Information on EC 3.6.3.32 - quaternary-amine-transporting ATPase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.6.3.32
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RECOMMENDED NAME
GeneOntology No.
quaternary-amine-transporting ATPase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + H2O + quaternary amine/out = ADP + phosphate + quaternary amine/in
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric ester
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-
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SYSTEMATIC NAME
IUBMB Comments
ATP phosphohydrolase (quaternary-amine-importing)
ABC-type (ATP-binding cassette-type) ATPase, characterized by the presence of two similar ATP-binding domains. Does not undergo phosphorylation during the transport process. A bacterial enzyme that imports betaine and glycine.
CAS REGISTRY NUMBER
COMMENTARY hide
9000-83-3
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
OpuA
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Manually annotated by BRENDA team
strain NZ9000
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Manually annotated by BRENDA team
strain 10403S
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Manually annotated by BRENDA team
ATP-binding protein, gene PSPTO4575, OpuCA; pv. tomato, strain DC3000
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
ADP + H2O
AMP + phosphate
show the reaction diagram
-
OpuAA protein, ATPase activity
-
-
?
AMP + H2O
adenine + phosphate
show the reaction diagram
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OpuAA protein, ATPase activity, similar substrate affinities to monomeric and dimeric states
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-
?
ATP + H2O
ADP + phosphate
show the reaction diagram
ATP + H2O + betaine/out
ADP + phosphate + betaine/in
show the reaction diagram
ATP + H2O + betonicine/out
ADP + phosphate + betonicine/in
show the reaction diagram
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-
-
-
?
ATP + H2O + dimethylsulfonioacetate/out
ADP + phosphate + dimethylsulfonioacetate/in
show the reaction diagram
ATP + H2O + glycine betaine/out
ADP + phosphate + glycine betaine/in
show the reaction diagram
ATP + H2O + L-proline betaine/out
ADP + phosphate + L-proline betaine/in
show the reaction diagram
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-
-
-
?
ATP + H2O + proline betaine/out
ADP + phosphate + proline betaine/in
show the reaction diagram
ATP + H2O + quaternary amine/out
ADP + phosphate + quaternary amine/in
show the reaction diagram
additional information
?
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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
ATP + H2O
ADP + phosphate
show the reaction diagram
ATP + H2O + dimethylsulfonioacetate/out
ADP + phosphate + dimethylsulfonioacetate/in
show the reaction diagram
P46922
OpuA is a classic ABC importer that relies on a substrate binding protein priming the transporter with specificity and selectivity
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-
?
ATP + H2O + glycine betaine/out
ADP + phosphate + glycine betaine/in
show the reaction diagram
ATP + H2O + quaternary amine/out
ADP + phosphate + quaternary amine/in
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ba2+
bivalent cations more effective activators than monovalent cations
K2SO4
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100 mosmol/kg osmolality, stimulates OpuA activity
K3PO4
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100 mosmol/kg osmolality, pH 7.0, stimulates OpuA activity. 200 mM, stimulates ATPase activity
KCl
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100 mM, activates OpuA instantaneous and reversible
Na2SO4
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100 mosmol/kg osmolality, stimulates OpuA activity
NaCl
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100 mosmol/kg osmolality, stimulates OpuA activity
NH4+
less effective than KCl, in the presence of the protonophore SF6847 much more effective
Sodium phosphate
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100 mosmol/kg osmolality, pH 7.0, stimulates OpuA activity
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ADP
-
inhibits ATPase activity
Betaine aldehyde
weakly inhibits betaine uptake
BusR
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acts as a repressor of busA transcription
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choline
slightly inhibits betaine uptake
Cs+
potent inhibitor
K3PO4
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above 200 mM, inhibits ATPase activity
NaCl
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0.5 M, inhibits
proline betaine
competitive
Rb+
potent inhibitor
Tetracaine
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low concentrations of the cationic amphipath decrease the activation of OpuA
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
anionic lipids
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absence of phosphatidylglycerol and/or phosphatidylserine results in trapping of the protein in an inactive state
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dioleoyl-phosphatidylglycerol
needed for full osmoregulatory activity
glycerol
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at 270 mM, the amount of glycine betaine taken up after 30 seconds is 30% higher than that of the iso-osmotic control sample
negatively charged lipid
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phosphate ion-containing buffer
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phosphatidylethanolamine
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non-bilayer lipid essential for high activity of OpuA
Tetracaine
instantaneously activates inside-out oriented OpuA, reconstitued in membranes with 40% of anionic lipid, 1mM equally effective than 0.2 mM KCl
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.25 - 5.4
ATP
0.0017 - 0.272
betaine
0.0012 - 0.0157
glycine betaine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.004 - 0.25
ATP
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
12
ADP
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30ºC
0.065
carnitine
pH 7.0, 25°C
0.198
proline
pH 7.0, 25°C
0.048
proline betaine
pH 7.0, 25°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.005
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ATPase activity, pH 7.0, 200 mM K3PO4, 360 mosmol/kg
0.009
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ATPase activity, pH 7.0, 50 mM K3PO4, 290 mM sucrose, 450 mosmol/kg
0.018
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ATPase activity, pH 7.0, 50 mM K3PO4, 90 mosmol/kg
0.023
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ATPase activity, pH 7.0, 100 mM K3PO4, 180 mosmol/kg
0.095
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ATPase activity, pH 7.0, 50 mM K3PO4, 200 mM NaCl, 450 mosmol/kg
0.099
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ATPase activity, pH 7.0, 50 mM K3PO4, 200 mM KCl, 450 mosmol/kg
0.117
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ATPase activity, pH 7.0, 150 mM K3PO4, 270 mosmol/kg
0.124
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ATPase activity, pH 7.0, 350 mM K3PO4, 630 mosmol/kg
0.146
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ATPase activity, pH 7.0, 250 mM K3PO4, 450 mosmol/kg
0.148
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ATPase activity, pH 7.0, 50 mM K3PO4, 160 mM K2SO4, 450 mosmol/kg
0.18
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ATPase activity, pH 7.0, 200 mM K3PO4, 360 mosmol/kg
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9
for betaine uptake in Aphanothece halophytica cells
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
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30°C, 300 mM KCl
30
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30°C, 600 mM sucrose
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 35
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10°C: about 45% of maximal activity, 35°C: about 55% of maximal activity, at 30 mM KCl
15 - 40
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about 45% of maximal activity at 15°C and at 40°C, at 600 mM sucrose
20 - 24
assay at
37 - 52
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reduced growth rate at higher temperatures, addition of 1 mM glycine betaine, homobetaine, carnitine, butyrobetaine, crotonobetaine, DMSP, choline, or glutamate have a clear thermoprotective effect at 52°C, heat protectants are taken up under heat stress via the OpuA, OpuC, and OpuD transporters, despite the strongly reduced glycine betaine transport rate at 52°C, substantial glycine betaine accumulation, but it is not increased in comparison to cells grown at 37°C, thermoprotection by glutamate does not depend on an increased cellular pool of this amino acid
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
28000
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substrate-binding domain, 1 * 28000, SDS-PAGE
45679
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x * 45679, BusAA + x * 61966, BusAB + x * ?, BusAC, calculation from nucleotide sequence
47700
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OpuAA monomer, calculated from cDNA
56130
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x * 56130, calculation from nucleotide sequence
61966
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x * 45679, BusAA + x * 61966, BusAB + x * ?, BusAC, calculation from nucleotide sequence
64660
calculated from sequence analysis
65000
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OpuAA, gel filtration
95400
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OpuAA dimer, calculated from cDNA
additional information
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the OpuA component of the wild-type enzyme runs at a lower molecular weight than that of W484C and the substrate binding domain-less mutant
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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nucleotide-binding protein domain, displays lower ATPase activity than the dimeric form
monomer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
lipoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by hanging-drop method, structures of substrate-binding domain in complex with glycine betaine solved at 2.0 A resolution and in complex with glycine proline at 2.8 A resolution, structures show a substrate-binding protein-dependent-typical class II fold, structural differences of complexes occur within the ligand-binding pocket as well as across the domain-domain interface, explaining the differences in affinity of the substrate-binding domain-glycine betaine complex with KD = 0.017 mM, and substrate-binding domain-proline betaine complex with KD = 0.295 mM
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substrate-binding domain in complex with glycine betaine solved at 2.0 A resolution and in complex with glycine proline at 2.8 A resolution
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substrate-binding protein OpuBC in complex with choline, to 1.6 A resolution. The positively charged trimethylammonium head group of choline is wedged into an aromatic cage formed by four tyrosine residues and is bound via cation-pi interactions. The hydroxyl group of choline protrudes out of this aromatic cage and makes a single interaction with residue Gln19. A water network stabilizes choline within its substrate-binding site and promotes indirect interactions between the two lobes of the OpuBC protein. Disruption of this intricate water network strongly reduces choline binding affinity or abrogates ligand binding
substrate-binding protein OpuCC in the apo-form and in complex with carnitine, glycine betaine, choline and ectoine to 2.3, 2.7, 2.4, 1.9 and 2.1 A resolution, respectively. OpuCC is composed of two alpha/beta/alpha globular sandwich domains linked by two hinge regions, with a substrate-binding pocket located at the interdomain cleft. Upon substrate binding, the two domains shift towards each other to trap the substrate
crystallization in an open and closed-liganded conformation, to 1.9 and 2.3 A resolution, respectively. Solutes like proline and carnitine bind with affinities that are 3 to 4 orders of magnitude lower than affinities of substrates glycine betaine or proline betaine. The low affinity substrates are not noticeably transported by membrane-reconstituted OpuA. The binding pocket is formed by three tryptophans coordinating the quaternary ammonium group of glycine betaine in the closed-liganded structure
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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enzyme is activated by decreasing temperature within the range of 15°C to 4°C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
both monomeric and dimeric OpuA species are stabilized at 1 M NaCl
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OpuAA forms a stable dimer in the nucleotide-free state in solution
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity chromatography and size-exclusion chromatography of solubilized membrane fractions
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affinity chromatography of solubilized membranes
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by affinity tag as first step and gel filtration
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by Ni2+-nitrilotriacetic acid resin
gel filtration, recombinant protein
glycine betaine binding protein
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isolated transmembrane subunits purified by Strep-tag affinity purification
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nickel-Sepharose column chromatography
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OpuAA, purified to homogeneity, affinity chromatography and gel filtration
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substrate-binding domain, to apparent homogeneity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned into pApBetT, transfered to Escherichia coli and then to MKH13 cells, overexpression in freshwater Synechococcus sp. strain PCC7942 cells
cloning of the regulatory region of the glycine betaine transport system
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expressed in Escherichia coli BL21 cells
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expressed in Escherichia coli, cysteine mutants generated and inserted into pBAD33
expressed in Escherichia coli, DH5-alpha used as host for cloning, MG1655 used as host for mutagenesis
expression in Escherichia coli
expression in Lactococcus lactis
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expression of OpuAA protein in Escherichia coli
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into the pTOPO vector
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nucleotide-binding protein domain cloned as an N- or C-terminal His-tagged fusion protein and overexpressed in Escherichia coli BL21 (DE3) under the control of an arabinose-inducible promoter, overexpression of the transmembrane domain only possible in Walker strains BL21(DE3)C41 and C43 under the control of an ITPG- or arabinose-inducible promoter as N- and C-terminal His- and Strep-tagged versions
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substrate-binding domain into vector pBKB76, expression in Escherichia coli BL21 (DE3)
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transmembrane subunits cloned into vector pBAD33 or pET21a, expression in Escherichia coli
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
enzyme expression is induced in response to an osmotic upshock and sustained high salinity
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D149A
decrease in choline binding affinity by approximately 18fold
D149A/L155A
decrease in choline binding affinity by approximately 38fold
D74A
mutant is unable to bind choline
E171Q
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the monomer is the preferred species for the nucleotide-free state in solution
F19W
mutant analyzed
G161C
single cysteine mutants generated by site-directed mutagenesis
L155A
decrease in choline binding affinity by approximately 25fold
M21A
decrease in choline binding affinity by approximately 3fold
N115A
mutant is unable to bind choline
Q19A
decrease in choline binding affinity by approximately 15fold
Q19A/L155A
mutant is unable to bind choline
S171C
single cysteine mutants generated by site-directed mutagenesis
S45C
single cysteine mutants generated by site-directed mutagenesis
T94D
shares a quite similar pattern of fluorescence spectrum to that of the paralogue OpuBC. Only choline can trigger obvious changes of fluorescence intensity of mutant T94D, whereas carnitine, GB and ectoine cannot
Y91A
complete loss of binding affinity
Y91F
slight decrease in binding affinity
Y91W
slight decrease in binding affinity
D149A
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decrease in choline binding affinity by approximately 18fold
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D74A
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mutant is unable to bind choline
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L155A
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decrease in choline binding affinity by approximately 25fold
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N115A
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mutant is unable to bind choline
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Q19A
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decrease in choline binding affinity by approximately 15fold
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S24C
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mutations in the amphipathic alpha-helix fused to the core of the transmembrane domain of the OpuABC subunit. Mutation does not have distant structural effects on the overall conformation of the transporter
T23C
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mutations in the amphipathic alpha-helix fused to the core of the transmembrane domain of the OpuABC subunit. Mutation does not have distant structural effects on the overall conformation of the transporter
T25C
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mutations in the amphipathic alpha-helix fused to the core of the transmembrane domain of the OpuABC subunit. Mutation does not have distant structural effects on the overall conformation of the transporter
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
molecular biology
additional information
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