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Reductive cleavage of S-adenosylmethionine by biotin synthase from Escherichia coli

Reductive cleavage of S-adenosylmethionine by biotin synthase from Escherichia coli
Reductive cleavage of S-adenosylmethionine by biotin synthase from Escherichia coli
Biotin synthase (BioB) catalyzes the insertion of a sulfur atom between the C6 and C9 carbons of dethiobiotin. Reconstituted BioB from Escherichia coli contains a [4Fe-4S](2+/1+) cluster thought to be involved in the reduction and cleavage of S-adenosylmethionine (AdoMet), generating methionine and the reactive 5'-deoxyadenosyl radical responsible for dethiobiotin H-abstraction. Using EPR and Mössbauer spectroscopy as well as methionine quantitation we demonstrate that the reduced S = 1/2 [4Fe-4S](1+) cluster is indeed capable of injecting one electron into AdoMet, generating one equivalent of both methionine and S = 0 [4Fe-4S](2+) cluster. Dethiobiotin is not required for the reaction. Using site-directed mutagenesis we show also that, among the eight cysteines of BioB, only three (Cys-53, Cys-57, Cys-60) are essential for AdoMet reductive cleavage, suggesting that these cysteines are involved in chelation of the [4Fe-4S](2+/1+) cluster.
iron-sulfur cluster, pyruvate formate-lyase, anaerobic ribonucleotide reductase, electron-paramagnetic-resonance, lysine 2, 3-aminomutase, radical mechanisms, activating enzyme, single turnover, in-vitro, protein
0021-9258
13449-13454
Ollagnier-de Choudens, Sandrine
2a7fdf40-6851-4342-8bb7-15257d38e007
Sanakis, Yiannis
b3ee6ad6-22b5-4288-8127-8526d1d510d6
Hewitson, Kirsty S.
b5020a62-d602-4ab5-8d74-68346b7b076e
Roach, Peter
ca94060c-4443-482b-af3e-979243488ba9
Münck, Eckard
e81bf79d-b96e-4e14-bf31-40b87b573c9b
Fontecave, Marc
6293f4f2-e3b0-4e56-8eb6-eb5a252b6a19
Ollagnier-de Choudens, Sandrine
2a7fdf40-6851-4342-8bb7-15257d38e007
Sanakis, Yiannis
b3ee6ad6-22b5-4288-8127-8526d1d510d6
Hewitson, Kirsty S.
b5020a62-d602-4ab5-8d74-68346b7b076e
Roach, Peter
ca94060c-4443-482b-af3e-979243488ba9
Münck, Eckard
e81bf79d-b96e-4e14-bf31-40b87b573c9b
Fontecave, Marc
6293f4f2-e3b0-4e56-8eb6-eb5a252b6a19

Ollagnier-de Choudens, Sandrine, Sanakis, Yiannis, Hewitson, Kirsty S., Roach, Peter, Münck, Eckard and Fontecave, Marc (2002) Reductive cleavage of S-adenosylmethionine by biotin synthase from Escherichia coli. The Journal of Biological Chemistry, 277 (16), 13449-13454. (doi:10.1074/jbc.M111324200).

Record type: Article

Abstract

Biotin synthase (BioB) catalyzes the insertion of a sulfur atom between the C6 and C9 carbons of dethiobiotin. Reconstituted BioB from Escherichia coli contains a [4Fe-4S](2+/1+) cluster thought to be involved in the reduction and cleavage of S-adenosylmethionine (AdoMet), generating methionine and the reactive 5'-deoxyadenosyl radical responsible for dethiobiotin H-abstraction. Using EPR and Mössbauer spectroscopy as well as methionine quantitation we demonstrate that the reduced S = 1/2 [4Fe-4S](1+) cluster is indeed capable of injecting one electron into AdoMet, generating one equivalent of both methionine and S = 0 [4Fe-4S](2+) cluster. Dethiobiotin is not required for the reaction. Using site-directed mutagenesis we show also that, among the eight cysteines of BioB, only three (Cys-53, Cys-57, Cys-60) are essential for AdoMet reductive cleavage, suggesting that these cysteines are involved in chelation of the [4Fe-4S](2+/1+) cluster.

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More information

Published date: 19 April 2002
Keywords: iron-sulfur cluster, pyruvate formate-lyase, anaerobic ribonucleotide reductase, electron-paramagnetic-resonance, lysine 2, 3-aminomutase, radical mechanisms, activating enzyme, single turnover, in-vitro, protein

Identifiers

Local EPrints ID: 19832
URI: http://eprints.soton.ac.uk/id/eprint/19832
ISSN: 0021-9258
PURE UUID: 6bd66560-1702-4fa9-9d0a-ff328994910c
ORCID for Peter Roach: ORCID iD orcid.org/0000-0001-9880-2877

Catalogue record

Date deposited: 22 Feb 2006
Last modified: 08 Jan 2022 06:46

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Contributors

Author: Sandrine Ollagnier-de Choudens
Author: Yiannis Sanakis
Author: Kirsty S. Hewitson
Author: Peter Roach ORCID iD
Author: Eckard Münck
Author: Marc Fontecave

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