Literature summary for 7.6.2.8 extracted from

Santos, J.A.; Rempel, S.; Mous, S.T.; Pereira, C.T.; Ter Beek, J.; de Gier, J.W.; Guskov, A.; Slotboom, D.J.
Functional and structural characterization of an ECF-type ABC transporter for vitamin B12 (2018), eLife, 7, e35828 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of ECF-CbrT complements in an Escherichia coli strain lacking its endogenous vitamin B12 transporter (Escherichia coli strain DELTAFEC, with btuF and btuC also deleted , thus abolished endogenous Cbl-uptake, from parental strain JW0154). Recombinant ECF-CbrT supports cobalamin-dependent growth of an Escherichia coli deletion strain	Lactobacillus delbrueckii subsp. bulgaricus

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified ECF-CbrT complex in detergent (n-dodecyl-beta-D-maltopyranoside) solution, sitting drop vapour diffusion method, mixing of protein solution containing 50 mM HEPES pH 8, 150 mM NaCl, 1% polyoxyethylene(10)dodecyl ether, with precipitant solution containing 0.2 M KCl, 0.1 M sodium citrate, pH 5.5, 37% v/v pentaerythritol propoxylate, X-ray diffraction structure determination and analysis at 3.4 A resolution, molecular replacement with the structure of the folate transporter, ECF-FolT2, from Lactobacillus delbrueckii as a search model	Lactobacillus delbrueckii subsp. bulgaricus

Crystallization (Comment)

Organism

purified ECF-CbrT complex in detergent (n-dodecyl-beta-D-maltopyranoside) solution, sitting drop vapour diffusion method, mixing of protein solution containing 50 mM HEPES pH 8, 150 mM NaCl, 1% polyoxyethylene(10)dodecyl ether, with precipitant solution containing 0.2 M KCl, 0.1 M sodium citrate, pH 5.5, 37% v/v pentaerythritol propoxylate, X-ray diffraction structure determination and analysis at 3.4 A resolution, molecular replacement with the structure of the folate transporter, ECF-FolT2, from Lactobacillus delbrueckii as a search model

Lactobacillus delbrueckii subsp. bulgaricus

Inhibitors

Inhibitors	Comment	Organism
adenosyl-cobalamin	is less effective causing about 25% inhibition of cobalmin uptake	Lactobacillus delbrueckii subsp. bulgaricus
cobinamide	addition of a 250fold excess of cobalamin decreases the uptake of radiolabeled cobalamin to about 25%	Lactobacillus delbrueckii subsp. bulgaricus
cyano-cobalamin	a 250fold excess of unlabeled CN-Cbl inhibits the uptake of the radiolabeled substrate almost completely	Lactobacillus delbrueckii subsp. bulgaricus
hydroxycobalamin	inhibits uptake to a similar extent as cyano-cobalamin	Lactobacillus delbrueckii subsp. bulgaricus
methylcobalamin	inhibits uptake to a similar extent as cyano-cobalamin	Lactobacillus delbrueckii subsp. bulgaricus
additional information	hemin does not compete with cobalamin-uptake	Lactobacillus delbrueckii subsp. bulgaricus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	binding kinetics of substrates and competing ligands	Lactobacillus delbrueckii subsp. bulgaricus

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
membrane	integral membrane enzyme	Lactobacillus delbrueckii subsp. bulgaricus	16020	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Lactobacillus delbrueckii subsp. bulgaricus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]	Lactobacillus delbrueckii subsp. bulgaricus	-	ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]	Lactobacillus delbrueckii subsp. bulgaricus DSM 20081	-	ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]	Lactobacillus delbrueckii subsp. bulgaricus NBRC 13953	-	ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]	Lactobacillus delbrueckii subsp. bulgaricus JCM 1002	-	ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]	Lactobacillus delbrueckii subsp. bulgaricus NCIMB 11778	-	ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]	Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842	-	ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]	Lactobacillus delbrueckii subsp. bulgaricus	-	ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]	Lactobacillus delbrueckii subsp. bulgaricus DSM 20081	-	ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]	Lactobacillus delbrueckii subsp. bulgaricus NBRC 13953	-	ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]	Lactobacillus delbrueckii subsp. bulgaricus JCM 1002	-	ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]	Lactobacillus delbrueckii subsp. bulgaricus NCIMB 11778	-	ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]	Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842	-	ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]	-	?

Organism

Organism	UniProt	Comment	Textmining
Lactobacillus delbrueckii subsp. bulgaricus	Q1GBI8	-	-
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842	Q1GBI8	-	-
Lactobacillus delbrueckii subsp. bulgaricus DSM 20081	Q1GBI8	-	-
Lactobacillus delbrueckii subsp. bulgaricus JCM 1002	Q1GBI8	-	-
Lactobacillus delbrueckii subsp. bulgaricus NBRC 13953	Q1GBI8	-	-
Lactobacillus delbrueckii subsp. bulgaricus NCIMB 11778	Q1GBI8	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]	-	Lactobacillus delbrueckii subsp. bulgaricus	ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]	-	Lactobacillus delbrueckii subsp. bulgaricus DSM 20081	ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]	-	Lactobacillus delbrueckii subsp. bulgaricus NBRC 13953	ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]	-	Lactobacillus delbrueckii subsp. bulgaricus JCM 1002	ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]	-	Lactobacillus delbrueckii subsp. bulgaricus NCIMB 11778	ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]	-	Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842	ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]	-	Lactobacillus delbrueckii subsp. bulgaricus	ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]	-	Lactobacillus delbrueckii subsp. bulgaricus DSM 20081	ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]	-	Lactobacillus delbrueckii subsp. bulgaricus NBRC 13953	ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]	-	Lactobacillus delbrueckii subsp. bulgaricus JCM 1002	ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]	-	Lactobacillus delbrueckii subsp. bulgaricus NCIMB 11778	ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]	-	Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842	ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]	-	?
additional information	hemin does not compete with cobalamin-uptake. Although promiscuous among cobalamin variants and cobalamin precursors, ECF-CbrT is a dedicated vitamin B12 transporter	Lactobacillus delbrueckii subsp. bulgaricus	?	-	?
additional information	hemin does not compete with cobalamin-uptake. Although promiscuous among cobalamin variants and cobalamin precursors, ECF-CbrT is a dedicated vitamin B12 transporter	Lactobacillus delbrueckii subsp. bulgaricus DSM 20081	?	-	?
additional information	hemin does not compete with cobalamin-uptake. Although promiscuous among cobalamin variants and cobalamin precursors, ECF-CbrT is a dedicated vitamin B12 transporter	Lactobacillus delbrueckii subsp. bulgaricus NBRC 13953	?	-	?
additional information	hemin does not compete with cobalamin-uptake. Although promiscuous among cobalamin variants and cobalamin precursors, ECF-CbrT is a dedicated vitamin B12 transporter	Lactobacillus delbrueckii subsp. bulgaricus JCM 1002	?	-	?
additional information	hemin does not compete with cobalamin-uptake. Although promiscuous among cobalamin variants and cobalamin precursors, ECF-CbrT is a dedicated vitamin B12 transporter	Lactobacillus delbrueckii subsp. bulgaricus NCIMB 11778	?	-	?
additional information	hemin does not compete with cobalamin-uptake. Although promiscuous among cobalamin variants and cobalamin precursors, ECF-CbrT is a dedicated vitamin B12 transporter	Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842	?	-	?

Subunits

Subunits	Comment	Organism
More	ECF-transporters are multi-subunit membrane complexes that consist of two ATPases, similar to the ATPases of ABC transporters, and two membrane embedded proteins, not related to any other protein family	Lactobacillus delbrueckii subsp. bulgaricus

Synonyms

Synonyms	Comment	Organism
Cbl transporter	-	Lactobacillus delbrueckii subsp. bulgaricus
cobalamin-specific ECF-type ABC transporter	-	Lactobacillus delbrueckii subsp. bulgaricus
ECF-CbrT	-	Lactobacillus delbrueckii subsp. bulgaricus
ECF-type ABC transporter for vitamin B12	-	Lactobacillus delbrueckii subsp. bulgaricus
energy coupling factor (ECF-) type ABC transporter	-	Lactobacillus delbrueckii subsp. bulgaricus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Lactobacillus delbrueckii subsp. bulgaricus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Lactobacillus delbrueckii subsp. bulgaricus

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Lactobacillus delbrueckii subsp. bulgaricus

General Information

General Information	Comment	Organism
evolution	ECF-transporters are multi-subunit membrane complexes that consist of two ATPases, similar to the ATPases of ABC transporters, and two membrane embedded proteins, not related to any other protein family	Lactobacillus delbrueckii subsp. bulgaricus
physiological function	cobalamin-specific ECF-type ABC transporter from Lactobacillus delbrueckii, ECF-CbrT, mediates the specific, ATP-dependent uptake of cobalamin. Cobalamin (vitamin B12) is the most complex B-type vitamin and is synthetized exclusively in a limited number of prokaryotes. Its biologically active variants contain rare organometallic bonds, which are used by enzymes in a variety of central metabolic pathways such as L-methionine synthesis and ribonucleotide reduction. Enzyme ECF-CbrT catalyzes ATP-dependent transport of cobalamin and cobinamide	Lactobacillus delbrueckii subsp. bulgaricus