BRENDA - Reference to 7.6.2.8; Id = 752221

recombinant expression of N-terminally His10-tagged BtuCD in Escherichia coli, recombinant expression of wild-type and mutant His6-tagged BtuF variants in Escherichia coli

Escherichia coli

analysis of the cobinamide (Cbi)-bound BtuF crystal structure model, PDB ID 5M29, crystal structures of Cbi-bound BtuF mutants W66F, W66Y and W66L, sitting drop vapor diffusion technique, mixing of 20 mg/ml protein in 10 mM Tris pH 8 and 100 mM NaCl, with precipitant solution containing 1% w/v tryptone, 50 mM HEPES, pH 7.0, and 12% w/v PEG 3350, 1-2 weeks, 20°C, X-ray diffraction structure determination and analysis at 1.5-1.7 A resolution, molecular replacement using the BtuF structure (PDB ID 1N2Z) as search model

Escherichia coli

additional information

site-directed mutagenesis of tryptophan residue W66 in the substrate binding cleft , the affinity for cobinamide of the W66X mutants is lower except for W66F. Three mutants with impaired Cbi binding (W66A, W66R, and W66E) and one with high binding affinity (W66F) are used for transport assays. Despite having lower Cbi binding affinities, Cbi transport is hardly affected by W66X substitution

Escherichia coli

W66A

site-directed mutagenesis, reduces the affinity for cobinamide severalfold compared to wild-type

Escherichia coli

W66E

site-directed mutagenesis, reduces the affinity for cobinamide severalfold compared to wild-type

Escherichia coli

W66F

site-directed mutagenesis, does not reduce the affinity for cobinamide severalfold compared to wild-type

Escherichia coli

W66H

site-directed mutagenesis, reduces the affinity for cobinamide 10fold compared to wild-type

Escherichia coli

W66L

site-directed mutagenesis, reduces the affinity for cobinamide 3fold compared to wild-type

Escherichia coli

W66R

site-directed mutagenesis, reduces the affinity for cobinamide 10fold compared to wild-type

Escherichia coli

W66Y

site-directed mutagenesis, reduces the affinity for cobinamide severalfold compared to wild-type

Escherichia coli

Mg2+

required

Escherichia coli

ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]

Escherichia coli

-

ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]

-

?

ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]

Escherichia coli

-

ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]

-

?

Escherichia coli

P06609 AND P06611 AND P37028

genes btuC, btuD, and btuF encoding for vitamin B12 import system permease protein BtuC, vitamin B12 import ATP-binding protein BtuD, and vitamin B12-binding protein BtuF

-

recombinant N-terminally His10-tagged BtuCD from Escherichia coli and recombinant wild-type and mutant His6-tagged BtuF variants from Escherichia coli by nickel affinity chromatography

Escherichia coli

liposome reconstitution of recombinant purified BtuCD

Escherichia coli

ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]

-

752221

Escherichia coli

ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]

-

?

ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]

-

752221

Escherichia coli

ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]

-

?

additional information

BtuCD-F catalyzes the uptake of cobinamide, a cobalamin precursor, and cobalamin. BtuCD-catalyzed in vitro transport of cyano-cobinamide and of cobalamin is ATP- and BtuF-dependent. Tryptophan residue W66 of BtuF is involved in the substrate binding of cobalamin

752221

Escherichia coli

?

-

BtuCD-F

-

Escherichia coli

vitamin B12 transporter

-

Escherichia coli

22

-

assay at

Escherichia coli

7.5

-

assay at

Escherichia coli

ATP

-

Escherichia coli

recombinant expression of N-terminally His10-tagged BtuCD in Escherichia coli, recombinant expression of wild-type and mutant His6-tagged BtuF variants in Escherichia coli

Escherichia coli

ATP

-

Escherichia coli

analysis of the cobinamide (Cbi)-bound BtuF crystal structure model, PDB ID 5M29, crystal structures of Cbi-bound BtuF mutants W66F, W66Y and W66L, sitting drop vapor diffusion technique, mixing of 20 mg/ml protein in 10 mM Tris pH 8 and 100 mM NaCl, with precipitant solution containing 1% w/v tryptone, 50 mM HEPES, pH 7.0, and 12% w/v PEG 3350, 1-2 weeks, 20°C, X-ray diffraction structure determination and analysis at 1.5-1.7 A resolution, molecular replacement using the BtuF structure (PDB ID 1N2Z) as search model

Escherichia coli

additional information

site-directed mutagenesis of tryptophan residue W66 in the substrate binding cleft , the affinity for cobinamide of the W66X mutants is lower except for W66F. Three mutants with impaired Cbi binding (W66A, W66R, and W66E) and one with high binding affinity (W66F) are used for transport assays. Despite having lower Cbi binding affinities, Cbi transport is hardly affected by W66X substitution

Escherichia coli

W66A

site-directed mutagenesis, reduces the affinity for cobinamide severalfold compared to wild-type

Escherichia coli

W66E

site-directed mutagenesis, reduces the affinity for cobinamide severalfold compared to wild-type

Escherichia coli

W66F

site-directed mutagenesis, does not reduce the affinity for cobinamide severalfold compared to wild-type

Escherichia coli

W66H

site-directed mutagenesis, reduces the affinity for cobinamide 10fold compared to wild-type

Escherichia coli

W66L

site-directed mutagenesis, reduces the affinity for cobinamide 3fold compared to wild-type

Escherichia coli

W66R

site-directed mutagenesis, reduces the affinity for cobinamide 10fold compared to wild-type

Escherichia coli

W66Y

site-directed mutagenesis, reduces the affinity for cobinamide severalfold compared to wild-type

Escherichia coli

Mg2+

required

Escherichia coli

ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]

Escherichia coli

-

ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]

-

?

ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]

Escherichia coli

-

ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]

-

?

recombinant N-terminally His10-tagged BtuCD from Escherichia coli and recombinant wild-type and mutant His6-tagged BtuF variants from Escherichia coli by nickel affinity chromatography

Escherichia coli

liposome reconstitution of recombinant purified BtuCD

Escherichia coli

ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]

-

752221

Escherichia coli

ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]

-

?

ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]

-

752221

Escherichia coli

ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]

-

?

additional information

BtuCD-F catalyzes the uptake of cobinamide, a cobalamin precursor, and cobalamin. BtuCD-catalyzed in vitro transport of cyano-cobinamide and of cobalamin is ATP- and BtuF-dependent. Tryptophan residue W66 of BtuF is involved in the substrate binding of cobalamin

752221

Escherichia coli

?

-

22

-

assay at

Escherichia coli

7.5

-

assay at

Escherichia coli

malfunction

substitution of W66 in BtuF with tyrosine or leucine reduced the affinity 3fold compared to wild-type, and a change to histidine or arginine reduces it more than 10fold

Escherichia coli

additional information

the crystal structure of cobinamide-bound BtuF reveals a conformational change of a tryptophan residue W66 in the substrate binding cleft compared to the structure of cobalamin-bound BtuF, molecular dynamics simulations. BtuF is a class III periplasmic substrate binding protein

Escherichia coli

physiological function

ATP-binding cassette (ABC) transporters are a large family of integral membrane proteins and involved in nutrient uptake, drug extrusion, and lipid homeostasis. They use the energy of ATP binding and hydrolysis to power substrate transport across the lipid bilayer. BtuCD-F is an ABC transporter that mediates cobalamin (Cbl) uptake into Escherichia coli, Escherichia coli is unable to synthesize Cbl de novo. BtuCD-F might also be involved in the uptake of cobinamide, a cobalamin precursor. Precursor cobinamide (Cbi) lacks the 5,6-dimethylbenzimidazole (DMB) moiety and sugar-phosphate linker and is therefore smaller than Cbl. BtuCD-catalyzed in vitro transport of cyano-cobinamide is ATP- and BtuF-dependent. BtuF residue W66 is important for high affinity Cbi binding, but not for substrate delivery or transport

Escherichia coli

malfunction

substitution of W66 in BtuF with tyrosine or leucine reduced the affinity 3fold compared to wild-type, and a change to histidine or arginine reduces it more than 10fold

Escherichia coli

additional information

the crystal structure of cobinamide-bound BtuF reveals a conformational change of a tryptophan residue W66 in the substrate binding cleft compared to the structure of cobalamin-bound BtuF, molecular dynamics simulations. BtuF is a class III periplasmic substrate binding protein

Escherichia coli

physiological function

ATP-binding cassette (ABC) transporters are a large family of integral membrane proteins and involved in nutrient uptake, drug extrusion, and lipid homeostasis. They use the energy of ATP binding and hydrolysis to power substrate transport across the lipid bilayer. BtuCD-F is an ABC transporter that mediates cobalamin (Cbl) uptake into Escherichia coli, Escherichia coli is unable to synthesize Cbl de novo. BtuCD-F might also be involved in the uptake of cobinamide, a cobalamin precursor. Precursor cobinamide (Cbi) lacks the 5,6-dimethylbenzimidazole (DMB) moiety and sugar-phosphate linker and is therefore smaller than Cbl. BtuCD-catalyzed in vitro transport of cyano-cobinamide is ATP- and BtuF-dependent. BtuF residue W66 is important for high affinity Cbi binding, but not for substrate delivery or transport

Escherichia coli

749978

Agarwal

Mechanistic basis of vitamin ...

Vibrio cholerae serotype O1, Vibrio cholerae serotype O1 ATCC 39541, Vibrio cholerae serotype O1 Classical Ogawa 395, Vibrio cholerae serotype O1 O395

Biochim. Biophys. Acta

1867

140-151

2019

-

1

-

1

-

16

-

6

-

20

-

1

-

1

-

1

-

1

-

1

-

16

-

20

-

1

-

1

-

1

-

749821

Okamoto

Characterization of human ATP ...

Homo sapiens

Biochem. Biophys. Res. Commun.

496

1122-1127

2018

-

1

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1

-

2

1

-

1

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3

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1

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2

-

1

-

1

-

1

-

1

-

1

-

2

1

-

1

-

1

-

2

-

1

-

1

-

2

-

750412

Santos

Functional and structural cha ...

Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, Lactobacillus delbrueckii subsp. bulgaricus DSM 20081, Lactobacillus delbrueckii subsp. bulgaricus JCM 1002, Lactobacillus delbrueckii subsp. bulgaricus NBRC 13953, Lactobacillus delbrueckii subsp. bulgaricus NCIMB 11778

eLife

7

e35828

2018

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1

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6

1

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12

-

8

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18

1

5

1

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1

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1

-

1

-

6

-

1

-

12

-

18

1

-

1

-

2

-

751644

Schmitt

Vitamin B12 import is all abo ...

Escherichia coli

Nat. Chem. Biol.

14

640-641

2018

-

1

-

1

-

1

-

1

-

1

-

2

-

1

-

1

-

1

-

1

-

1

-

2

-

751670

Rempel

Cysteine-mediated decyanation ...

Thiobacillus denitrificans

Nat. Commun.

9

3038

2018

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1

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1

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2

-

1

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1

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4

-

4

1

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1

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1

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1

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1

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2

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1

-

4

-

1

-

1

-

3

-

751661

Goudsmits

Single-molecule visualization ...

Escherichia coli

Nat. Commun.

8

1652

2017

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1

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5

1

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1

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1

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8

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1

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1

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5

1

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2

1

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1

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1

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2

1

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3

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752215

Mireku

Structural basis of nanobody- ...

Escherichia coli

Sci. Rep.

7

14296

2017

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1

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1

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1

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1

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752221

Mireku

Conformational change of a tr ...

Escherichia coli

Sci. Rep.

7

41575

2017

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8

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1

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8

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750063

Priess

Release of entropic spring re ...

Escherichia coli

Biophys. J.

110

2407-2418

2016

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751956

Pan

ATP hydrolysis induced confor ...

Escherichia coli

PLoS ONE

11

e0166980

2016

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752200

Kawaguchi

Translocation of the ABC tran ...

Homo sapiens

Sci. Rep.

6

30183

2016

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734003

Su

Conformational motions and fun ...

Escherichia coli

Int. J. Mol. Sci.

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17933-17951

2015

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Joseph

Conformational cycle of the vi ...

Escherichia coli

J. Biol. Chem.

289

3176-3185

2014

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734760

Korkhov

Structure of AMP-PNP-bound Btu ...

Escherichia coli

Nat. Struct. Mol. Biol.

21

1097-1099

2014

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2

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1

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734820

Gopinath

A vitamin B12 transporter in M ...

Mycobacterium tuberculosis

Open Biology

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120175

2013

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Korkhov

Asymmetric states of vitamin B ...

Escherichia coli

FEBS Lett.

586

972-976

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Weng

-

The conformational transition ...

Escherichia coli

PLoS ONE

7

e305465

2012

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734771

Korkhov

Structure of AMP-PNP-bound vit ...

Escherichia coli

Nature

490

367-372

2012

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Di Bartolo

In vitro folding and assembly ...

Escherichia coli

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286

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719987

Joseph

Transmembrane gate movements i ...

Escherichia coli

J. Biol. Chem.

286

41008-41017

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Lewinson

A distinct mechanism for the A ...

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332-338

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Kandt

Holo-BtuF stabilizes the open ...

Escherichia coli

Proteins

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738-753

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713505

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Molecular dynamics simulation ...

Escherichia coli

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620-630

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Asymmetric conformational flex ...

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1918-1930

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Distinct gate conformations of ...

Escherichia coli

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Study on the mechanism of the ...

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The conformational coupling an ...

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Dynamics and function in a bac ...

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Simulation of the coupling bet ...

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Asymmetry in the structure of ...

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Opening and closing motions in ...

Escherichia coli

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In vitro functional characteri ...

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Vitamin B12 transport in Esche ...

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Vitamin B12 transport in Esche ...

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Nucleotide sequence of the btu ...

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Identification of the btuCED p ...

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Transport of vitamin B12 in Es ...

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-

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-

1

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-

Conformational change of a tryptophan residue in BtuF facilitates binding and transport of cobinamide by the vitamin B12 transporter BtuCD-F

Data extracted from this reference: