Literature summary for 7.6.2.8 extracted from

Rempel, S.; Colucci, E.; de Gier, J.W.; Guskov, A.; Slotboom, D.J.
Cysteine-mediated decyanation of vitamin B12 by the predicted membrane transporter BtuM (2018), Nat. Commun., 9, 3038 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene Tbd_2719 or btuM, recombinant expression of codon-optimized enzyme in Escherichia coli strain MC1061 and in Escherichia coli strain JW0154 (DELTAbtuF::KmR::DELTA FEC) lacking active BtuF and BtuC, overexpression of His-tagged enzyme and of EPEA-tagged enzyme	Thiobacillus denitrificans

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant His-tagged enzyme BtuM with natively bound cobalmin and anomalously bound cobalmin, sitting drop vapour diffusion method, mixing of 0.002 ml of protein in 50mM Tris-HCl, pH 7.5, or in 50 mM HEPES-NaOH pH 8.0, 100 mM NaCl, 0.005 mM cyano-Cbl and 0.35% detergent, with 0.002 ml of precipitant solution containing 25 mM Tris, pH 8.5, and 25-30% v/v PEG 400 or 50 mM Tris, pH 8.5, and 27-30% v/v PEG 400, or 75 mM Tris, pH 8.5, and 29-30% v/v PEG 400, at 8°C, 3-4 weeks, X-ray diffraction structure determination and analysis at 2.0-2.5 A resolution, structure modeling	Thiobacillus denitrificans

Crystallization (Comment)

Organism

purified recombinant His-tagged enzyme BtuM with natively bound cobalmin and anomalously bound cobalmin, sitting drop vapour diffusion method, mixing of 0.002 ml of protein in 50mM Tris-HCl, pH 7.5, or in 50 mM HEPES-NaOH pH 8.0, 100 mM NaCl, 0.005 mM cyano-Cbl and 0.35% detergent, with 0.002 ml of precipitant solution containing 25 mM Tris, pH 8.5, and 25-30% v/v PEG 400 or 50 mM Tris, pH 8.5, and 27-30% v/v PEG 400, or 75 mM Tris, pH 8.5, and 29-30% v/v PEG 400, at 8°C, 3-4 weeks, X-ray diffraction structure determination and analysis at 2.0-2.5 A resolution, structure modeling

Thiobacillus denitrificans

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
inner membrane	integral membrane enzyme	Thiobacillus denitrificans	-	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Thiobacillus denitrificans

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]	Thiobacillus denitrificans	-	ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]	-	?
additional information	Thiobacillus denitrificans	BtuM binds vitamin B12 in its base-off conformation, in which the 5,6-dimethylbenzimidazole moiety does not bind to the cobalt ion, but with a cysteine residue as axial ligand of the corrin cobalt ion, BtuMTd binds cobalamin (Cbl) using cysteine ligation. In contrast, at physiological pH the conformation of free Cbl in aqueous solution is base-on with the 5,6-dimethylbenzimidazole moiety coordinated to the cobalt ion in the alpha-axial position	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Thiobacillus denitrificans	Q3SFD8	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme to homogeneity by nickel affnity chromatography in presence of 0.35% n-nonyl-beta-D-glucopyranoside, gel filtration, and ultrafiltration, recombinant EPEA-tagged enzyme by affinity chromatography and ultrafiltration	Thiobacillus denitrificans

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + H2O + cobinamide-[cobalamin-binding protein][side 1]	-	Thiobacillus denitrificans	ADP + phosphate + cobinamide[side 2] + [cobalamin-binding protein][side 1]	-	?
ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]	-	Thiobacillus denitrificans	ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]	-	?
additional information	BtuM binds vitamin B12 in its base-off conformation, in which the 5,6-dimethylbenzimidazole moiety does not bind to the cobalt ion, but with a cysteine residue as axial ligand of the corrin cobalt ion, BtuMTd binds cobalamin (Cbl) using cysteine ligation. In contrast, at physiological pH the conformation of free Cbl in aqueous solution is base-on with the 5,6-dimethylbenzimidazole moiety coordinated to the cobalt ion in the alpha-axial position	Thiobacillus denitrificans	?	-	?
additional information	BtuM binds vitamin B12 in its base-off conformation, in which the 5,6-dimethylbenzimidazole moiety does not bind to the cobalt ion, but with a cysteine residue as axial ligand of the corrin cobalt ion, BtuMTd binds cobalamin (Cbl) using cysteine ligation. In contrast, at physiological pH the conformation of free Cbl in aqueous solution is base-on with the 5,6-dimethylbenzimidazole moiety coordinated to the cobalt ion in the alpha-axial position. The membrane environment also appears to preclude Cbl binding to purified BtuMTd, as binding is observed only when the substrate is added before solubilisation. BtuMTd also catalyses cysteine-mediated decyanation of vitamin B12	Thiobacillus denitrificans	?	-	?

Synonyms

Synonyms	Comment	Organism
BtuM	-	Thiobacillus denitrificans
BtuMTd	-	Thiobacillus denitrificans
Tbd_2719	-	Thiobacillus denitrificans

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
22	-	assay at room temperature	Thiobacillus denitrificans

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Thiobacillus denitrificans

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Thiobacillus denitrificans

General Information

General Information	Comment	Organism
evolution	the enzyme is a ABC transporter, all ABC-type ATPases encoded by the organism are predicted to be part of classical ABC transporters, and not ECF transporters. The organism does not encode an ECF-module. BtuM homologues (apart from one exception) are found exclusively in organisms lacking an ECF-module. But BtuMTd structurally resembles the S-components of ECF transporters	Thiobacillus denitrificans
additional information	function and structure of BtuMTd, cobalamin binding structure, overview	Thiobacillus denitrificans
physiological function	the membrane protein BtuM acts as transporter for uptake of essential vitamin B12, i.e. cobalamin, one of the most complex cofactors known, and used by enzymes catalyzing for instance methyl-group transfer and ribonucleotide reduction reactions. BtuMTd likely combines two functions: transport of the substrate into the bacterial cell, and chemical modification of the substrate. A cobalt-cysteine interaction allows for chemical modification of the substrate prior to translocation, which is a rare feature among uptake systems. BtuM homologues are small membrane proteins of about 22 kDa, and found predominantly in Gram-negative species, distributed mostly over alpha-, beta-, and gamma-proteobacteria	Thiobacillus denitrificans