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Information on EC 1.1.1.27 - L-lactate dehydrogenase and Organism(s) Geobacillus stearothermophilus and UniProt Accession P00344
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1.1.1.27 L-lactate dehydrogenaseIUBMB Comments
Also oxidizes other (S)-2-hydroxymonocarboxylic acids. NADP+ also acts, more slowly, with the animal, but not the bacterial, enzyme.
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The taxonomic range for the selected organisms is: Geobacillus stearothermophilus
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
lactate dehydrogenase, lactic dehydrogenase, ldh-a, lactate dehydrogenase a, l-lactate dehydrogenase, ldh-5, lactic acid dehydrogenase, ldh-1, pfldh, alpha-hydroxybutyrate dehydrogenase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
dehydrogenase, lactate
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Epsilon crystallin
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epsilon-crystallin
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-
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Immunogenic protein p36
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L(+)-nLDH
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L-(+)-lactate dehydrogenase
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L-lactic acid dehydrogenase
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L-lactic dehydrogenase
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L-LDH
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-
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lactate dehydrogenase
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lactate dehydrogenase NAD-dependent
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lactic acid dehydrogenase
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lactic dehydrogenase
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LDH
NAD-lactate dehydrogenase
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proteins, specific or class, anoxic stress response, p34
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LDH
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(S)-lactate + NAD+ = pyruvate + NADH + H+
the NAD (H)-dependent L-LDH catalyzes the reduction of pyruvate by an ordered catalytic mechanism, catalytic cycle, overview
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
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oxidation
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reduction
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PATHWAY SOURCE
PATHWAYS
KEGG
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-, -, -, -, -, -
SYSTEMATIC NAME
IUBMB Comments
(S)-lactate:NAD+ oxidoreductase
Also oxidizes other (S)-2-hydroxymonocarboxylic acids. NADP+ also acts, more slowly, with the animal, but not the bacterial, enzyme.
CAS REGISTRY NUMBER
COMMENTARY
9001-60-9
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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
(S)-lactate + NADP+
pyruvate + NADPH + H+
wild-type enzyme is specific for NAD+. Mutant enzyme F16Q/I37K/D38SC81S/N85R utilizes NADP+ better than wild-type enzyme, prefers NADP+ to NAD+. Mutant F16Q/C81S/N85R utilizes NAD+ better than wild-type enzyme, weakly active with NADP+
-
-
?
(S)-lactate + NAD+
pyruvate + NADH + H+
wild-type enzyme is specific for NAD+. Mutant enzyme F16Q/I37K/D38SC81S/N85R utilizes NADP+ better than wild-type enzyme, prefers NADP+ to NAD+. Mutant F16Q/C81S/N85R utilizes NAD+ better than wild-type enzyme, weakly active with NADP+
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-
?
(S)-lactate + NAD+
pyruvate + NADH + H+
residues I12, R81, M85, G210, and V214 determine the enzyme's substrate specificity
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-
r
pyruvate + NADH + H+
(S)-lactate + NAD+
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study of the dependence of the chemical reaction mechanism of lactate dehydrogenase on the protonation state of titratable residues and on the level of the quantum mechanical description by means of hybrid quantum-mechanical methods
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-
?
additional information
?
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substrate analogue oxamate is isoelectric and isosteric to pyruvate and has binding kinetics very similar to that of pyruvate. As the substrate approaches the catalytic site, a catalytically key surface loop (residues 98-110) closes over the ligand, bringing residue Arg109 into hydrogen bonding contact with ligand, water leaves the pocket, and the pocket geometry rearranges to allow for favorable interactions between the cofactor and the ligand, which facilitates on-enzyme catalysis
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-
?
additional information
?
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substrate analogue oxamate is isoelectric and isosteric to pyruvate and has binding kinetics very similar to that of pyruvate. As the substrate approaches the catalytic site, a catalytically key surface loop (residues 98-110) closes over the ligand, bringing residue Arg109 into hydrogen bonding contact with ligand, water leaves the pocket, and the pocket geometry rearranges to allow for favorable interactions between the cofactor and the ligand, which facilitates on-enzyme catalysis
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-
?
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
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NADP+
wild-type enzyme is specific for NAD+. Mutant enzyme F16Q/I37K/D38SC81S/N85R utilizes NADP+ better than wild-type enzyme, prefers NADP+ to NAD+. Mutant F16Q/C81S/N85R utilizes NAD+ better than wild-type enzyme, weakly active wth NADP+
NAD+
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the coenzyme is in an open conformation and the adenine ribose ring of it is surrounded by Asp 38, Val 39, and Gly 99. Asp 38 and Gly 99 give some specificity to the adenine orientation, overview
NAD+
wild-type enzyme is specific for NAD+. Mutant enzyme F16Q/I37K/D38SC81S/N85R utilizes NADP+ better than wild-type enzyme, prefers NADP+ to NAD+. Mutant F16Q/C81S/N85R utilizes NAD+ better than wild-type enzyme, weakly active wth NADP+
NADH
kinetic modelling of NADH binding to the enzyme, overview
NADH
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the coenzyme is in an open conformation and the adenine ribose ring of it is surrounded by Asp 38, Val 39, and Gly 99. Asp 38 and Gly 99 give some specificity to the adenine orientation, Asp 38 is an important residue in stabilizing NADH binding, overview
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
D-fructose 1,6-bisphosphate
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slightly inhibits activity of hybrid enzyme constructed from fragments of the LDH genes from Bacillus stearothermophilus (coding for aa 15-100) and Bacillus megaterium (coding for aa 101-331)
NAD+
-
substrate inhibition due to an abortive NAD+-pyruvate complex reducing the steady state concentration of functional LDH
pyruvate
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substrate inhibition due to an abortive NAD+-pyruvate complex reducing the steady state concentration of functional LDH
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
D-fructose 1,6-diphosphate
activates wild-type enzyme and mutant enzymes F16Q/I37K/D38SC81S/N85R and F16Q/C81S/N85R
fructose 1,6-bisphosphate
assures that the protein forms tetrameric uniformity and serves as an allosteric activator of the enzyme
D-fructose 1,6-bisphosphate
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activates the pyruvate reduction
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
4.7
NADP+
25°C, pH 8, mutant enzyme F16Q/I37K/D38SC81S/N85R, activated by fructose 1,6-diphosphate
0.06
pyruvate
pH 6.0, 25°C, recombinant wild-type enzyme
0.0081
NAD+
25°C, pH 8, mutant enzyme F16Q/C81S/N85R, activated by fructose 1,6-diphosphate
0.105
NAD+
25°C, pH 8, wild-type enzyme, activated by fructose 1,6-diphosphate
0.05
NADH
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measured without D-fructose 1,6-bisphosphate, wild-type enzyme
0.08
NADH
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measured with D-fructose 1,6-bisphosphate, wild-type enzyme
0.19
NADH
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measured without D-fructose 1,6-bisphosphate, hybrid enzyme constructed from fragments of the LDH genes from Bacillus stearothermophilus (coding for aa 15-100) and Bacillus megaterium (coding for aa 101-331)
0.2
pyruvate
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measured with D-fructose 1,6-bisphosphate, wild-type enzyme
2.76
pyruvate
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pH 6.0, 25°C, recombinant wild-type enzyme in presence of fructose 1,6-bisphosphate
3.7
pyruvate
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pH 6.0, 25°C, recombinant mutant D38R in presence of fructose 1,6-bisphosphate
6.8
pyruvate
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measured with D-fructose 1,6-bisphosphate, hybrid enzyme constructed from fragments of the LDH genes from Bacillus stearothermophilus (coding for aa 15-100) and Bacillus megaterium (coding for aa 101-331)
7
pyruvate
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measured without D-fructose 1,6-bisphosphate, hybrid enzyme constructed from fragments of the LDH genes from Bacillus stearothermophilus (coding for aa 15-100) and Bacillus megaterium (coding for aa 101-331)
13
pyruvate
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measured without D-fructose 1,6-bisphosphate, wild-type enzyme
19.3
pyruvate
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pH 6.0, 25°C, recombinant mutant D38R in absence of fructose 1,6-bisphosphate
32
pyruvate
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pH 6.0, 25°C, recombinant wild-type enzyme in absence of fructose 1,6-bisphosphate
additional information
additional information
Michaelis-Menten kinetic modelling, detailed overview. NADH can bind only to the open-loop apoenzyme, substrate analogue oxamate can bind only to the bsLDH·NADH binary complex in the open-loop conformation, and oxamate binding is followed by closing of the active site loop preventing oxamate unbinding. The open and closed states of the loop are in dynamic equilibrium and interconvert on the submillisecond time scale. This interconversion strongly accelerates with an increase in temperature because of significant enthalpy barriers. Binding of NADH to bsLDH results in minor changes of the loop dynamics and does not shift the open-closed equilibrium, but binding of the oxamate substrate mimic shifts this equilibrium to the closed state. At high excess oxamate concentrations where all active sites are nearly saturated with the substrate mimic, all active site mobile loops are mainly closed, kinetic analysis, overview
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additional information
additional information
-
Michaelis-Menten kinetic modelling, detailed overview. NADH can bind only to the open-loop apoenzyme, substrate analogue oxamate can bind only to the bsLDH·NADH binary complex in the open-loop conformation, and oxamate binding is followed by closing of the active site loop preventing oxamate unbinding. The open and closed states of the loop are in dynamic equilibrium and interconvert on the submillisecond time scale. This interconversion strongly accelerates with an increase in temperature because of significant enthalpy barriers. Binding of NADH to bsLDH results in minor changes of the loop dynamics and does not shift the open-closed equilibrium, but binding of the oxamate substrate mimic shifts this equilibrium to the closed state. At high excess oxamate concentrations where all active sites are nearly saturated with the substrate mimic, all active site mobile loops are mainly closed, kinetic analysis, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
250
pyruvate
pH 6.0, 25°C, recombinant wild-type enzyme
23
NAD+
25°C, pH 8, mutant enzyme F16Q/C81S/N85R, activated by fructose 1,6-diphosphate
23
NAD+
25°C, pH 8, mutant enzyme F16Q/I37K/D38SC81S/N85R, activated by fructose 1,6-diphosphate
32
NAD+
25°C, pH 8, wild-type enzyme, activated by fructose 1,6-diphosphate
21.1
pyruvate
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pH 6.0, 25°C, recombinant wild-type enzyme in presence of fructose 1,6-bisphosphate
178.4
pyruvate
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pH 6.0, 25°C, recombinant mutant D38R in presence of fructose 1,6-bisphosphate
194.9
pyruvate
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pH 6.0, 25°C, recombinant mutant D38R in absence of fructose 1,6-bisphosphate
327.2
pyruvate
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pH 6.0, 25°C, recombinant wild-type enzyme in absence of fructose 1,6-bisphosphate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
10.9
pyruvate
-
pH 6.0, 25°C, recombinant wild-type enzyme in presence of fructose 1,6-bisphosphate
31.1
pyruvate
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pH 6.0, 25°C, recombinant mutant D38R in presence of fructose 1,6-bisphosphate
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structure of a mutant into which an additional loop has been engineered in order to prevent tetramerization
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
F16Q/C81S/N85R
catalytic efficiency is higher than that of wild-type enzyme, utilizes NAD+ better than wild-type enzyme, weakly active wth NADP+
F16Q/I37K/D38S/C81S/N85R
utilizes NADP+ better than wild-type enzyme, prefers NADP+ to NAD+
D38E
-
site-directed mutagenesis, the mutant shows a twofold reduced substrate inhibition by pyruvate compared to the wild-type enzyme
D38R
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site-directed mutagenesis, the mutant shows a threefold reduced substrate inhibition by pyruvate compared to the wild-type enzyme
S100M
-
a hybrid gene is constructed from fragments of the LDH genes from Bacillus stearothermophilus (coding for aa 15-100) and Bacillus megaterium (coding for aa 101-331). The hybrid LDH, named S100M, is more thermostable than Bacillus megaterium LDH, less thermostable than Bacillus stearothermophilus LDH and unlike the two wild-type enzymes, it can not be activated by D-fructose 1,6-bisphosphate
additional information
additional information
the malate dehydrogenase, EC 1.1.1.37, mutant I12V/R81Q/M85E/G210A/V214I shows a substrate specificity that is switched from malate dehydrogenase to that of lactate dehydrogenase, overview
additional information
-
a mutant into which an additional loop has been engineered in order to prevent tetramerization
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
58
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30 min, stable up to A hybrid gene is constructed from fragments of the LDH genes from Bacillus stearothermophilus (coding for aa 15-100) and Bacillus megaterium (coding for aa 101-331). The hybrid LDH is named S100M
additional information
-
a hybrid gene is constructed from fragments of the LDH genes from Bacillus stearothermophilus (coding for aa 15-100) and Bacillus megaterium (coding for aa 101-331). The hybrid LDH, named S100M, is more thermostable than Bacillus megaterium LDH, less thermostable than Bacillus stearothermophilus LDH and unlike the two wild-type enzymes, it can not be activated by D-fructose 1,6-bisphosphate
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant wild-type and mutant enzymes from Escherichia coli strain JM109 by ion-exchange chromatography and affinity chromatography, respectively
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
a chimeric bifunctional enzyme composing of galactose dehydrogenase from Pseudomonas fluorescens and lactate dehydrogenase from Bacillus stearothermophilus is successfully constructed and expressed in Escherichia coli
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expression in Escherichia coli HB101 using a pEMBL vector. The gene is strongly expressed in the vector used if the orientation of the insert allows the LDH promoter and the vector's lac promoter to direct transcription in the same direction
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expression of wild-type and mutant enzymes in Escherichia coli strain JM109
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
biotechnology
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a chimeric bifunctional enzyme composing of galactose dehydrogenase from Pseudomonas fluorescens and lactate dehydrogenase from Bacillus stearothermophilus is successfully constructed. The chimeric enzyme is able to recycle NAD with a continuous production of lactate without any externally added NADH
synthesis
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the enzyme has a commercial significance, as it can be used to produce chiral building blocks for the synthesis of key pharmaceuticals and agrochemicals, optimization of enzyme reaction by engineering to eliminate the substrate inhibition
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Cameron, A.D.; Roper, D.I.; Moreton, K.M.; Muirhead, H.; Holbrook, J.J.; Wigley, D.B.
Allosteric activation in Bacillus stearothermophilus lactate dehydrogenase investigated by an X-ray crystallographic analysis of a mutant designed to prevent tetramerization of the enzyme
J. Mol. Biol.
238
615-625
1994
Geobacillus stearothermophilus
Ferrer, S.; Silla, E.; Tunon, I.; Oliva, M.; Moliner, V.; Williams, I.H.
Dependence of enzyme reaction mechanism on protonation state of titratable residues and QM level description: lactate dehydrogenase
Chem. Commun. (Camb.)
2005
5873-5875
2005
Geobacillus stearothermophilus
Prachayasittikul, V.; Ljung, S.; Isarankura-Na-Ayudhya, C.; Buelow, L.
NAD(H) recycling activity of an engineered bifunctional enzyme galactose dehydrogenase/lactate dehydrogenase
Int. J. Biol. Sci.
2
10-16
2006
Geobacillus stearothermophilus
Flores, H.; Ellington, A.D.
A modified consensus approach to mutagenesis inverts the cofactor specificity of Bacillus stearothermophilus lactate dehydrogenase
Protein Eng. Des. Sel.
18
369-377
2005
Geobacillus stearothermophilus (P00344), Geobacillus stearothermophilus
Waldvogel, S.; Weber, H.; Zuber, H.
Structure and function of L-lactate dehydrogenase from thermophilic and mesophilic bacteria VII
Biol. Chem. Hoppe-Seyler
368
1391-1399
1987
Geobacillus stearothermophilus, Priestia megaterium
Binay, B.; Karagueler, N.G.
Attempting to remove the substrate inhibition of L-lactate dehydrogenase from Bacillus stearothermophilus by site-directed mutagenesis
Appl. Biochem. Biotechnol.
141
265-272
2007
Geobacillus stearothermophilus
Yin, Y.; Kirsch, J.F.
Identification of functional paralog shift mutations: conversion of Escherichia coli malate dehydrogenase to a lactate dehydrogenase
Proc. Natl. Acad. Sci. USA
104
17353-17357
2007
Escherichia coli, Geobacillus stearothermophilus (P00344)
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