Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a [4Fe–4S] protein

AbstractThe last enzyme (LytB) of the methylerythritol phosphate pathway for isoprenoid biosynthesis catalyzes the reduction of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into isopentenyl diphosphate and dimethylallyl diphosphate. This enzyme possesses a dioxygen-sensitive [4Fe–4S] cluster. This prosthetic group was characterized in the Escherichia coli enzyme by UV/visible and electron paramagnetic resonance spectroscopy after reconstitution of the purified protein. Enzymatic activity required the presence of a reducing system such as flavodoxin/flavodoxin reductase/reduced nicotinamide adenine dinucleotide phosphate or the photoreduced deazaflavin radical

Functional analysis of the Arabidopsis thaliana GCPE protein involved in plastid isoprenoid biosynthesis

AbstractPlastid isoprenoids are synthesized via the 2-C-methyl-D-erythritol 4-phosphate pathway. A few years after its discovery, most of the Escherichia coli genes involved in the pathway have been identified, including gcpE. In this work, we have identified an Arabidopsis thaliana protein with homology to the product of this gene. The plant polypeptide, GCPE, contains two structural domains that are absent in the E. coli protein: an N-terminal extension and a central domain of 30 kDa. We demonstrate that the N-terminal region targets the Arabidopsis protein to chloroplasts in vivo, consistent with its role in plastid isoprenoid biosynthesis. Although the presence of the internal extra domain may have an effect on activity, the Arabidopsis mature GCPE was able to complement a gcpE-defective E. coli strain, indicating the plant protein is a true functional homologue of the bacterial gcpE gene product