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J. Bacteriol. doi:10.1128/JB.01678-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

The C-terminal extension of ferrochelatase is critical for enzyme activity and for functioning of the tetrapyrrole pathway in Synechocystis PCC 6803

Institute of Microbiology, Department of Autotrophic Microorganisms, Opatovicky mlyn, 379 81 Trebon, Czech Republic; Institute of Physical Biology, University of South Bohemia, 373 33 Nove Hrady, Czech Republic; Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Sheffield S10 2TN, United Kingdom; Faculty of Science, University of South Bohemia, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic

^* To whom correspondence should be addressed. Email: sobotka{at}alga.cz.

	Abstract

Heme and chlorophyll (Chl) share a common biosynthetic pathway up to the branch point where magnesium chelatase and ferrochelatase (FeCH) insert either magnesium for Chl biosynthesis or ferrous iron for heme biosynthesis. A distinctive feature of ferrochelatases in cyanobacteria is their C-terminal extension, which forms a putative transmembrane segment containing a chlorophyll-binding motif. We analysed the H324 strain of Synechocystis sp. PCC 6803, which contains a truncated FeCH enzyme lacking this C-terminal domain. Truncated FeCH was localized to the membrane fraction suggesting that the C-terminal domain is not necessary for membrane association of the enzyme. Measurements of enzyme activity and complementation experiments revealed that the H324 mutation dramatically reduced activity of the FeCH, which resulted in highly upregulated 5-aminolevulinic acid synthesis in the H324 mutant implying a direct role for heme in the regulation of flux through the pathway. Moreover, the H324 mutant accumulated a large amount of protoporphyrin IX and levels of Chl precursors were also significantly increased, suggesting that some, but not all, of the ‘extra’ flux can be diverted down Chl branch. Analysis of the recombinant full-length and truncated FeCHs demonstrated that the C-terminal extension is critical for activity of the FeCH and it is strictly required for oligomerization of this enzyme. The observed changes in tetrapyrrole trafficking and the role of the C-terminus in functioning of FeCH are discussed.