Information on EC 3.6.3.33 - vitamin B12-transporting ATPase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.6.3.33
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RECOMMENDED NAME
GeneOntology No.
vitamin B12-transporting ATPase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + H2O + vitamin B12/out = ADP + phosphate + vitamin B12/in
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric ester
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transmembrane transport
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SYSTEMATIC NAME
IUBMB Comments
ATP phosphohydrolase (vitamin B12-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 cobalamin derivatives.
CAS REGISTRY NUMBER
COMMENTARY hide
9000-83-3
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
encoded by btuD gene; K12
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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gene disruption eliminates the ability of Mycobacterium tuberculosis to use exogenous vitamin B12 in vitro
physiological function
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the enzyme is required for the assimilation of exogenous vitamin B12 to enable methylmalonyl-CoA pathway function and is essential for corrinoid transport
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + H2O + vitamin B12/out
ADP + phosphate + vitamin B12/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 + vitamin B12/out
ADP + phosphate + vitamin B12/in
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
periplasmic binding protein BtuF
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4fold decrease of activity in n-alkyl-phosphocholine
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sodium ortho-vanadate
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sodium orthovanadate
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2 mM, the ATPase activity of BtuCD complex and of subunit BtuD is reduced by 96%
additional information
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not inhibitory: N-ethylmaleimide
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
periplasmic binding protein BtuF
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0.02 mmol, 16% increase in basal activity in lauryl dimethylamine-N-oxide, 50% increase in activity in the presence of vitamin B12 in TX-100
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.053 - 0.077
ATP
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.3 - 1.39
ATP
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.18
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in the absence of Btu-F and vitamin B12 in proteoliposomes
0.33
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in the presence of Fos-choline
0.44
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in the presence of 0.02 mmol BtuF in proteoliposomes
0.98
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in the presence of lauryl dimethylamine-N-oxide
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20000
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btuE, nucleotide sequence
20470
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btuE, nucleotide sequence
26000
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btuC , nucleotide sequence
27090
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btuD, nucleotide sequence
29000
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btuD, nucleotide sequence
31000
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x * 31000, subunit BtuD, SDS-PAGE
31680
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btuC, nucleotide sequence
66000
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btuB
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 31000, subunit BtuD, SDS-PAGE
dimer
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BtuCD structure analysis, overview
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
BtuCD-F complex analyzed at a resolution of 2.6 A, substantial conformational changes observed as compared with previously reported structures of BtuCD and BtuF
catalytically impaired BtuCD mutant E159Q in complex with BtuF, to 3.5 A resolution. The BtuC subunits adopts a distinct asymmetric conformation. The structure suggests that BtuF does not discriminate between, or impose, asymmetric conformations of BtuCD
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crystal structure analysis of the crystal structure of the BtuCD-F complex, modelling, overview
elucidation of gating mechanism by EPR spectroscopy. The translocation gates of the BtuCDF complex undergo conformational changes in line with a two-state alternating access model. Binding of ATP drives the gates to an inward-facing conformation. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B12 promotes the reopening of the gates toward the periplasm and the dislodgement of BtuF from the transporter
in complex with beta-gamma-imidoadenosine 5'-triphosphate, sitting drop vapor diffusion method, using 20-30% (w/v) PEG 400, 100 mM N-(2-acetamido)-iminodiacetic acid, pH 6.8, 100 mM sodium potassium citrate
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molecular dynamics simulations to explore the atomic details of the conformational transitions of BtuCD importer. The outward-facing to inward-facing transition is initiated by the conformational movement of nucleotide-binding domains. The subsequent reorientation of the substrate translocation pathway at transmembrane domains begins with the closing of the periplasmic gate, followed by the opening of the cytoplasmic gate in the last stage of the conformational transition due to the extensive hydrophobic interactions at this region, consistent with the functional requirement of unidirectional transport of the substrates. The reverse inward-facing to outward-facing transition exhibits intrinsic diversity of the conformational transition pathways and significant structural asymmetry
mutant E159Q/N162C in complex with adenylyl imidodiphosphate, sitting drop vapor diffusion method, using 100 mM ADA buffer, pH 6.9, 1.2 M NaCl, and 14-18% (w/v) PEG 2000 MME
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BtuCD-F complex, and HI470/1 X-ray diffraction structure analysis using PDB-IDs 2NQ2, 1L7V and 2QI9, overview
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
BtuCD-F complex is stable in lauryl dimethylamine-N-oxide, dodecyl maltoside and Triton X-100 when storing the protein for several days at either 4C or room temperature
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA column chromatography
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NiNTA affinity chromatography, in the absence of vitamin B12
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purified in 0.1% n-dodecyl-N,N-dimethylamine-N-oxide
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recombinant His-tagged wild-type and mutant enzymes by nickel affinity chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
bacteriophage p1; cloning of the btuCD region
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btuB gene; expressed in Escherichia coli
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expressed as a C-terminal Flag-tagged fusion protein
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expressed in Escherichia coli BL21(DE3)Gold cells
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expressed in Escherichia coli strain BL21 (DE3)
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expression in Escherichia coli
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genes btuC and btuD form a cluster, encoding permease protein btuC and ATP-binding protein btuD, forming the ABC transporter BtuCD, expression of wild-type and utant enzymes as His-tagged proteins
phage M13mp8 or M13mp9
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E159Q
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mutation in subunit BtuD, results in abolished ATP hydrolysis activity of BtuCDF. Mutant is still able to bind nucleotides and binding protein BtuF in a manner similar to the wild-type protein
E159Q/N162C
L85C
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the mutant shows a 6.5fold reduction in ATPase activity compared to the wild type enzyme
S141C
site-directed mutagenesis, structure analysis
S143C
site-directed mutagenesis, structure analysis
T168C
site-directed mutagenesis, structure analysis
additional information
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a system consisting of the BtuC subunit embedded in a palmitoyloleoyl phosphatidylcholine lipid bilayer is constructed and a more-than-57ns MD simulation is performed to study the functional motions of BtuC at the atomic level: results show that a stable protein-lipid bilayer is obtained and the palmitoyloleoyl phosphatidylcholine lipid bilayer is able to adjust its thickness to match the embedded BtuC which undergo relatively complicated motions
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
BtuCD transporter can be re-assembled from both prefolded and partly unfolded, urea denatured BtuC and BtuD subunits. The in vitro re-assembly leads to a BtuCD complex with the correct, native, BtuC and BtuD subunit stoichiometry. The highest rates of ATP hydrolysis are achieved for BtuCD re-assembled from partly unfolded subunits. This supports the idea of cooperative folding and assembly of the constituent protein subunits of the BtuCD transporter
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