Phenyllactate
0.084 - 0.4
Phenyllactate
6
potassium ferricyanide
-
pH 9.5, temperature not specified in the publication
7.4 - 20
pyruvate
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
13.2 - 372
(S)-Lactate
6 - 184
cytochrome c
185 - 225
FAD
0.011 - 207
ferricytochrome c
0.2 - 2.8
Fluoropyruvate
0.031 - 473
L-lactate
0.02 - 8.5
L-Mandelate
0.29 - 16.5
Phenyllactate
additional information
additional information
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
170
acetate
-
in pre-steady-state kinetic studies of the FDH domain reduction and reoxidation, acetate acts as a competitive inhibitor of L-lactate
450
chloride
-
in pre-steady-state kinetic studies of the FDH domain reduction and reoxidation, chloride acts as a competitive inhibitor of L-lactate
1.4 - 6
D-lactate
2.2
ethane nitronate
-
-
0.006 - 0.0088
Ferrocytochrome
45
L-lactate
-
-
0.0026
L-Mandelate
-
-
13 - 27
malate
0.4 - 1.3
oxalate
0.3
oxlate
-
-
-
28
Propionate
-
-
2 - 40
pyruvate
0.0014
sulfite
-
-
0.0073
zinc-substituted cytochrome c
-
pH 7.5, 25C, wild-type enzyme
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.055
-
(S)-lactate with cytochrome c as electron acceptor
0.4
-
+/- 0.02, was found in the cell-free extract
0.415
-
partially purified enzyme with 2,6-dichloroindophenol as electron acceptor
0.43
-
+/-0.05, cell-free extract
0.51
-
+/- 0.03, cell-free extract
0.66
-
+/- 0.06, the cell-free extract
0.72
-
6-dichloroindophenol as electron acceptor
3
-
cell-free extract from recombinant Hansenula polymorpha cell line expressing gene CYB2
3.2
-
recombinant enzyme in cells of Hansenula polymorpha
9
-
purified native enzyme, pH 7.5, 20C
895
-
purified enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.3 - 7.5
-
assay at
7 - 7.5
-
assay at
7.2 - 8.4
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-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.2
-
almost no activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 24
-
assay at
20
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assay at
25 - 30
-
assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
optimization of culture conditions for recombinant cells of strain C-105 expressing gene CYB2 and showing increased FCb2 activity
Manually annotated by BRENDA team
-
two isozymes
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10500
-
cytochrome domain, SDS-PAGE
56200
-
calculated from amino acid sequence, cleavage product from 64259 precursor
220000
230000
-
cleaved enzyme
235000
-
X-ray diffraction studies
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 47000, recombinantly expressed flavocytochrome b2 flavin-binding domain, SDS-PAGE
homotetramer
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of flavocytochrome b2 solved at 3.0 A resolution by the method of multiple isomorphous replacement with anomalous scattering
-
crystal structure of mutant H723Q bound with pyruvate is determined at 2.8 A
-
crystallization of flavin binding domain and intact enzyme, hanging drop vapor diffusion method
-
crystallization of Y143F mutant, vapor diffusion technique in presence of PEG 4000
-
Fcb2 free and in complex with sulfite, X-ray diffraction structure analysis at 2.3-2.6 A resolution
-
hanging drop vapor diffusion method, using 20% polyethylene glycol 4000, 0.1 M MES, pH 6.5, and 0.2 M MgCl2, at 18C
-
mutant L230A in complex with phenylglyoxalate, mutant A198G/L230A in complex with sulfite, mutant A198G/L230A in complex with pyruvate
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used as essential purification step, crystallized as DNA complex
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 10
-
cytochrome domain stable
348067
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
thermostable enzyme
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
intact enzyme unstable at low ionic strength
-
PMSF stabilizes, purification in absence of PMSF yields cleaved form of enzyme
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-180C, long term storage
-
-70C in 0.1 M phosphate buffer, 1 mM EDTA, pH 7, 10 mM DL-lactate is added to the Fcb2 preparations to keep the enzyme in the reduced state
-
-70C, in 0.1 M phosphate buffer, 1 mM EDTA, pH 7, 10 mM DL-lactate is added to the Fcb2 preparations to keep the enzyme in the reduced state
-
-70C, purified enzymes are stored in 100 mM potassium phosphate, 1 mM EDTA, and 20 mM D,L-lactate at pH 7.5
-
-80C as saturated solution
-
-80C, 0.1 M phosphate buffer, 1 mM EDTA, 1 mM PMSF
-
20C, 0.2 M phosphate buffer, pH 7.2, 1 mM EDTA, protected from light, 50% loss of activity within 10-20 days
-
4C, 70% saturated ammonium sulfate, under nitrogen, 2 months, 20% loss of activity of purified enzyme
-
4C, precipitate from 70% saturated ammonium sulfate, under nitrogen, stable for several weeks
-
4C, saturated ammonium sulfate, under nitrogen, protected from light, stabe
-
under nitrogen, 1 month, no loss of activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cytochrome domain
-
flavin and heme domain from cloned Escherichia coli
-
native enzyme to homogeneity
-
partial purification of cytochrome b2 core
-
purification in presence and absence of PMSF results intact or cleaved enzyme
-
purification of different enzyme fragments
-
recombinantly expressed flavocytochrome b2 flavin-binding domain
-
wild type and mutant enzymes
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cytochrome domain in Escherichia coli
-
expressed in Saccharomyces cerevisiae YN1 strain (flavocytochrome b2 deletion mutant of strain XS560-1)
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expression of flavocytochrome b2 flavin-binding domain in Escherichia coli
-
flavin and heme domain expressed in Escherichia coli
-
gene CYB2, expression in the recipient strain Hansenula polymorpha C-105 impaired in glucose repression and devoid of catalase activity, under the control of the strong Hansenula polymorpha alcohol oxidase promoter in a plasmid for multicopy integration, optimization of culture conditions for recombinant cells with increased FCb2 activity
-
in Escherichia coli
-
in Escherichia coli AR120
-
in Escherichia coli JM 101
in Escherichia coli MM294
-
in Escherichia coli TG1
-
overexpression in Hansenula polymorpha leading to enhanced L-lactate-dependent respiration compared to the wild-type cells
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recombinant wild-type Fcb2 and its recombinant FDH domain (FMN-binding domain) are expressed in Escherichia coli
-
recombinant wild-type Fcb2 and its recombinant flavin domain are expressed in Escherichia coli
-
recombinant wild-type Fcb2 and its recombinant FMN-binding domain (FDH domain) are expressed in Escherichia coli
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
flavocytochrome b2 production is induced by the presence of oxygen and L-lactate
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
-
a L-lactate-selective microbial biosensor is developed using permeabilized cells of gene-engineered thermotolerant methylotrophic yeast Hansenula polymorpha, over-producing FCb2. The HpCYB2 gene, encoding FCb2, under the control of the strong Hansenula polymorpha alcohol oxidase promoter in the frame of a plasmid for multicopy integration is transformed to the recipient strain Hansenula polymorpha C-105 (gcr1 catX) impaired in glucose repression and devoid of catalase activity. The biosensor based on recombinant yeast cells exhibit a higher Km value (Km: 3.02 mM) and hence expanded linear range toward l-lactate as compared to a similar sensor based on the initial cells of Hansenula polymorpha C-105 (Km: 0.33 mM)
A198G
-
turnover reduced to 50%
A198G/L230A
E91K
-
mutation has no effect on the rate of cytochrome c reduction, no significantly different behavior with regard to inhibition by ferrocytochrome c
F52A
-
mutation has no effect on the rate of cytochrome c reduction
L230A/A198G
-
the double mutant enzyme has a 6fold greater catalytic efficiency with L-mandelate than with L-lactate
R289K
-
kcat (1/sec) (substrate: L-lactate): 8.6 (in 200 mM phosphate: 9.2, in 400 mM potassium acetate: 8.8, in 400 mM KCl: 9.2, in 400 mM KBr: 7.8), Km (mM) (substrate: L-lactate): 7.0 (in 200 mM phosphate: 8.7, in 400 mM potassium acetate: 9.2, in 400 mM KCl: 6.5, in 400 mM KBr: 5.8). Mutant is not sensitive for excess lactate concentration. In contrast to the wild-type enzyme high concentrations of acetate, phosphate, chloride and bromide show no influence on the mutant enzyme
R376K
-
mutant enzyme shows no activity
R38E
-
activity and inhibitory profile similar to wild type
Y254del
-
deletion mutant
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
environmental protection
synthesis
-
the enzyme is potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods
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