Literature summary for 7.6.2.8 extracted from

Mireku, S.A.; Ruetz, M.; Zhou, T.; Korkhov, V.M.; Kraeutler, B.; Locher, K.P.
Conformational change of a tryptophan residue in BtuF facilitates binding and transport of cobinamide by the vitamin B12 transporter BtuCD-F (2017), Sci. Rep., 7, 41575 .

Cloned(Commentary)

Cloned (Comment)	Organism
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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants

Protein Variants	Comment	Organism
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

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
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]	-	?

Organism

Organism	UniProt	Comment	Textmining
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	-

Purification (Commentary)

Purification (Comment)	Organism
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

Renatured (Commentary)

Renatured (Comment)	Organism
liposome reconstitution of recombinant purified BtuCD	Escherichia coli

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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]	-	?
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	Escherichia coli	?	-	?

Synonyms

Synonyms	Comment	Organism
BtuCD-F	-	Escherichia coli
vitamin B12 transporter	-	Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
22	-	assay at	Escherichia coli

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Escherichia coli

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Escherichia coli

General Information

General Information	Comment	Organism
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