Cyanobacterial Non-mevalonate Pathway

Abstract

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE), which catalyzes the conversion of 2-Cmethyl-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 Stransferase (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.

Cloning and Characterization of the 2-C-Methyl-D-erythritol 4-Phosphate (MEP) Pathway Genes of a Natural-Rubber Producing Plant,Hevea brasiliensis

“…[30][31][32][33][34][35][36] In addition, insertion sequences, whose function remains unknown, are highly conserved in plant HDS and HDR. 37) These insertion sequences were also highly conserved in the HDS and HDR of H. brasiliensis.…”

Possible direct involvement of the active‐site [4Fe–4S] cluster of the GcpE enzyme from Thermus thermophilus in the conversion of MEcPP

“…Sequence alignments of the available GcpE sequences [13][14][15][16][17] show that there are only three highly conserved cysteine residues that in principle can coordinate a [4Fe-4S] cluster that contains a unique Fe atom or a [3Fe-4S] cluster. In the case of enzyme from Thermosynechococcus elongatus the reconstitution procedure resulted in an enzyme preparation that showed an absorption spectrum with bands at 305, 395 and 585 nm, which was interpreted as being due to a [4Fe-4S] + cluster [12]. No additional methods were used to confirm this.…”

Isotope tracer investigations of natural products biosynthesis: the discovery of novel metabolic pathways

“…To date, it is known that a variety of eubacteria 10 including Actinomycetes (soil bacteria) 2,11 and cyanobacteria proficiently utilize this pathway. 12,13 In fact, a number of bacteria solely utilize the MEP pathway for the biosynthesis of isoprenoids and do not carry the genetic information for the mevalonate pathway. 14 Many eukaryotic organisms, including a number of protists (i.e.…”