Publications

• Cyanobacterial non-mevalonate pathway: (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase interacts with ferredoxin in Thermosynechococcus elongatus BP-1.

  Okada K., Hase T.

  (E)-4-Hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE), which catalyzes the conversion of 2-C-methyl-D-erythritol cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP), is an essential enzyme of the non-mevalonate (2-C-methyl-D-erythritol-4-phosphate (MEP)) pathw ... >> More

  (E)-4-Hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE), which catalyzes the conversion of 2-C-methyl-D-erythritol cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP), is an essential enzyme of the non-mevalonate (2-C-methyl-D-erythritol-4-phosphate (MEP)) pathway for isoprenoid biosynthesis. The terminal steps of the MEP pathway are still not fully understood, although this pathway is necessary for survival in various organisms such as cyanobacteria, plastids of algae and higher plants, and the apicoplast of human malaria parasites. To determine the efficient redox partner for thermophilic cyanobacterial GcpE, We have expressed the gcpE and petF genes in Escherichia coli and studied the protein-protein interaction of GcpE protein with ferredoxin I (PetF) from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. Recombinant GcpE protein was purified by an N-terminal His(6) tag and reconstituted as a [4Fe-4S](2+) metalloprotein. GcpE was shown to interact strongly with PetF via the bacterial two-hybrid system designed to detect protein-protein interactions. Moreover, a direct protein-protein interaction between PetF and GcpE was confirmed in an in vitro glutathione S-transferase (GST) pull-down assay. To investigate electron transfer activity from PetF to GcpE, we also constructed a NADPH-dependent reducing shuttle system with purified recombinant ferredoxin-NADP(+) oxidoreductase (PetH) and PetF. The result demonstrated that PetF has the ability to transfer electrons to GcpE. Thus, the combined data provide the first evidence that GcpE is a ferredoxin-dependent enzyme in T. elongatus BP-1. << Less

  J. Biol. Chem. 280:20672-20679(2005) [PubMed] [EuropePMC]

• Isoprenoid biosynthesis in chloroplasts via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE) from Arabidopsis thaliana is a [4Fe-4S] protein.

  Seemann M., Wegner P., Schuenemann V., Bui B.T., Wolff M., Marquet A., Trautwein A.X., Rohmer M.

  The mevalonate-independent methylerythritol phosphate pathway is widespread in bacteria. It is also present in the chloroplasts of all phototrophic organisms. Whereas the first steps, are rather well known, GcpE and LytB, the enzymes catalyzing the last two steps have been much less investigated. ... >> More

  The mevalonate-independent methylerythritol phosphate pathway is widespread in bacteria. It is also present in the chloroplasts of all phototrophic organisms. Whereas the first steps, are rather well known, GcpE and LytB, the enzymes catalyzing the last two steps have been much less investigated. 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate is transformed by GcpE into 4-hydroxy-3-methylbut-2-enyl diphosphate, which is converted by LytB into isopentenyl diphosphate or dimethylallyl diphosphate. Only the bacterial GcpE and LytB enzymes have been investigated to some extent, but nothing is known about the corresponding plant enzymes. In this contribution, the prosthetic group of GcpE from the plant Arabidopsis thaliana and the bacterium Escherichia coli has been fully characterized by Mossbauer spectroscopy after reconstitution with (57)FeCl(3), Na(2)S and dithiothreitol. It corresponds to a [4Fe-4S] cluster, suggesting that both plant and bacterial enzymes catalyze the reduction of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate via two consecutive one-electron transfers. In contrast to the bacterial enzyme, which utilizes NADPH/flavodoxin/flavodoxin reductase as a reducing shuttle system, the plant enzyme could not use this reduction system. Enzymatic activity was only detected in the presence of the 5-deazaflavin semiquinone radical. << Less

  J. Biol. Inorg. Chem. 10:131-137(2005) [PubMed] [EuropePMC]

• Isoprenoid biosynthesis in plant chloroplasts via the MEP pathway: direct thylakoid/ferredoxin-dependent photoreduction of GcpE/IspG.

  Seemann M., Tse Sum Bui B., Wolff M., Miginiac-Maslow M., Rohmer M.

  In the methylerythritol phosphate pathway for isoprenoid biosynthesis, the GcpE/IspG enzyme catalyzes the conversion of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate. This reaction requires a double one-electron transfer involving a [4Fe-4S] cluster ... >> More

  In the methylerythritol phosphate pathway for isoprenoid biosynthesis, the GcpE/IspG enzyme catalyzes the conversion of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate. This reaction requires a double one-electron transfer involving a [4Fe-4S] cluster. A thylakoid preparation from spinach chloroplasts was capable in the presence of light to act as sole electron donor for the plant GcpE Arabidopsis thaliana in the absence of any pyridine nucleotide. This is in sharp contrast with the bacterial Escherichia coli GcpE, which requires flavodoxin/flavodoxin reductase and NADPH as reducing system and represents the first proof that the electron flow from photosynthesis can directly act in phototrophic organisms as reducer in the 2-C-methyl-d-erythritol 4-phosphate pathway, most probably via ferredoxin, in the absence of any reducing cofactor. In the dark, the plant GcpE catalysis requires in addition of ferredoxin NADP(+)/ferredoxin oxido-reductase and NADPH as electron shuttle. << Less

  FEBS Lett. 580:1547-1552(2006) [PubMed] [EuropePMC]