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Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions
Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions
Lipoyl cofactors are essential for living organisms and are produced by the insertion of two sulfur atoms into the relatively unreactive C–H bonds of an octanoyl substrate. This reaction requires lipoyl synthase, a member of the radical S-adenosylmethionine (SAM) enzyme superfamily. In the present study, we solved crystal structures of lipoyl synthase with two [4Fe–4S] clusters bound at opposite ends of the TIM barrel, the usual fold of the radical SAM superfamily. The cluster required for reductive SAM cleavage conserves the features of the radical SAM superfamily, but the auxiliary cluster is bound by a CX4CX5C motif unique to lipoyl synthase. The fourth ligand to the auxiliary cluster is an extremely unusual serine residue. Site-directed mutants show this conserved serine ligand is essential for the sulfur insertion steps. One crystallized lipoyl synthase (LipA) complex contains 5?-methylthioadenosine (MTA), a breakdown product of SAM, bound in the likely SAM-binding site. Modelling has identified an 18 Å (1 Å=0.1 nm) deep channel, well-proportioned to accommodate an octanoyl substrate. These results suggest that the auxiliary cluster is the likely sulfur donor, but access to a sulfide ion for the second sulfur insertion reaction requires the loss of an iron atom from the auxiliary cluster, which the serine ligand may enable.
1470-8728
123-133
Harmer, Jenny
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Hiscox, Martyn J.
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Dinis, Pedro C.
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Fox, Stephen J.
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Iliopoulos, Andreas
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Hussey, James E.
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Sandy, James
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Van Beek, Florian T.
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Essex, Jonathan W.
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Roach, Peter L.
ca94060c-4443-482b-af3e-979243488ba9
Harmer, Jenny
1a640f52-b660-461d-9cf8-17fd80b247d0
Hiscox, Martyn J.
803a1ab7-c693-45c6-98c8-9458e7642042
Dinis, Pedro C.
40a6c6d3-c9f3-44fc-809b-c865d203ce74
Fox, Stephen J.
47218de5-e554-47a2-af51-f045c6720fd9
Iliopoulos, Andreas
7bf6acc7-0bcb-41d6-8ea0-6e2fb28701ba
Hussey, James E.
bdc1d857-e592-44ca-b26e-4ce43439fcb7
Sandy, James
2f22431e-dd2b-4fcf-a68a-d5d068ac478c
Van Beek, Florian T.
7d92997c-fcc0-41f0-895a-c6428b3b549e
Essex, Jonathan W.
1f409cfe-6ba4-42e2-a0ab-a931826314b5
Roach, Peter L.
ca94060c-4443-482b-af3e-979243488ba9

Harmer, Jenny, Hiscox, Martyn J., Dinis, Pedro C., Fox, Stephen J., Iliopoulos, Andreas, Hussey, James E., Sandy, James, Van Beek, Florian T., Essex, Jonathan W. and Roach, Peter L. (2014) Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions. Biochemical Journal, 464 (1), 123-133. (doi:10.1042/BJ20140895).

Record type: Article

Abstract

Lipoyl cofactors are essential for living organisms and are produced by the insertion of two sulfur atoms into the relatively unreactive C–H bonds of an octanoyl substrate. This reaction requires lipoyl synthase, a member of the radical S-adenosylmethionine (SAM) enzyme superfamily. In the present study, we solved crystal structures of lipoyl synthase with two [4Fe–4S] clusters bound at opposite ends of the TIM barrel, the usual fold of the radical SAM superfamily. The cluster required for reductive SAM cleavage conserves the features of the radical SAM superfamily, but the auxiliary cluster is bound by a CX4CX5C motif unique to lipoyl synthase. The fourth ligand to the auxiliary cluster is an extremely unusual serine residue. Site-directed mutants show this conserved serine ligand is essential for the sulfur insertion steps. One crystallized lipoyl synthase (LipA) complex contains 5?-methylthioadenosine (MTA), a breakdown product of SAM, bound in the likely SAM-binding site. Modelling has identified an 18 Å (1 Å=0.1 nm) deep channel, well-proportioned to accommodate an octanoyl substrate. These results suggest that the auxiliary cluster is the likely sulfur donor, but access to a sulfide ion for the second sulfur insertion reaction requires the loss of an iron atom from the auxiliary cluster, which the serine ligand may enable.

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Manuscript for Biochem J v11 merged with figs single.pdf - Accepted Manuscript
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e-pub ahead of print date: 15 November 2014
Published date: 15 November 2014
Organisations: Computational Systems Chemistry

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Local EPrints ID: 390564
URI: http://eprints.soton.ac.uk/id/eprint/390564
ISSN: 1470-8728
PURE UUID: e8090d24-7c19-43e7-ab77-76bd517eed65
ORCID for Jonathan W. Essex: ORCID iD orcid.org/0000-0003-2639-2746
ORCID for Peter L. Roach: ORCID iD orcid.org/0000-0001-9880-2877

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Date deposited: 04 Apr 2016 16:02
Last modified: 05 Nov 2019 02:04

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Contributors

Author: Jenny Harmer
Author: Martyn J. Hiscox
Author: Pedro C. Dinis
Author: Stephen J. Fox
Author: Andreas Iliopoulos
Author: James E. Hussey
Author: James Sandy
Author: Florian T. Van Beek
Author: Peter L. Roach ORCID iD

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