Cyanobacterial non-mevalonate pathway: (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase interacts with ferredoxin in Thermosynechococcus elongatus BP-1.
(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.
Okada
Ken
K
Division of Enzymology, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. okadak@protein.osaka-u.ac.jp
Hase
Toshiharu
T
D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology* / Q000378:metabolism; D004579:Electron Transport; D004798:Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D007506:Iron-Sulfur Proteins; D008798:Mevalonic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology; D011994:Recombinant Proteins; D016415:Sequence Alignment; D014169:Transformation, Bacterial
D004798:Enzymes; D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; C421242:hydroxymethylbutenyl 4-diphosphate synthase; D009249:NADP; D019883:Alkyl and Aryl Transferases; D005982:Glutathione Transferase; D008798:Mevalonic Acid