Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center
The ring contraction process that occurs during cobalamin (vitamin B12) biosynthesis is mediated via the action of two enzymes, CobG and CobJ. The first of these generates a tertiary alcohol at the C-20 position of precorrin-3A by functioning as a monooxygenase, a reaction that also forms a gamma la...
| Main Authors: | , , , , , , , , , |
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| Format: | Journal Article |
| Published: |
The American Society for Biochemistry and Molecular Biology Inc
2009
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| Online Access: | http://hdl.handle.net/20.500.11937/31299 |
| _version_ | 1848753340155953152 |
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| author | Schroeder, S. Lawrence, A. Biedendieck, R. Rose, R. Deery, E. Graham, Ross McLean, K. Munro, A. Rigby, S. Warren, M. |
| author_facet | Schroeder, S. Lawrence, A. Biedendieck, R. Rose, R. Deery, E. Graham, Ross McLean, K. Munro, A. Rigby, S. Warren, M. |
| author_sort | Schroeder, S. |
| building | Curtin Institutional Repository |
| collection | Online Access |
| description | The ring contraction process that occurs during cobalamin (vitamin B12) biosynthesis is mediated via the action of two enzymes, CobG and CobJ. The first of these generates a tertiary alcohol at the C-20 position of precorrin-3A by functioning as a monooxygenase, a reaction that also forms a gamma lactone with the acetic acid side chain on ring A. The product, precorrin-3B, is then acted upon by CobJ, which methylates at the C-17 position and promotes ring contraction of the macrocycle by catalyzing a masked pinacol rearrangement. Here, we report the characterization of CobG enzymes from Pseudomonas denitrificans and Brucella melitensis. We show that both contain a [4Fe-4S] center as well as a mononuclear non-heme iron. Although both enzymes are active in vivo, the P. denitrificans enzyme was found to be inactive in vitro. Further analysis of this enzyme revealed that the mononuclear non-heme iron was not reducible, and it was concluded that it is rapidly inactivated once it is released from the bacterial cell. In contrast, the B. melitensis enzyme was found to be fully active in vitro and the mononuclear non-heme iron was reducible by dithionite. The reduced mononuclear non-heme was able to react with the oxygen analogue NO, but only in the presence of the substrate precorrin-3A. The cysteine residues responsible for binding the Fe-S center were identified by site-directed mutagenesis. A mechanism for CobG is presented. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. |
| first_indexed | 2025-11-14T08:22:57Z |
| format | Journal Article |
| id | curtin-20.500.11937-31299 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| last_indexed | 2025-11-14T08:22:57Z |
| publishDate | 2009 |
| publisher | The American Society for Biochemistry and Molecular Biology Inc |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | curtin-20.500.11937-312992017-09-13T15:21:51Z Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center Schroeder, S. Lawrence, A. Biedendieck, R. Rose, R. Deery, E. Graham, Ross McLean, K. Munro, A. Rigby, S. Warren, M. The ring contraction process that occurs during cobalamin (vitamin B12) biosynthesis is mediated via the action of two enzymes, CobG and CobJ. The first of these generates a tertiary alcohol at the C-20 position of precorrin-3A by functioning as a monooxygenase, a reaction that also forms a gamma lactone with the acetic acid side chain on ring A. The product, precorrin-3B, is then acted upon by CobJ, which methylates at the C-17 position and promotes ring contraction of the macrocycle by catalyzing a masked pinacol rearrangement. Here, we report the characterization of CobG enzymes from Pseudomonas denitrificans and Brucella melitensis. We show that both contain a [4Fe-4S] center as well as a mononuclear non-heme iron. Although both enzymes are active in vivo, the P. denitrificans enzyme was found to be inactive in vitro. Further analysis of this enzyme revealed that the mononuclear non-heme iron was not reducible, and it was concluded that it is rapidly inactivated once it is released from the bacterial cell. In contrast, the B. melitensis enzyme was found to be fully active in vitro and the mononuclear non-heme iron was reducible by dithionite. The reduced mononuclear non-heme was able to react with the oxygen analogue NO, but only in the presence of the substrate precorrin-3A. The cysteine residues responsible for binding the Fe-S center were identified by site-directed mutagenesis. A mechanism for CobG is presented. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. 2009 Journal Article http://hdl.handle.net/20.500.11937/31299 10.1074/jbc.M807184200 The American Society for Biochemistry and Molecular Biology Inc unknown |
| spellingShingle | Schroeder, S. Lawrence, A. Biedendieck, R. Rose, R. Deery, E. Graham, Ross McLean, K. Munro, A. Rigby, S. Warren, M. Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center |
| title | Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center |
| title_full | Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center |
| title_fullStr | Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center |
| title_full_unstemmed | Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center |
| title_short | Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center |
| title_sort | demonstration that cobg, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin b12) biosynthetic pathway, contains an fe-s center and a mononuclear non-heme iron center |
| url | http://hdl.handle.net/20.500.11937/31299 |