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Information on EC 4.99.1.B2 - vitamin B12 decyanase

for references in articles please use BRENDA:EC4.99.1.B2

preliminary BRENDA-supplied EC number

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EC Tree

4 Lyases

4.99 Other lyases

4.99.1 Sole sub-subclass for lyases that do not belong in the other subclasses

4.99.1.B2 vitamin B12 decyanase

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The expected taxonomic range for this enzyme is: Thiobacillus denitrificans

Reaction Schemes

hydroxyl-cobalamin

Synonyms

BtuM, BtuMTd, Tbd_2719, vitamin B12 decyanase transporter, show all synonyms

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SYNONYM

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

BtuM

Thiobacillus denitrificans

Q3SFD8

751670

BtuMTd

Thiobacillus denitrificans

Q3SFD8

751670

Tbd_2719

Thiobacillus denitrificans

Q3SFD8

751670

vitamin B12 decyanase transporter

Thiobacillus denitrificans

Q3SFD8

751670

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REACTION

REACTION DIAGRAM

COMMENTARY

ORGANISM

UNIPROT

LITERATURE

cyano-cobalamin + H2O = hydroxyl-cobalamin + cyanide

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SUBSTRATE

PRODUCT

REACTION DIAGRAM

ORGANISM

UNIPROT

COMMENTARY
(Substrate)

LITERATURE
(Substrate)

COMMENTARY
(Product)

LITERATURE
(Product)

Reversibility
r=reversible
ir=irreversible
?=not specified

cyano-cobalamin + H2O

hydroxyl-cobalamin + cyanide

Thiobacillus denitrificans

Q3SFD8

cyano-Cbl, binding and decyanation

751670

dicyano-cobinamide + H2O

hydroxyl-cobinamide + cyanide

Thiobacillus denitrificans

Q3SFD8

cyano-Cbi, binding and decyanation

751670

additional information

2 entries

additional information

Thiobacillus denitrificans

Q3SFD8

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

751670

additional information

Thiobacillus denitrificans

Q3SFD8

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

751670

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NATURAL SUBSTRATE

NATURAL PRODUCT

REACTION DIAGRAM

ORGANISM

UNIPROT

COMMENTARY
(Substrate)

LITERATURE
(Substrate)

COMMENTARY
(Product)

LITERATURE
(Product)

REVERSIBILITY
r=reversible
ir=irreversible
?=not specified

cyano-cobalamin + H2O

hydroxyl-cobalamin + cyanide

Thiobacillus denitrificans

Q3SFD8

cyano-Cbl, binding and decyanation

751670

additional information

Thiobacillus denitrificans

Q3SFD8

751670

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pH OPTIMUM

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

7.5

Thiobacillus denitrificans

Q3SFD8

assay at

751670

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TEMPERATURE OPTIMUM

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

Thiobacillus denitrificans

Q3SFD8

decyanation assay at room temperature

751670

Thiobacillus denitrificans

Q3SFD8

growth assay at

751670

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ORGANISM

COMMENTARY

LITERATURE

UNIPROT

SEQUENCE DB

SOURCE

Thiobacillus denitrificans

751670

Q3SFD8

UniProt

brenda

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LOCALIZATION

ORGANISM

UNIPROT

COMMENTARY

GeneOntology No.

LITERATURE

SOURCE

inner membrane

Thiobacillus denitrificans

Q3SFD8

integral membrane enzyme

751670

brenda

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GENERAL INFORMATION

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

evolution

Thiobacillus denitrificans

Q3SFD8

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

751670

physiological function

Thiobacillus denitrificans

Q3SFD8

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

751670

additional information

2 entries

additional information

Thiobacillus denitrificans

Q3SFD8

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

751670

additional information

Thiobacillus denitrificans

Q3SFD8

function and structure of BtuMTd, cobalamin binding structure, overview

751670

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UNIPROT

ENTRY NAME

ORGANISM

NO. OF AA

NO. OF TRANSM. HELICES

MOLECULAR WEIGHT[Da]

SOURCE

SEQUENCE

LOCALIZATION PREDICTION

The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).

Q3SFD8 pBLAST

Q3SFD8_THIDA

Thiobacillus denitrificans (strain ATCC 25259)

198

21751

TrEMBL

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CRYSTALLIZATION (Commentary)

ORGANISM

UNIPROT

LITERATURE

crystal structure PDB ID 6FFV

Thiobacillus denitrificans

Q3SFD8

751670

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PROTEIN VARIANTS

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

D67A

Thiobacillus denitrificans

Q3SFD8

site-directed mutagenesis, the mutant cannot be purified

751670

H28A

Thiobacillus denitrificans

Q3SFD8

site-directed mutagenesis, binding of cobalamin to the mutant is accompanied by decyanation, indicating that the conserved residue is not essential for the reaction

751670

R153A

Thiobacillus denitrificans

Q3SFD8

site-directed mutagenesis, binding of cobalamin to the mutant is accompanied by decyanation, indicating that the conserved residue is not essential for the reaction

751670

Y85L

Thiobacillus denitrificans

Q3SFD8

site-directed mutagenesis, binding of cobalamin to the mutant is accompanied by decyanation, indicating that the conserved residue is not essential for the reaction

751670

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CLONED (Commentary)

ORGANISM

UNIPROT

LITERATURE

recombinant expression of C-terminally His8-tagged wild-type and mutant enzymes in Escherichia coli strain MC1061

Thiobacillus denitrificans

Q3SFD8

751670

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REF.

AUTHORS

TITLE

JOURNAL

VOL.

PAGES

YEAR

ORGANISM (UNIPROT)

PUBMED ID

SOURCE

751670

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

Nat. Commun.

3038

2018

Thiobacillus denitrificans (Q3SFD8)

30072686

brenda

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EXTERNAL LINKS (specific for EC-Number 4.99.1.B2)

ExplorEnz (official portal for IUBMB Enzyme Nomenclature)

Expasy Enzyme Nomenclature Database

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MetaCyc

SABIO-RK

NCBI: PubMed, Protein, Nucleotide, Structure, Gene, OMIM

Enzyme Nomenclature (alternative site)

UniProt

PDB

PROSITE Database of protein families and domains

InterPro (database of protein families, domains and functional sites)

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