Literature summary for 4.99.1.B2 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 .

Search for more literature for 4.99.1.B2

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of C-terminally His8-tagged wild-type and mutant enzymes in Escherichia coli strain MC1061	Thiobacillus denitrificans

Crystallization (Commentary)

Crystallization (Comment)	Organism
crystal structure PDB ID 6FFV	Thiobacillus denitrificans

Protein Variants

Protein Variants	Comment	Organism
D67A	site-directed mutagenesis, the mutant cannot be purified	Thiobacillus denitrificans
H28A	site-directed mutagenesis, binding of cobalamin to the mutant is accompanied by decyanation, indicating that the conserved residue is not essential for the reaction	Thiobacillus denitrificans
R153A	site-directed mutagenesis, binding of cobalamin to the mutant is accompanied by decyanation, indicating that the conserved residue is not essential for the reaction	Thiobacillus denitrificans
Y85L	site-directed mutagenesis, binding of cobalamin to the mutant is accompanied by decyanation, indicating that the conserved residue is not essential for the reaction	Thiobacillus denitrificans

Localization

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

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
cyano-cobalamin + H2O	Thiobacillus denitrificans	cyano-Cbl, binding and decyanation	hydroxyl-cobalamin + cyanide	-	?
additional information	Thiobacillus denitrificans	BtuMTd is merely a decyanating enzyme, and the potential reaction product hydroxyl-Cbl is subsequently transported by another protein. BtuMTd also mediates uptake of hydroxyl-Cbl in the growth assay. Mass spectrometric ligand analysis. 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	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
cyano-cobalamin + H2O	cyano-Cbl, binding and decyanation	Thiobacillus denitrificans	hydroxyl-cobalamin + cyanide	-	?
dicyano-cobinamide + H2O	cyano-Cbi, binding and decyanation	Thiobacillus denitrificans	hydroxyl-cobinamide + cyanide	-	?
additional information	BtuMTd is merely a decyanating enzyme, and the potential reaction product hydroxyl-Cbl is subsequently transported by another protein. BtuMTd also mediates uptake of hydroxyl-Cbl in the growth assay. Mass spectrometric ligand analysis. 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	a cobalt-cysteine interaction allows for chemical modification of the substrate prior to translocation across the inner membrane, which is a rare feature among uptake systems. BtuM binds vitamin B12 in its base-off conformation (in which the 5,6-dimethylbenzimidazole moiety does not bind to the cobalt ion), with a cysteine residue as axial ligand of the corrin cobalt ion, the unusual thiolate coordination allows for decyanation of vitamin B12, a unique feature for a vitamin B12-transport protein. BtuMTd binds cobalamin using cysteine ligation followed by BtuMTd catalysed decyanation reaction. Cys80 is required for decyanation of cyano-cobinamide, and binding and modification of the substrate are separate events, the cysteine is also required for base-on to base-off conversion	Thiobacillus denitrificans	?	-	?

Synonyms

Synonyms	Comment	Organism
BtuM	-	Thiobacillus denitrificans
BtuMTd	-	Thiobacillus denitrificans
Tbd_2719	-	Thiobacillus denitrificans
vitamin B12 decyanase transporter	-	Thiobacillus denitrificans

Temperature Optimum [°C]

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

pH Optimum

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

General Information

General Information	Comment	Organism
evolution	BtuM homologues (apart from one exception) are found exclusively in organisms lacking an ECF-module. In contrast to BtuM proteins, ECF-type ABC transporters are predominantly found in Gram-positive bacteria. They are multi-subunit complexes consisting of two peripheral ATPases and two transmembrane components (EcfT and S-component). EcfT and the ATPases together form the socalled ECF-module. S-Components bind the transported substrate, and dynamically associate with the ECF-module to allow substrate translocation. Intriguingly, no homologues of EcfT can be found in Thiobacillus denitrificans	Thiobacillus denitrificans
additional information	BtuMTd structure function relationship analysis, detailed overview. The proposed decyanation mechanism differs from the mechanism used by CblC, where a flavin acts as reducing agent. In CblC, the flavin donates two electrons resulting in the reductive decyanation (CN-) and the reduction of the Co2+ ion. For BtuMTd, cysteine-catalysed reductive decyanation only results in the release of CN-, but not in the reduction of the Co2+-ion	Thiobacillus denitrificans
additional information	function and structure of BtuMTd, cobalamin binding structure, overview	Thiobacillus denitrificans
physiological function	potential alternative inner membrane vitamin B12 transporter BtuM is a predicted bacterial vitamin B12 uptake system. Uptake of vitamin B12 is essential for many prokaryotes. BtuMTd supports vitamin B12-dependent growth. Cyano-cobalamin (Cbl) contains a cyano-group as the beta-ligand. Cyano-Cbl is the most stable form of vitamin B12 but, despite the tight binding of the beta-ligand, in the crystal structure the cyano-group is absent indicating protein-mediated decyanation, in place of the cyano-group, the imidazole group of His207 from a neighbouring BtuMTd molecule in the crystal is located at the beta-axial position	Thiobacillus denitrificans

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Release 2023.1 (January 2023)