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Catalytic Site Atlas Version 2.2.12
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CSA entry for 1r30
Original Entry
Title:
Transferase
Compound:
Biotin synthase
Mutant:
No
UniProt/Swiss-Prot:
P12996-BIOB_ECOLI
EC Class:
2.8.1.6
Other CSA Entries:
Overview of all sites for 1r30
Homologues of 1r30
Entries for UniProt/Swiss-Prot: P12996
Entries for EC: 2.8.1.6
Other Databases:
PDB entry: 1r30
PDBsum entry: 1r30
UniProt/Swiss-Prot: P12996
IntEnz entry: 2.8.1.6
Literature Report:
Introduction:
Biotin synthase (BioB) is a member of the radical-SAM superfamily. It catalyses the insertion of an S atom between the C6 and C9 carbon atoms of dethiobiotin (DTB).

BioB contains [4Fe-4S] clusters involved in the reduction and cleavage of S-adenosyl-L-methionine (AdoMet), generating methionine and a primary 5'-deoxyadenosyl radical responsible for dethiobiotin H-abstraction.

i.e. BioB is involved in the activation of AdoMet in the biotin synthesis pathway and the incorporation of S atoms into DTB.

An oxygen sensitive Fe/S cluster is bound to each polypeptide chain and is chelated by three catalytic Cys residues. The cluster provides electrons for AdoMet cleavage and can adopt both +1 and +2 redox states.
Mechanism:
Formation of biotin from dethiobiotin (DTB) requires the abstraction of 2 hydrogen atoms from the DTB C6 and C9 carbons. This is carried out by the product of AdoMet reduction, the 5'-deoxyadenosyl radical. Two hydrogen atoms are selectively removed from the C9 methyl and C6 methylene positions of DTB.

Radicals of C6 and C9 can then react with sulpur atoms , leading to C-S bond formations at these positions.

[4Fe-4S] clusters of +1 oxidation state are required for this process. Since the Fe/S clusters are essential for catalysis, the 3 cluster-chelating Cys residues (Cys 53, Cys 57 and Cys 60) also have a catalytic effect, and are arranged in a conserved C-X3-C-X2-C cysteine triad motif. A fourth cluster ligand, Arg 260 is also essential for catalysis.

The +1 iron-sulphur cluster injects one electron into the AdoMet bound in its close proximity. The resulting, unstable suphuranyl radical breaks down into methionine and the 5'-deoxyadenosyl radical. This subsequently abstracts a hydrogen from a specific carbon atom of a glycine residue in the polypeptide chain.
Sites:

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Found by:
Literature reference 

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
CYSA 53 53Sidechain
ElectrostaticCofactor
Stabilisation by chelation of the iron-sulphur cluster.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11834738 Current protein Conservation of residue
PubMed ID 11834738 Current protein Mutagenesis of residue
PubMed ID 11862544 Current protein Conservation of residue
PubMed ID 11862544 Current protein Mutagenesis of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
CYSA 57 57Sidechain
ElectrostaticCofactor
Stabilisation by chelation of the iron-sulphur cluster.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11834738 Current protein Conservation of residue
PubMed ID 11834738 Current protein Mutagenesis of residue
PubMed ID 11862544 Current protein Conservation of residue
PubMed ID 11862544 Current protein Mutagenesis of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
CYSA 60 60Sidechain
ElectrostaticCofactor
Stabilisation by chelation of the iron-sulphur cluster.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11834738 Current protein Mutagenesis of residue
PubMed ID 11862544 Current protein Mutagenesis of residue
PubMed ID 11862544 Current protein Conservation of residue
PubMed ID 11834738 Current protein Conservation of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ARGA 260 260Sidechain
ElectrostaticCofactor
The unusual Arg 260 environment is important for preserving charge neutrality within the core by reducing the overall net negative charge of the buried cluster. Arg 260 plays an important role in redox modulation. The Arg 260 side chain can rearrange to bridge the two Fe atoms and facilitate proposed S transfer.
Evidence from paper Evidence concerns Evidence type
PubMed ID 14704425 Current protein Conservation of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
FESA 402 0Sidechain
Radical formationSubstrate
feS cluster acts as a cofactor by taking part in a redox reaction to produce an active radical, which can then go on to form carbon radicals at C6 and C9.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11862544 Current protein Residue is covalently bound to intermediate, based on structural data
PubMed ID 11862544 Current protein Conservation of residue
PubMed ID 11862544 Current protein Structural similarity to homologue of known mechanism
Notes:
Site directed mutagenesis studies have also shown that three other cysteine residues (Cys 97, Cys 128 and Cys 188) are important for catalysis. Their exact function is unknown, but it is thought that catalytic dependence on these residues may be to the chelation of a second Fe/S cluster.
References:
1
The iron-sulfur center of biotin synthase: site-directed mutants.
K. S. Hewitson and S. Ollagnier-de Choudens and Y. Sanakis and N. M. Shaw and J. E. Baldwin and E. Münck and P. L. Roach and M. Fontecave
J Biol Inorg Chem 7, (1-2) 83-93, (2002).
11862544
2
Reductive cleavage of S-adenosylmethionine by biotin synthase from Escherichia coli.
S. Ollagnier-de Choudens and Y. Sanakis and K. S. Hewitson and P. Roach and E. Münck and M. Fontecave
J Biol Chem 277, (16) 13449-54, (2002).
11834738
3
Role of the [2Fe-2S] cluster in recombinant Escherichia coli biotin synthase.
G. N. Jameson and M. M. Cosper and H. L. Hernández and M. K. Johnson and B. H. Huynh
Biochemistry 43, (7) 2022-31, (2004).
14967042
4
Crystal structure of biotin synthase, an S-adenosylmethionine-dependent radical enzyme.
F. Berkovitch and Y. Nicolet and J. T. Wan and J. T. Jarrett and C. L. Drennan
Science 303, (5654) 76-9, (2004).
14704425
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