The purple phototrophic bacterium Rhodobacter sphaeroides utilises bacteriochlorophyll a for light harvesting and photochemistry. The synthesis of this photopigment includes the reduction of a vinyl group at the C8 position to an ethyl group, catalysed by a C8-vinyl reductase. An active form of this enzyme has not been identified in R. sphaeroides, but its genome contains two candidate ORFs (open reading frames) similar to those reported to encode C8-vinyl reductases in the closely related Rhodobacter capsulatus (bchJ), and in plants and green sulfur bacteria (rsp_3070). To determine which gene encodes the active enzyme, knock-out mutants in both genes were constructed. Surprisingly, mutants in which one or both genes were deleted still retained the ability to synthesize C8-ethyl bacteriochlorophyll. These genes were subsequently expressed in a cyanobacterial mutant that cannot synthesize C8-ethyl chlorophyll a. R. sphaeroides rsp_3070 was able to restore synthesis of the WT (wild-type) C8-ethyl chlorophyll a in the mutant, whereas bchJ did not. The results of the present study demonstrate that Rsp_3070 is a functional C8-vinyl reductase and that R. sphaeroides utilises at least two enzymes to catalyse this reaction, indicating the existence of a third class, while there remains no direct evidence for the activity of BchJ as a C8-vinyl reductase.
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Research Article|
February 15 2013
Identification of an 8-vinyl reductase involved in bacteriochlorophyll biosynthesis in Rhodobacter sphaeroides and evidence for the existence of a third distinct class of the enzyme
Daniel P. Canniffe;
Daniel P. Canniffe
*Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, U.K.
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Philip J. Jackson;
Philip J. Jackson
*Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, U.K.
†ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, U.K.
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Sarah Hollingshead;
Sarah Hollingshead
*Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, U.K.
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Mark J. Dickman;
Mark J. Dickman
†ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, U.K.
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C. Neil Hunter
C. Neil Hunter
1
*Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, U.K.
1To whom correspondence should be addressed (email c.n.hunter@sheffield.ac.uk).
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Publisher: Portland Press Ltd
Received:
November 14 2012
Revision Received:
December 06 2012
Accepted:
December 19 2012
Accepted Manuscript online:
December 19 2012
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2013 Biochemical Society
2013
Biochem J (2013) 450 (2): 397–405.
Article history
Received:
November 14 2012
Revision Received:
December 06 2012
Accepted:
December 19 2012
Accepted Manuscript online:
December 19 2012
Citation
Daniel P. Canniffe, Philip J. Jackson, Sarah Hollingshead, Mark J. Dickman, C. Neil Hunter; Identification of an 8-vinyl reductase involved in bacteriochlorophyll biosynthesis in Rhodobacter sphaeroides and evidence for the existence of a third distinct class of the enzyme. Biochem J 1 March 2013; 450 (2): 397–405. doi: https://doi.org/10.1042/BJ20121723
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